Functional Health

Anti-Aging 


Anti-Aging Stem Cell Treatment

Aging is a rather complicated process in which the cells eventually die after becoming progressively damaged over time. However, this process can be reversed or at least impeded by fresh stem cells. These cells have a special anti-aging effect by repairing and regenerating the different organs which have been damaged by stress and various toxic substances which we come across in our day-to-day lives. With stem cell anti-aging we can help repopulate the body with younger cells.  They also help to improve our immune functions significantly.  

While the indications of aging usually begin at around the age of 25, this may vary depending on lifestyle choices. For example, smokers are going to experience it much earlier because of their exposure to toxic elements. Here´s some indications of aging:
Poor concentration, loss of memory, tiredness, loss of energy, general fatigue, mood swings, loss of hair, wrinkles, age spots, reduced sex drive, insomnia, general aches and pains, loss of skin texture as well as degenerative diseases.


Stem Cells Treatment

Stem cells are the supplier of healthy “replacement cells” which you are born with. They are assigned by your body to replace the damaged cells or those which are either old or in the process of dying. With the advent of time, when you start losing healthy cells, the system´s recovery process becomes harder and longer. Eventually, your body loses its ability to heal properly due to age, life illnesses and traumas.


It is at this point that some chronic problems may start. During the stem cell therapy, you are going to be examined by our specialists regarding your present condition and they will also review your medical background comprehensively along with other information in order to get the best outcome.

Results of stem cell anti-aging therapy



Overall improvements

You can expect improvements after stem cell therapy, including but not limited to:

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Physical improvements

Less head/neck aches

Decreased soreness in neck, arms and legs

Reduced stiffness in joints

Far less tiredness or fatigue

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Aesthetic improvements

The skin on the face and hands becomes tighter

Fewer wrinkles

Looking younger – general younger appearance

Change in color of hair from grey to black/normal

Hair thickens

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Mental and Emotional improvements

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Improvements in Energy Levels

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Improvement in the Overall Quality of Life

Reduction of the effects of the degenerative ailments, fewer wrinkles, reduced fatigue, reduced joint stiffness, mental and emotional improvement as well as improved vitality and libido.

How stem cell anti-aging treatment works?


During a typical stem cell anti-aging treatment, 50 to 100 millions umbilical cord-derived donor mesenchymal stem cells are given to the patient by IV infusions and local tissue injections. This young optimised pool of stem cells works in a number of ways

  • • Repopulating of ageing stem cells pools
  • • Regeneration of degraded tissue
  • • Modulation of immune system
  • • Anti-inflammatory mechanisms
  • • Cell-to-Cell stimulatory effects

Consequently, your body becomes renewed and rejuvenated following the active cell replenishment


What is stem cell therapy and why umbilical cord MSCs?

Stem cell therapy refers to the use of stem cells in a therapeutic treatment. Ageing is the decline of stem cells in the body which are responsible for regeneration. Anti-aging is possible by repopulating your regenerative stem cell pools with younger and optimised stem cells. In our opinion the younger acting the stem cell the better, but only in a safe manner. To follow both these aspects we select donor umbilical cord tissue-derived mesenchymal stem cells (MSCs). Umbilical cord MSCs are naive juveneille stem cells which holder greater regenerative capabilities then aged adult stem cells and also produce a unique portfolio of growth factors & RNA which support the stem cell rejuvenation process. Keeping the stem cells young is key to achieve real anti-aging. Many laboratories & stem cell treatment providers offer super huge numbers of stem cells for low prices.





Chronic Back Pain Treatment


Heart disease will ultimately lead to heart failure and it´s a condition in which the heart is not able to pump enough blood throughout the body for its normal function. The cause of heart disease can be either functional or structural disorder of the heart. Common causes of heart failure include cardiomyopathy, hypertension, heart valve problems and coronary artery disease. There is no cure to heart failure, though we provide heart stem cell treatment and therapy for cardiovascular diseases that can improve conditions and symptoms for the patient.

Our program combines the following

  • • Adult Stem Cell injections: Adipose-derived stem cells or Umbilical cord stem cells
  • • Shock Wave Therapy
  • • IV Laser therapy
  • • Tissue laser therapy
  • • Growth factor injections
  • • Peptide therapy
  • • Nutrition infusions
  • • Enzyme injection therapy





Diabetes


Diabetes Stem Cell Treatment

Diabetes is a medical condition that occurs when the body is unable to regulate the sugar concentration in the blood; the concentration is regulated by insulin which is triggered by b-cells when there is an increase in blood glucose.

When patients suffer from diabetes, their b-cells are dysfunctional or are just not producing enough insulin. It is linked to several medical conditions and increases the risk for diseases like heart disease, kidney disease, amputations, blindness and more.



Types of diabetes

  • 1. Type 1 Diabetes is the result of a person’s immune system fighting and destroying the b-cells, it usually begins in the early stages of life and accounts for 10% of the total cases of diabetes. People with this type have to take insulin shots daily in order to survive.
  • 2. Type 2 Diabetes results from b-cell performance decline and an increase in insulin resistance; this type is linked with genetic factors and obesity, hence this type can be prevented by living a healthy lifestyle.
  • 3. There is another type of diabetes that affects pregnant women, it is called Gestational Diabetes where the hormones released by the placenta to sustain the pregnancy, make the pregnant woman’s cells more resistant to insulin. Once the pancreas is unable to overcome the resistance, the patient develops diabetes.

What causes diabetes?


It is not known exactly why the body immune system fights the b-cells and it causes Type 1 diabetes. It´s assumed that this could have been caused by genetic vulnerability and certain environmental factors but there is no absolute clear reason why this happens.


Type 2 diabetes occurs when the cells resist insulin action and the insulin produced is not enough to overcome this resistance, the reason behind this resistance is also unknown but it is strongly assumed that obesity is directly linked to this type of diabetes

Gestational diabetes risks

A woman who develops gestational diabetes has a higher risk of developing Type 2 diabetes. The risk factors for gestational diabetes include:

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Age

Older women are more likely to get gestational diabetes

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Family/personal history

If you have a family member with diabetes, you are more likely to get gestational diabetes and if you have had gestational diabetes in your previous pregnancy, gave birth to a large baby or had a stillbirth, you are at higher risk

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Weight

Being obese prior to the pregnancy increases your risk

Symptoms of diabetes

The symptoms vary depending on sugar concentration; sometimes people with Type 2 diabetes may initially have no symptoms while those with Type 1 diabetes experience symptoms more quickly and more severely.

Some of the typical signs and symptoms of diabetes include:

  • • Patient urinates frequently
  • • Patient gets really hungry
  • • Patient gets thirst too quickly
  • • Losing a lot of weight
  • • The urine has ketones
  • • Patient gets tired really fast
  • • Patient is very irritable
  • • Patient cannot see clearly
  • • Sores heal slowly
  • • Patient gets too many infections e.g. vaginal infections

Diagnosis of diabetes

The diagnosis of diabetes involves testing the concentration of sugar in the blood which can be done by using a variety of tests like the glycated hemoglobin test, random blood test, fasting blood sugar test, and oral glucose tolerance test. Urine can also be tested for the presence of ketones to check if the patient has auto-antibodies.



Diabetes Stem Cell Treatment

Mesenchymal stem cells (MSCs) are self-renewing multipotent cells that have the capacity to secrete multiple biologic factors that can restore and repair injured tissues.

It is known that MSCs play a crucial role in healing damaged tissues. They can differentiate to replace the dead cells as well as secrete stimulant factors to activate surrounding cells in the microenvironment, enhancing the tissue repair process. Therefore, MSCs can be applied to treat tissues impaired by chronic hyperglycemia. MSC transplantation can increase beta cell mass via the following effects: 

(1) beta cell replacement through cellular differentiation;

(2) local microenvironment modification by production of cytokines, chemokines and factors to stimulate endogenous regeneration;

(3) reduction or prevention of autoimmunity to beta cells. Immunomodulatory and inflammatory effects of MSCs also contribute to the reduction of insulin resistance.

Results achieved with stem cell therapy

  • • Significant decrease in fasting blood sugars and the level of Hemoglobin A1C
  • • Significant decrease in Triglyceride levels
  • • Measurable improvement in kidney function with a decrease in creatinine levels

Protect Your Heart with Astra oil Red

  • Be able to prevent and restore macular degeneration, help to treat eye strain, help to treat macular degeneration which is considered as top reason of vision loss in elderly. 
  • Protect skin and eyes from UV ray. Apart from that, it is also found that, Astaxanthin can restore healthy skin and reduce wrinkles 
  • Help to relieve tiredness after workout and playing sport
  • Help to protect brain cells and slowdown memory disorder and Alzheimer's disease
  • Increase blood circulation, reduce blood pressure, increase HLD, reduce triglyceride in blood and help to facilitate function of mitochondrial which produce energy in cardiac muscle cells 
  • Help to balance immunity in people who suffer from allergy, autoimmune disease, weakened immune system, and people who suffer from chronic viral infections, such as HIV, hepatitis virus, herpes and Herpes Zoster. 
  • Prevent liver degeneration in diabetes patients
  • Help to make sperm stronger and help keep men's prostate healthy
  • Help to reduce body's inflammation
  • Help relieve gastroesophageal reflux disease, indigestion, gastritis, both H. pylori infected and uninfected.





Visual /Ocular Diseases 


Sight is arguably our most important sense; we rely upon it to navigate through our surroundings with ease. Loss of vision can have a huge impact on a person’s life, but many of the disorders that cause blindness are currently difficult or impossible to treat. Researchers are now using stem cell technology to explore possible new approaches to treatments for loss of vision.

Treatments for most disorders that cause vision loss are difficult or not yet possible.

Specialised cells in the eye serve specific functions to focus light and turn what is being seen into signals sent to the brain. The eye contains several types of stem cells that constantly replace specialised cell that become worn out or damaged.

Many diseases that cause blindness are still not treatable. Researchers are working to understand what causes these diseases, what other types of stem cells reside in the eye and how stem cells might be used to repair or even restore vision to patients. Many of these studies are still in the early years. 

Heart Stem Cell Treatment

When looking at the heart, stem cells have the ability to perform two main roles:

Home into the damaged areas
• Stimulate multiple biological events which cause healing of the heart muscle

For example, studies have demonstrated that stem cell therapy will cause new muscle cells to be formed through stimulation of dormant stem cells that are already inside the heart muscle. In these studies, the administered stem cells also transformed into new heart muscle cells.

Our heart stem cell treatment protocol for heart failure involves the administration of mesenchymal stem cells harvested from human umbilical cord tissue or from adipose tissue from the patient in the early stage of the disease.

These stem cells along with specific peptides and stem cell growth factors can show real benefits to patients suffering from heart disease.

Supportive therapies such as Biophoton blood laser cleaning and blood oxygen therapy also support the body’s healing process. 





Osteoarthritis Stem Cell Treatment



Osteoarthritis is a degenerative disease where the cartilage that covers the end of bones in a joint wears off, leading to pain and swelling when moving; in worse cases, the bones break down developing a growth and the broken bits of bone float around the joint.
Osteoarthritis is a chronic disease that affects the joints. It can affect any joint mostly in knees, lower back, hips, fingers, neck and others.

What causes osteoarthritis?


In a normal joint, there is a tissue that allows frictionless joint movement. In osteoarthritis, the smooth surface of cartilage becomes rough and it eventually wears down; it might even lead to bone to bone friction which will lead to wearing of the bones. The cartilage can become rough due to old age, overusing joints, genes, bone deformities, and joint injuries.

Some of the factors that may increase your risk of osteoarthritis include:

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Aging

The older the person, the higher the risk of osteoarthritis.

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Gender

For reasons still unknown, women are more susceptible to osteoarthritis than men.

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Obesity

Being overweight increases your risk of osteoarthritis in various ways; your weight may cause too much stress on weight-bearing joints and also the fat tissue may release proteins that may lead to inflammation in and around the joints.

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 Injuries

If you keep getting joint injuries or have ever had a joint injury that healed, you at a greater risk of osteoarthritis.

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Work, Sport

If you have a job that includes tasks that place a lot of continuous stress on joints or if you also do a lot of exercises that put a lot of strain on your joints, you are at a higher risk.

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Genes

If you have a close family member who has osteoarthritis, you may be at higher risk of osteoarthritis.

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Deformities

People born with any kind of bone deformity are at higher risk of osteoarthritis.

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Other types of joint diseases

People with other joint diseases like rheumatoid arthritis and gout are at greater risk of osteoarthritis.

Symptoms of osteoarthritis

Symptoms vary from person to person. A typical symptom is a reaction to external natural elements like the weather, some people will experience different pain intensity based on the weather. Some of the signs and symptoms of osteoarthritis include:

1. Stiff joints
When you are at rest or still, your joints will feel stiff and then it wears off once you start to move.

2. Pain
You feel pain when you are moving the joints or at the end of the day; the pain becomes more severe as the osteoarthritis worsens.

3. Grating sensation
Due to bone friction, your bones may produce a creaking sound when you move or use the affected joint.

4. Problem using a joint
You may experience trouble when using your joint; it might not move freely or it may be less stable.

5. Bone lumps
When bones break down, the bits float around the joint forming lumps. 

How is osteoarthritis diagnosed?

Once you have experienced some of the symptoms described above, you may want to check with your doctor for osteoarthritis. During the check-up, the doctor tests your blood to rule out other types of arthritis. Then, you take an x-ray scan which may show the bone spurs or even show if you have any calcium deposits around your joints.


Osteoarthritis stem cell treatment

Stem cells have the ability to grow into various tissues, cells or even cartilages, and can help manage the symptoms of osteoarthritis. Stem cells are administered into the body using injections; once the stem cells are in the joint their objective is to fight off inflammation, to reduce pain, and to increase lubrication of joints by increasing production of synovial fluid.
Patients experience improvements typically soon after the stem cell injections are applied to the affected area.

Results for knee osteoarthritis include:

  • Patients feel the relief.
  • Pain and stiffness are gone.
  • There is no restriction in movement.
  • Improved flexibility.
  • Improved mobility.
  • Cartilage repair.

OSTEOARTHRITIS KNEE STEM CELL TREATMENT PROGRAM OPTIONS (7-14 days)

Option 1) 200,000,000 purified Mesenchymal stem cells
Option 2) 100,000,000 purified Mesenchymal stem cells

  • Interarticular MSC injections / IV infusions MSC
  • Bioquark peptide
  • Cartilage cell growth factors
  • Shockwave therapy
  • Interarticular Laser therapy
  • Stem Cell stimulation Laser therapy
  • Oxygen therapy
  • Enzyme + Nutriton course





Spinal Cord Injury


Spinal cord injury (SCI) occurs when the spinal cord becomes damaged, most commonly, when motor vehicle accidents, falls, acts of violence, or sporting accidents fracture vertebrae and crush or transect the spinal cord.

Damage to the spinal cord usually results in impairments or loss of muscle movement, muscle control, sensation and body system control.


Can stem cells help treat spinal cord injury?


Presently, post-accident care for spinal cord injury patients focuses on extensive physical therapy, occupational therapy, and other rehabilitation therapies; teaching the injured person how to cope with their disability.

A number of published papers and case studies support the feasibility of treating spinal cord injury with allogeneic human umbilical cord tissue-derived stem cells and autologous bone marrow-derived stem cells.

Through administration of umbilical cord tissue-derived mesenchymal stem cells, we have observed improvements in spinal cord injury patients treated at our facilities. 


How is osteoarthritis diagnosed?Which types of stem cells are used to treat spinal cord injuries and how are they collected?

The adult stem cells used to treat spinal cord injuries at the Stem Cell Institute come from two sources: the patient’s own bone marrow (autologous mesenchymal and CD34+) and human umbilical cord tissue(allogeneic mesenchymal). Umbilical cords are donated by mothers after normal, healthy births.

A licensed anesthesiologist harvests bone marrow from both hips under light general anesthesia in a hospital operating room. This procedure takes about 1 1/2 – 2 hours. Before they are administered to the patient, these bone marrow-derived stem cells must pass testing for quality, bacterial contamination (aerobic and anaerobic) and endotoxin.

All donated umbilical cords are screened for viruses and bacteria to International Blood Bank Standards.
Only a small percentage of donated umbilical cords pass our rigorous screening process.


What are the advantages of treating spinal cord injury with allogeneic umbilical cord tissue-derived stem cells?

  • • Since HUCT mesenchymal stem cells are immune system privileged, cell rejection is not an issue and Human Leukocyte Antigen (HLA) matching is not necessary.
  • • The stem cells with the best anti-inflammatory activity, immune modulating capacity, and ability to stimulate regeneration can be screened and selected.
  • • Allogeneic stem cells can be administered multiple times over the course of days in uniform dosages that contain high cell counts.
  • • Umbilical cord tissue provides an abundant supply of mesenchymal stem cells.
  • • There is a growing body of evidence showing that umbilical cord-derived mesenchymal stem cells are more robust than mesenchymal stem cells from other sources.
  • • No need to administer chemotherapy drugs like Granulocyte-colony stimulating factor (G-CSF or GCSF) to stimulate the bone marrow to produce granulocytes and stem cells and release them into the bloodstream.





Visual /Ocular Diseases 


Sight is arguably our most important sense; we rely upon it to navigate through our surroundings with ease. Loss of vision can have a huge impact on a person’s life, but many of the disorders that cause blindness are currently difficult or impossible to treat. Researchers are now using stem cell technology to explore possible new approaches to treatments for loss of vision.

Treatments for most disorders that cause vision loss are difficult or not yet possible.

Specialised cells in the eye serve specific functions to focus light and turn what is being seen into signals sent to the brain. The eye contains several types of stem cells that constantly replace specialised cell that become worn out or damaged.

Many diseases that cause blindness are still not treatable. Researchers are working to understand what causes these diseases, what other types of stem cells reside in the eye and how stem cells might be used to repair or even restore vision to patients. Many of these studies are still in the early years.


Can stem cells help treat spinal cord injury?


Presently, post-accident care for spinal cord injury patients focuses on extensive physical therapy, occupational therapy, and other rehabilitation therapies; teaching the injured person how to cope with their disability.

A number of published papers and case studies support the feasibility of treating spinal cord injury with allogeneic human umbilical cord tissue-derived stem cells and autologous bone marrow-derived stem cells.

Through administration of umbilical cord tissue-derived mesenchymal stem cells, we have observed improvements in spinal cord injury patients treated at our facilities. 


Replacing retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells have a number of important jobs, including looking after the adjacent retina. If these cells stop working properly due to damage or disease, then certain parts of the retina die. As the retina is the component of the eye responsible for detecting light, this leads to the onset of blindness. RPE cells can be damaged in a variety of diseases such as: age-related macular degeneration (AMD), retinitis pigmentosa and Leber’s congenital aneurosis.

One way to treat these diseases would be to replace the damaged RPE cells with transplanted healthy cells. Unfortunately, it is not possible to take healthy RPE cells from donors so it is necessary to find another source of cells for transplantation. Scientists have recently produced new RPE cells from both embryonic stem cells and iPS cells in the lab. The safety of embryonic stem cell-derived RPE cells has been tested in phase I/II clinical trials for patients with Stargardt’s macular dystrophy, and for thse affected by AMD by a stem cell biotech company called Advanced Cell Technologies. The results of the trial, published in 2014 , demonstrated safety and showed engraftment of the transplanted RPE cells. However, some participants experienced adverse side effects from the immunosuppression and the transplantation procedure itself. Interestingly, despite not being an endpoint of this trial, several patients also reported an improvement in vision.

A second Phase I/II trial exploring the use of RPEs derived from human embryonic stem cells for people with wet AMD is currently underway in the United Kingdom. The first patient received their transplant in September 2015. This work, led by Prof Pete Coffey, is ongoing and is being carried out at Moorfields Eye Hospital as part of the London Project to Cure Blindness.

Finally, Japanese researcher, Dr Masayo Takahashi is leading a clinical trial in Japan which transplants RPE cells made from iPS cells into patients with wet AMD. The trial was put on hold for several months due to regulatory changes in Japan and concerns about mutations in an iPS cell product to be used in the trial. The trial has recommenced June 2016 and many await the results.

There are several other phase I or I/II clinical trials using pluripotent stem cells world-wide involving small numbers of participants. These trials are examining primarily the safety, but in some cases also the effectiveness, of the use of RPEs developed from pluripotent stem cells in dry and wet AMD and Stargardt’s macular degeneration.

Replacement of damaged RPE cells will only be effective in patients who still have at least part of a working retina, and therefore some level of vision (i.e. at early stages of the disease). This is because the RPE cells are not themselves responsible for ‘seeing’, but are actually responsible for supporting the ‘seeing’ retina. Sight is lost in these types of diseases when the retina begins to degenerate because the RPE cells are not doing their job properly. So the RPE cells need to be replaced in time for them to support a retina that is still working. It is hoped that transplantation of new RPE cells will then permanently halt further loss of vision, and in some cases may even improve vision to some degree.





Replacing retinal pigment epithelial cells

therapies are being researched and tested in early clinical safety trials.

Replacing retinal cells
In many of the cases where vision is lost, we often find that the problem lies with malfunctioning retinal circuitry. Different disorders occur when particular, specialized cells in the circuit either stop working properly or die off. Despite the retina being more complicated than other components of the eye, it is hoped that if a source of new retinal cells can be found, we may be able to replace the damaged or dying cells to repair the retina. In addition, this approach may also help to repair damage caused to the optic nerve.

Again, scientists have turned to stem cell technology to provide the source of replacement cells. Several studies have now reported that both embryonic stem cells and iPS cells can be turned into different types of retinal cells in the lab. Within the eye, a type of cell called the Müller cell, which is found in the retina, is known to act as a stem cell in some species, such as the zebra fish. It has been suggested that this cell may also be able to act as a stem cell in humans, in which case it may provide another source of retinal cells for repair of the retina.

Unlike RPE cell transplantation, direct repair of the retina may allow patients who have already lost their vision to have it restored to some degree. This gives hope for patients with disorders like late-stage age-related macular degeneration, where the light-sensitive photoreceptor cells in the retina have already been lost. This type of research may also provide new treatments for people who suffer from retinal diseases like retinitis pigmentosa and glaucoma. However, despite encouraging evidence, such research is very much in its infancy. There are currently no patient clinical trials planned using this type of approach, as significant further research is still required first.

Stem cell technology holds great potential for improving the lives of people who suffer from visual disorders. A number of studies are currently being undertaken in order to develop new therapies to treat, and/or prevent a loss of vision. Central to this research is the development of our understanding of how different types of stem cells behave, and how best to harness their potential in the eye. A tailored approach is required, dependent upon the particular problem a patient is experiencing. Stem cells are not a one-stop, generic cure, but they do hold exciting potential for the production of new biological components that can be used to repair the eye.

Unlike RPE cell transplantation, direct repair of the retina may allow patients who have already lost their vision to have it restored to some degree. This gives hope for patients with disorders like late-stage age-related macular degeneration, where the light-sensitive photoreceptor cells in the retina have already been lost. This type of research may also provide new treatments for people who suffer from retinal diseases like retinitis pigmentosa and glaucoma. However, despite encouraging evidence, such research is very much in its infancy. There are currently no patient clinical trials planned using this type of approach, as significant further research is still required first.

Stem cell technology holds great potential for improving the lives of people who suffer from visual disorders. A number of studies are currently being undertaken in order to develop new therapies to treat, and/or prevent a loss of vision. Central to this research is the development of our understanding of how different types of stem cells behave, and how best to harness their potential in the eye. A tailored approach is required, dependent upon the particular problem a patient is experiencing. Stem cells are not a one-stop, generic cure, but they do hold exciting potential for the production of new biological components that can be used to repair the eye. 

Replacing the nerve cells of the retina

Current research aims to understand how to produce retinal nerve cells that could be used in future therapies.

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Did you know?

Blinking damages the surface of your eyes, but you do it about 12 times per minute. It's a good thing your eyes have stem cells to repair the damage!

Embryonic and induced pluripotent (iPS) stem cells cultured with growth factors become pigmented (brown in colour) and show characteristics of retinal pigment epithelium

Adding the right growth factors to Müller stem cells can make them become retinal nerve cells, as shown by the presence of a protein known as Islet 1 (pink).





VESTA CELL PRODUCTS

Stem cell therapy of the highest stem cell count and regenerative potentials.
Exceptional regenerative and repairative potentials and intelligence (more than 10 – 100 time regenerative potency of ordinary stem cell therapies) – A complete solution of degenerative diseases and rejuvenation. 


Introduction

Progenir is a stem cell research and therapy centre is a co-established by the world renowned stem cell scientistsfrom Europe, USA, Japan, Israel and Thailand, concentrating in the Research & Development of various protocols of stem cells therapy, e.g. separation of stem cells from different sources, stem cel therapies and cell based altertnative therapies. Progenir had made several remarkable achievements in stem cell therapy, especially in the cancer treatment which combines umbilical cord stem cells and Autologous Natural Killer Cells therapy, which achieve 80% total remission in 32 weeks. Progenir Stem Cell Therapies have also proven to improve all kinds of neurodegenerative diseases with total efficacies of 85%, and 90% success rate in stem cell based hair restoration…etc

Mesenchymal Stem Sell (MSC) 

A multipotent stem cells which can differentiate into various types of functional tissues in human body. MSC is the type of stem cell with the widest clinical applicationsand highest regenerative potentials. Clinical studies have proven that MSC implantation can lead to regeneration of cardiac muscles, neurons, bone marrows, liver, pancreatic Islet, bones, cartilages and other vital tissues, makes it an effective treatments for cardiomyopathy, coronary arteries disease, neurological and spinal cord trauma, neurodegenerative diseases, joints degenerations, muscular dystrophy, leukaemia…etc.

MSCs can be found in various body tissues as it is the ancestors of many body tissues. However, abundant of MSCs are discovered in adipose tissues, bone marrow and umbilical cord tissues. With the advancement in medical sciences, bone marrow aspiration, which is an invasive procedures yielding relatively low stem cell counts are no longer prevalent in stem cell isolation practices. More scientists are trying to isolate MSCs from umbilical cord tissues and adipose tissues. Many clinical studies has also proven the exceptional regenerative potentials of adipose and unblical cord derived stem cells, rendering a new hope for the cure of various chronic degenerative diseases. 

Progenir Stem Cell Isolation technique has resolved the technological bottlenecks and is able to isolate 50 millions to 200 millions MSCs from a single umbilical cord. The MSCs are genetically healthy with high regenerative and differentiation potentials which will exert regeneration and repair soon after infusion into the body. It has been proven clinically to be effective in several chronic diseases.

Stemcell Treatments for Chronic Diseases & Anti-Aging  

  • -Anti-Aging
  • -Chronic Back Pain
  • -Osteoarthritis
  • -Diabetes
  • -Heart
  • -Spinal Cord Injury
  • -Visual/Ocular
  • -Multiplesclerosis 
  • -Kidney
  • -Lung 
  • -Gastrointestinal disorders
  • -Liver 
  • -Rheumatoid arthritis & Other bones degeneration 
  • -Muscular Dystrophy
  • -Multiple System Atrophy 
  • -Wound Healing 
  • -Neurological disorder
  • -Urological disorder
  • -Stroke

Progenir Stem Cell therapy is indicated for various chronic degenerative conditions which are incurable to conventional medicines. For further enquiries, kindly contact us in the following methods with attached medical reports. 





Kidney


Mesenchymal stem cell treatment for chronic  renal failure

Chronic renal failure is an important clinical problem with significant socioeconomic impact worldwide. Despite advances in renal replacement therapies and organ transplantation, poor quality of life for dialysis patients and long transplant waiting lists remain major concerns for nephrologists treating this condition. There is therefore a pressing need for novel therapies to promote renal cellular repair and tissue remodeling. Over the past decade, advances in the field of regenerative medicine allowed development of cell therapies suitable for kidney repair. Mesenchymal stem cells (MSCs) are undifferentiated cells that possess immunomodulatory and tissue trophic properties and the ability to differentiate into multiple cell types. Studies in animal models of chronic renal failure have uncovered a unique potential of these cells for improving function and regenerating the damaged kidney. Nevertheless, several limitations pertaining to inadequate engraftment, difficulty to monitor, and untoward effects of MSCs remain to be addressed. Adverse effects observed following intravascular administration of MSCs include immune rejection, adipogenic differentiation, malignant transformation, and prothrombotic events. Nonetheless, most studies indicate a remarkable capability of MSCs to achieve kidney repair. This review summarizes the regenerative potential of MSCs to provide functional recovery from renal failure, focusing on their application and the current challenges facing clinical translation. 


Introduction

Chronic kidney disease (CKD) is a prevalent condition  (8 to 16%) associated with all cause and cardiovascular mortality. Importantly, CKD can progress towards end-stage renal disease (ESRD), requiring renal replacement therapy. ESRD currently accounts for 6.3% of the Medicare spending in the United States, and is projected  to increase by 85% by 2015. Furthermore, ESRD has 
a tremendous impact on quality of life and life expectancy of affected individuals. Therefore, it is imperative to develop therapeutic interventions to prevent, alleviate, or decelerate progression of renal failure.

Diabetes mellitus and hypertension represent major  causes of CKD and initiation of dialysis in the United 
States. In addition, glomerular diseases, malnutrition, infectious diseases, and acute kidney injury can progress to ESRD, contributing to the increased global burden of death associated with this condition. Current treatment modalities often fail to target the major underlying contributors for progression of renal disease. Chronic glomerular and tubulointerstitial fibrosis is a common pathway to ESRD, often associated with apoptosis, oxidative damage, and microvascular rarefaction. In fact, renal dysfunction is postulated to better correlate with the degree of tubulointerstitial than with glomerular damage.

Importantly, the kidney possesses intrinsic regenerative  capacity that allows the organ to recover after limited insults. Unfortunately, this regenerative potential is limited under chronic conditions and thus inefficient to prevent progressive glomerulosclerosis and tubulointerstial fibrosis. Treatment strategies that boost cellular regeneration might therefore offer good alternatives for patients with CKD.

Mesenchymal stem cells (MSCs) can be isolated from a  variety of tissues, differentiate into multiple cell lineages, and possess unique immunomodulatory properties that ameliorate inflammation and immune responses, constituting a promising tool to facilitate renal repair. MSCs are de fined by the presence of plastic-adherent cells under standard culture conditions, capacity to differentiate into osteoblasts, adipocytes and chondroblasts in vitro,expression of typical surface markers (CD29, CD44, CD73, CD90, CD105, and CD166), and the lack of CD45, CD34, CD14 or CD11b, CD79αor CD19 and HLA-DR surface molecules [10]. In recent years, experimental studies have uncovered the potential of MSCs to improve renal function in several models of CKD, and several clinical studies have indicated their safety and efficacy in CKD. Nevertheless, a number of hurdles need to be addressed before clinical translation. This review summarizes the current state of MSC transplantation for CKD, focusing on their mechanisms of renal repair, complications, obstacles for clinical translation, and potential approaches to overcome them 









Mesenchymal stem cells in experimental chronic kidney disease


Over the past few years, MSCs have been successfully applied in experimental models of CKD such as diabetes, hypertension, and chronic allograft nephropathy (Table 1). For example, a single intravenous dose of MSCs resulted in beta-pancreatic islet regeneration, prevented renal dam- age in streptozotocin-induced type 1 diabetes in C57BL/6 mice, and decreased hyperglycemia and glycosuria that persisted for 2 months after injection. Furthermore, MSC-treated diabetic mice showed histologically normal glomeruli, and albuminuria fell. Although the authors did not assess cellular mechanisms associated with MSC therapeutic effects, the long-lasting persistence of injected MSCs may suggest a direct effect to elicit kidney regeneration
Similarly, Lee and colleagues tested the effectiveness of intracardiac infusions of MSCs from human bone marrow in immunodeficient mice with type 2 diabetes produced with multiple low doses of streptozotocin. MSCs lowered blood glucose levels and decreased mesangial thickening and macrophage infiltration, suggesting their potential for improving renal lesions in subjects with diabetes mellitus. Interestingly, in kidneys of MSC-treated diabetic mice, a few injected human MSCs differentiated into glomerular endothelial cells. 

Additionally, in rats with modified 5/6 nephrectomy, a  single venous injection of MSCs 1 day after nephrectomy preserved renal function and attenuated  renal injury. Despite unchanged blood urea nitrogen and creatinine levels, MSC-treated animals showed at- 
tenuated progression of proteinuria. The scarce en graftment of MSCs in the kidneys of rats with chronic renal failure suggests that paracrine secretion 
of mediators, such as cytokines or growth factors, may have accounted for their beneficial effects. Indeed, vascular endothelial growth factor (VEGF) levels were substantially higher in MSC-treated animals 1 month after MSC injection 

Furthermore, a single dose of bone marrow-derived  MSCs 11 weeks after kidney transplantation in rats decreased interstitial fibrosis, tubular atrophy, T-cell and 
macrophage infiltration, and the expression of inflammatory cytokines. Interestingly, a decrease over time in the inflammatory and profibrotic cytokine levels in MSC-treated animals was associated with an increase in  the anti-inflammatory cytokine IL-10, although none of the injected MSCs were detected 7 days after delivery. These observations imply that the beneficial effect of these cells in this setting is primarily attributable to their paracrine immunomodulatory properties rather than long-term engraftment. 

We have previously shown in swine atherosclerotic renovascular disease that intrarenal delivery of MSCs isolated from subcutaneous adipose tissue protected the stenotic kidney despite sustained hypertension. Notably, MSCs also attenuated renal inflammation, endoplasmic-reticulum stress, and apoptosis through mechanisms involving cell contact. Furthermore, adjunctive MSCs improved renal function and structure after renal revascularization and 
reduced inflammation, oxidative stress, apoptosis, microvascular remodeling, and fibrosis in the stenotic kidney (Figure 1). This strategy also restores oxygen dependent tubular function in the stenotic-kidney medulla, extending their value to preserving medullary structure and function in chronic ischemic conditions

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Potential challenges for clinical translation

While preclinical studies have established the safety and efficacy of MSCs in different models of CKD, these results need cautious translation into routine clinical practice. Trials using MSCs for CKD patients may face various challenges, including selecting the optimal route of MSC delivery, scant homing and engraftment, immune rejection, ensuring thriving, and tracking of injected cells. Addressing these challenges may bolster the success of MSC therapy in improving renal function in CKD patients

Route of delivery 

The route of MSC delivery may influence the cells’capacity to home and engraft the damaged tissue, and thereby their efficacy for renal repair. Commonly used experimental methods to deliver MSCs include systemic intravenous, intra-arterial, or intraparenchymal delivery.When intravenously delivered in normal Sprague–Daw-ley rats, the majority of MSCs are initially trapped in thelungs, but in nonhuman primates the cells distribute broadly into the kidneys, skin, lung, thymus, and liver with estimated levels of engraftment ranging from 0.1 to 2.7%.
In contrast, direct delivery of MSCs into the renal artery of an ischemic kidney is associated with retention rates of 10 to 15%,, although the normal swine kidney retains only around 4%, due to the low tonic release of injury signals. However, injection of human MSCs into the mouse abdominal aorta may lead to occlusion in the distal vasculature due to their relatively large cell size (16 to 53 μm), suggesting that this approach should be used cautiously. Injections of MSCs into the renal parenchyma or their local subcapsular implantation confer renoprotective effect, but are difficult to implement in the human injured kidney

In experimental models of CKD, the optimal dose of MSCs is often empirical, with doses ranging from 0.5 × 〖10〗^6 to 10 × 〖10〗^6. Despite variability in dose regimens and route of delivery, the safety and beneficial effects of MSCs were consistent among studies. Nevertheless, the use of escalating doses is strongly recommended in clinical trials, and chronic adverse events should be evaluated prior to enrollment at the next dose level. 

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Homing

Circulating hematopoietic progenitor cells home to the damaged kidney by responding to injury signals that correspond to cognate surface receptors which they express. Accumulating evidence indicates that exogenously infused MSCs respond to similar homing signals. In mice, expression of CD44 and its major ligand hyaluronic acid mediates MSC migration to the injured kidney, and hyaluronic acid also promotes MSC dosedependent migration in vitro. Moreover, renal homing of intravenously injected MSCs was blocked by preincubation with the CD44 blocking antibody or by soluble hyaluronic acid, suggesting that CD44 and hyaluronic acid interactions recruit exogenous MSCs to the injured kidney. In addition, Liu and colleagues found that, when administered systemically, MSCs home to the ischemic kidney, improving renal function,accelerating mitogenic response, and reducing cell apoptosis, but these effect were abolished by either CXCR4 or CXCR7 inhibition,implicating the stromal derived factor-1CXCR4/CXCR7 axis in kidney repair.

Collectively, these observations suggest that strategies aimed to enhance MSC expression of homing signals may improve their capacity to attenuate renal dysfunction.Studies have shown that selective manipulation of MSCs before transplantation (preconditioning) enhances their ability to protect damaged tissues. The rationale underpinning this approach is that transplanted MSCs encounter a hostile microenvironment that mitigates their reparative capabilities and survival. Indeed, preconditioning with the mitogenic and prosurvival factor insulin-like growth factor (IGF)-1 before systemic infusion of bone marrow-derived MSCs (2 × 〖10〗^5) upregulates the expression of CXCR4 and restores normal renal function in a mice model of cisplatin-induced acute kidney injury 





Engraftment

Chronic renal failure is an important clinical problem with significant socioeconomic impact worldwide. Despite advances in renal replacement therapies and organ transplantation, poor quality of life for dialysis patients and long transplant waiting lists remain major concerns for nephrologists treating this condition. There is therefore a pressing need for novel therapies to promote renal cellular repair and tissue remodeling. Over the past decade, advances in the field of regenerative medicine allowed development of cell therapies suitable for kidney repair. Mesenchymal stem cells (MSCs) are undifferentiated cells that possess immunomodulatory and tissue trophic properties and the ability to differentiate into multiple cell types. Studies in animal models of chronic renal failure have uncovered a unique potential of these cells for improving function and regenerating the damaged kidney. Nevertheless, several limitations pertaining to inadequate engraftment, difficulty to monitor, and untoward effects of MSCs remain to be addressed. Adverse effects observed following intravascular administration of MSCs include immune rejection, adipogenic differentiation, malignant transformation, and prothrombotic events. Nonetheless, most studies indicate a remarkable capability of MSCs to achieve kidney repair. This review summarizes the regenerative potential of MSCs to provide functional recovery from renal failure, focusing on their application and the current challenges facing clinical translation. 

Some studies suggest that MSCs have the capacity to engraft the damaged tissue, integrate into tubular cells, and differentiate into mesangial cells. In swine renovascular disease, 4 weeks after intrarenal infusion, MSCs(10 × 〖10〗^6) were detected in all regions of the kidney, but mostly at the renal interstitium . On the other hand, intravenous infusion of bone marrow-derived MSCs (2 × 〖10〗^5) in mice with cisplatin-induced acute renal failure reduced the severity of renal injury, but none were detected within the renal tubules and only few cells within the renal interstitium at 1 to 4 days after infusion suggesting that MSC engraftment is not necessary to achieve renoprotection. Likewise, despite significant improvement in renal function, within 3 days of intracarotid infusion in a rat model of ischemia–reperfusion-induced acute renal failure, none of the MSCs differentiated into the tubular or endothelial cell phenotype, indicating that their beneficial effects are primarily mediated via paracrineactions rather than differentiation into target cells.

Methods to increase MSC engraftment may therefore enhance their utility in regenerative cellular therapy. Temporary obstruction of the renal artery following intrarenal delivery may prevent cell washout, and is associated with significant retention rates in the postischemic kidney. Alternatively, in a rat model of acute kidney injury s-nitroso N-acetyl penicillamine preconditioning enhances MSC engraftment, ultimately associated with a significant improvement in renal function.

Despite the crucial role attributed to MSC engraftment in potentiating the cells’beneficial effect at the site of in jury, there is currently consensus that the chief mechanism by which MSCs protect the damaged kidney is the release of growth factors, proangiogenic factors, and anti-inflammatory cytokines. Cultured MSCs release large amounts of the proangiogenic factor VEGF, which facilitates glomerular and tubular recovery MSCs can also produce IGF-1, while administration of IGF-1 gene-silenced MSCs limits their protective effect on renal function and tubular structure in murinecisplatin-induced kidney injury, indicating that MSCsexert their beneficial effects by producing IGF-1 .

Importantly, these paracrine actions of MSCs seem to mediate their immunomodulatory properties. In ischemia–reperfusion-induced acute kidney injury, infusion of MSCs downregulates renal expression of proinflammatory cytokines and adhesion molecules such as IL-1β, tumor necrosis factor alpha, interferon gamma, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1, but upregulates the expression of the anti-inflammatory IL-10 [26,33]. Likewise, we have shown in swine renovascular disease that intrarenal delivery of MSCs during renal revascularization decreased renal expression of tumor necrosis factor alpha and monocyte chemoattractant protein-1, but increased IL10 expression. Moreover, MSCs induced a shift in the macrophage phenotype from inflammatory (M1) to reparative (M2), uncovering their immunomodulatory potential. Taken together, these observations underscore the contribution of paracrine actions of MSCs to induce a shift from an inflammatory to an anti-
inflammatory microenvironment. It is not unlikely that the type, number, and expansion methods used to secure MSCs alter their engraftment capacity



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Rejection

For many years, MSCs have been considered immune privileged because of the lack of expression of co-stimulatory molecules and their capacity to decrease renal release and expression of inflammatory mediators. These attributes engendered the hope that MSCs could engraft in allogeneic nonimmunosuppressed recipients, and stimulated development of off-the-shelf allogeneic MSC products, which allow rapid generation of large amounts of cells from few donors. Nevertheless, in vivo and in vitro studies have demonstrated that MSCs may occasionally induce an immune switch transitioning from an immune privileged to an immunogenic phenotype that triggered cellular cytotoxicity or immune rejection. Moreover, implantation of murine MSCs engineered to release erythropoietin in major histocompatibility complexmismatched allogeneic mice increased the proportion of host-derived lymphoid CD8+ and natural killer infiltrating
cells, suggesting that MSCs are not intrinsically immune privileged. Taken together, these observations do not support the use of allogeneic MSCs as a universal cellular platform, at least until development of unequivocally immunoprivileged MSCs. Therefore, at this point, administration of autologous MSCs seems to be the safest
strategy

Thriving

An important feature of MSCs is their capacity to induce proliferation of renal glomerular and tubular cells, increasing cellular survival. By secreting proangiogenic and trophic factors, injected MSCs not only can enhance proliferation, but also can decrease apoptosis of tubular
cells. We have shown in swine renovascular disease that a single intrarenal delivery of MSCs in conjunction with renal revascularization increased proliferation of renal cells, and recently confirmed in vitro that MSCs blunt apoptosis by decreasing the expression of caspase-3.

However, whether MSCs remain in the circulation long enough to exert any long-lasting effect is a matter of debate. Ezquer and colleagues showed that intravenous MSCs home into the kidney of type 1 diabetic mice, and some donor MSCs remained in the kidney up to 2 months
later. Similarly, we found that 4 weeks after intrarenal delivery a significant number of MSCs were retained in the injected kidney, whereas by 12 weeks after cell transfer only a few cells were observed in the kidney, yet their beneficial effects were sustained. Longitudinal studies are needed to document the chronology of MSC retention and beneficial benefits in the kidney. Additionally, development of novel interventions such as preconditioning may enhance survival and potency of MSCs in renal failure. For instance, MSCs exposed to hypoxic conditions in culture sustain viability and function through preservation of oxidant status, and preconditioning with kallikrein or melatonin enhances their therapeutic potential

An important feature of MSCs is their capacity to induce proliferation of renal glomerular and tubular cells, increasing cellular survival. By secreting proangiogenic and trophic factors, injected MSCs not only can enhance proliferation, but also can decrease apoptosis of tubular
cells. We have shown in swine renovascular disease that a single intrarenal delivery of MSCs in conjunction with renal revascularization increased proliferation of renal cells, and recently confirmed in vitro that MSCs blunt apoptosis by decreasing the expression of caspase-3.
from exogenous MSCs illustrate their potential for transformation into tumors, underscoring the requirement for closely monitoring human MSCs in clinical studies. Alternatively, complications and maldifferentiation of live replicating MSCs warrant development of safer tactics and interventions.

Considerable evidence shows that MSCs release microvesicles which exhibit characteristics of their parental cells, and transfer proteins, lipids, and genetic material to target cells. We have recently shown that endothelial outgrowth cells release microvesicles,which may mediate their intercellular communications. Similarly, MSCs are avid producers of microvesicles (Figure 2) that shuttle functional components for their paracrine action. Delivery of microvesicles instead of their parent MSCs could avoid concerns about extensive expansion, cryopreservation, complications, and maldifferentiation of live replicating cells. Indeed, microvesicles derived from preconditioned MSCs promoted recovery in a rat hind-limb ischemia model. However, questions regarding their composition and potency relative to their parent MSCs remain unanswered,underscoring the need for studies to clarify the potential of this promising therapeutic modality.

Uremic conditions may also affect the efficacy of MSCs, limiting their potential use in patients with CKD. Uremia induced by partial kidney ablation in C57Bl/6 J mice leads to MSC functional incompetence, characterized by decreased expression of VEGF, VEGF receptor-1, and stromal derived factor-1, increased cellular senescence, and decreased proliferation. Conversely, MSCs isolated from subcutaneous adipose tissue of healthy controls and patients with renal disease show similar characteristics and functionality, underscoring the feasibility of autolo gous cell therapy in patients with renal disease [50]. Indeed, a recent meta-analysis of prospective clinical trials that used intravascular delivery of MSCs concluded that these cells have an excellent safety record.


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Interspecies differences in the biology of mesenchymal
stem cells


Although it is accepted that MSCs from different species are capable of differentiation into various lineages and express common MSC markers, species-dependent variability in their expression has been reported among different species. Furthermore, the mechanism of MSC-mediated immunosuppression varies among different species. For example, while immunosuppression by human-derived or monkey-derived MSCs is mediated by indoleamine 2,3dioxygenase, the molecular mechanisms underlying immunosuppression in mouse MSCs utilize nitric oxide. Several immune barriers have been also encountered in experimental xenotransplantation, the transplantation of MSCs from one species to another, warranting the development of genetic alternatives to overcome these obstacles. Clearly, results from experimental studies need to be carefully validated before clinical translation


Tracking

There is also a pressing need for better methods for detection and monitoring the fate of MSCs. Despite improvement in direct (fluorescent probe) and indirect (reporter genes) labeling techniques, questions regarding interactions of MSCs with tissue, differentiation,or migration remain unanswered. While fluorescent probes such as membrane tracers or microspheres need to be detected with histological techniques in a cell or organelle, reporter genes such as bioluminescence or fluores cent proteins can be used to identify different cell populations using imaging in vivo. However, these detection methods have little tissue penetration, limiting their use in large animal models or humans.

Conceivably, imaging modalities such as single-photonemission computed tomography or magnetic resonance imaging may address some of these deficiencies by providing high-resolution anatomical detail and tracking of cell viability [60,61]. Several types of agents are currently used for labeling MSCs for their detection with magnetic resonance imaging. Among them, superparamagnetic iron oxide particles are the most commonly applied, because of their capacity to induce changes in T2 relaxivity in vivo. However, the transfection agents used for superparamagnetic iron oxide particle internalization may also affect cell viability, and dying cells accumulate iron until dissolved or eliminated by phagocytosis, impeding their application as indices of cell viability. Further methods are therefore needed to better assess engraftment, survival,and function of MSCs in human subjects.


Clinical trials using mesenchymal stem cells for
renal repair




There are many theories about the different functions of Hydrogen Peroxide in the body and a great deal of scientific material supports almost every one. Hydrogen Peroxide is produced in the body in different amounts for different purposes. It is part of a system which helps you use the OXYGEN you breathe. It's is part of a system which helps your body regulate all living cell membranes. It is a hormonal regulator. necessary for your body to produce several hormonal substances such as estrogen. progesterone and thyroid. It is important in the regulation of blood sugar. and the production of energy in all cells. It helps regulate certain chemicals necessary to operate the brain and nervous system. It is used in the defense system of the body Lo kill bacteria. virus, yeast and parasites and has been found to be important in regulating the immune system. Scientists are discovering the function of Hydrogen Peroxide in the body is far more complex and important than previously realized.

Few clinical trials have tested safety and efficacy of MSCs for renal disease. Reinders and colleagues studied safety and feasibility in six kidney allograft recipients who received two intravenous infusions of expanded autologous bone marrow-derived MSCs (〖10〗^6cells/kg,  7 days apart) because of rejection and/or increased interstitial fibrosis and tubular atrophy [63]. Although the design of the study does not allow one to draw conclusions on efficacy, in two recipients with allograft rejection the renal biopsies after MSC treatment demonstrated resolution of tubulitis without interstitial fibrosis and tubular atrophy, whereas maintenance immune suppression remained unaltered, supporting the potential of MSCs in preventing allograft rejection. However, three patients developed an opportunistic infection, raising concerns regarding systemic immunosuppression after MSC infusions. Similarly, a recent prospective, open-label, randomized study demonstrated that, among patients undergoing renal transplant, intravenous infusion of marrow-derived autologous MSCs (1 × 〖10〗^6  to 2 × 〖10〗^6/kg) at kidney reperfusion and 2 weeks later decreased the in cidence of acute rejection and of opportunistic infection,and improved renal function at 1 year compared with  anti-IL-2 receptor antibody induction therapy. Importantly, delivery of autologous MSCs was not associated with adverse events, nor did it compromise graft survival. Likewise, autologous MSC infusion in two recipients of kidneys from living-related donors 7 days post-transplant restricted memory T-cell expansion and enlarged the T-regulatory cell population. These observations suggest safety and clinical feasibility of cell based therapy with MSCs in the context of kidney 
transplantation

Several clinical trials are currently underway to evaluate the therapeutic potential of autologous and allogeneic MSCs for treatment of renal diseases. For example, NCT01843387 investigates the safety,tolerability and efficacy of a single intravenous infusion of two doses of MSCs versus placebo in subjects with diabetic nephropathy and type 2 diabetes. NCT00659620 will test whether MSCs are effective in preventing organ rejection and maintaining kidney function in patients who develop chronic allograft nephropathy. In addition, NCT00659217 evaluates infusion of expanded autologous MSCs into patients with lupus nephritis. Finally, NCT01840540 is a phase I study of autologous MSCs in the treatment of atherosclerotic renal artery stenosis. While they aim primarily to test the feasibility and practical usefulness of MSCs in renal diseases, results from these clinical tri als may also shed light on the mechanisms responsible for MSC renal protection.


Conclusions and future Perspectives

Available experimental evidence confirms that MSCs contribute to cellular repair and ameliorate renal injur





Multiplesclerosis

Treatment with Umbilical Cord Stem Cells Safe with Sustained Benefits for MS, Trial Shows


Treatment with umbilical cord stem cells was found to be safe and leads to sustained improvements in disability and brain lesions of multiple sclerosis (MS) patients, according to a clinical trial.
The study, “Clinical feasibility of umbilical cord tissue-derived mesenchymal stem cells in the treatment of multiple sclerosis,” was published in the Journal of Translational Medicine.
Although current treatments for MS are able to reduce the frequency of flare-ups and slow disease progression, they are not able to repair the damage to nerve cells or the myelin sheath, the protective layer around nerve fibers.
MSCs may inhibit immune-mediated alterations. In particular, MSCs derived from the umbilical cord have a high ability to grow and multiply, increase the production of growth factors, and possess superior therapeutic activity, compared with other MSCs.
Diverse clinical studies have shown that MSCs can safely treat certain immune and inflammatory conditions, including MS.
The research team had previously demonstrated that MSCs can also improve cognitive and motor function.
Recent results with placenta or umbilical cord MSCs showed few mild or moderate adverse events, as well improvements in patients’ level of disability.
Researchers at the Stem Cell Institute in Panama have now completed a one-year Phase 1/2 clinical study (NCT02034188) to test the effectiveness and safety of umbilical cord MSCs for the treatment of MS.
The trial included 20 MS patients with a mean age of 41 years, 60 percent of whom were women. Fifteen participants had relapsing-remitting MS, four had primary progressive MS, and one had secondary progressive MS. Patients’ disease duration was a mean of 7.7 years.
Participants received seven intravenous infusions of 20×106 umbilical cord MSCs over seven days. The treatment’s effectiveness was evaluated at the start, at one month, and at one year after treatment.
Assessments included evaluating brain lesions with magnetic resonance imaging (MRI) and disability based on the Kurtzke Expanded Disability Status Scale (EDSS), as well as validated MS tests for neurological function, hand function, mobility, and quality of life.
Patients did not report any serious adverse events. Most mild adverse events possibly related to treatment were headaches, which are common after MSC infusions, and fatigue, which is common in MS patients, the authors observed.
Improvements were most evident at one month after treatment, namely in the level of disability, nondominant hand function, and average walk time, as well as bladder, bowel, and sexual dysfunction. Patients also reported improved quality of life.
MRI scans at one year after treatment revealed inactive lesions in 15 of 18 evaluated patients. One patient showed almost complete elimination of lesions in the brain, which “is a particularly encouraging finding,” the researchers wrote.
At the one year point, improvements in disability levels were also still present, and could translate into improved ability to walk and work without assistance.
“The potential durable benefit of UCMSC [umbilical cord MSC] at 1 month, and sustained in some measures to 1 year, is in stark contrast to current MS drug therapies, which are required to be taken daily or weekly,” the researchers wrote.
The safety of the treatment is another advantage over available MS therapies, the team said.
They concluded that “treatment with UCMSC intravenous infusions for subjects with MS is safe, and potential therapeutic benefits should be further investigated.”





Lungs

Potential of stem cell therapy to repair lung damage

A new study has found that stem cell therapy can reduce lung inflammation in an animal model of chronic obstructive pulmonary disease (COPD) and cystic fibrosis. Although, still at a pre-clinical stage, these findings have important potential implications for the future treatment of patients.

The findings were presented in Estoril, Portugal today (25 March, 2017) at the European Respiratory Society's Lung Science Conference.

Lung damage caused by chronic inflammation in conditions such as COPD and cystic fibrosis, leads to reduced lung function and eventually respiratory failure. Mesenchymal stem cell (MSC) therapy is currently being investigated as a promising therapeutic approach for a number of incurable, degenerative lung diseases. However, there is still limited data on the short and long-term effects of administering stem cell therapy in chronic respiratory disease.

The new research investigated the effectiveness of MSC therapy in a mouse model of chronic inflammatory lung disease, which reflects some of the essential features of diseases such as COPD and cystic fibrosis.

Researchers delivered stem cells intravenously to b-ENaC overexpressing mice at 4 and 6 weeks of age, before collecting samples tissue and cells from the lungs at 8 weeks. They compared these findings to a control group that did not receive the MSC therapy.

The results showed that inflammation was significantly reduced in the group receiving MSC therapy. Cells counts for both monocytic cells and neutrophils, both signs of inflammation, were significantly reduced after MSC therapy. Analysis of lung tissue revealed a reduction in the mean linear intercept and other measures of lung destruction in MSC treated mice. As well as reducing inflammation in the lung, MSC therapy also resulted in significant improvements in lung structure, suggesting that this form of treatment has the potential to repair the damaged lung.

Dr Declan Doherty, from Queens University Belfast, UK, commented: "These preliminary findings demonstrate the potential effectiveness of MSC treatment as a means of repairing the damage caused by chronic lung diseases such as COPD. The ability to counteract inflammation in the lungs by utilising the combined anti-inflammatory and reparative properties of MSCs could potentially reduce the inflammatory response in individuals with chronic lung disease whilst also restoring lung function in these patients. Although further research is needed to improve our understanding of how MSCs repair this damage, these findings suggest a promising role for MSC therapy in treating patients with chronic lung disease.

Professor Rachel Chambers, ERS Conferences and Research Seminars Director, commented: This paper offers novel results in a pre-clinical model which demonstrates the potential of MSC stem cell therapy for the treatment of long-term lung conditions with exciting potential implications for the future treatment of patients with COPD and cystic fibrosis. Although, still at an early stage in terms of translation to the human disease situation, this paper is one of many cutting-edge abstracts from the Lung Science Conference, which aims to provide an international platform to highlight novel experimental lung research with therapeutic potential. We rely on high quality basic and translational respiratory science, such as these latest findings, to develop novel therapeutic approaches for the millions of patients suffering from devastating and often fatal respiratory conditions. 





Gastrointestinal disorders

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Significance of Mesenchymal Stem Cells in Gastrointestinal Disorders

In this review we summarise the recent developments regarding the experimental and clinical use of mesenchymal stem cells (MSCs), focusing mainly on the treatment of gastrointestinal disorders. Next to their relevance in the field of regenerative medicine and immunology, this population of cells has also raised great expectations for possible applications in cancer therapy. While clinical trials were able to demonstrate the efficacy of MSCs in cases of inflammatory bowel disease and degenerative conditions of the liver, controversial results have been presented regarding their antineoplastic potential in gastrointestinal tumours. MSCs can differentiate into a large variety of specialised cells. They are capable of regulating both wound healing and immune responses through paracrine and endocrine signalling. Moreover, MSCs can be transfected with a great number of different therapeutic genes - considering their ability to selectively migrate towards neoplastic tissues, this feature allows for targeted therapy of solid tumours. Transfected genes can be designed so that they are expressed exclusively in the vicinity of the tumour, eventually triggering apoptosis in cancer cells. In this review, we demonstrate the natural distribution of exogenously applied MSCs in the host. Furthermore, we mention various methods of tracking MSCs in vivo and different parameters of administration that tend to influence therapeutic outcome (e.g., origin of MSCs, mode of application, or the potency of transfected genes). Finally, this review points out the hazards of MSC therapy, emphasising the risks related to their widespread clinical use.





Liver

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Mobilization of hepatic mesenchymal stem cells from human liver grafts.

Extensive studies have demonstrated the potential applications of bone marrow-derived mesenchymal stem cells (BM-MSCs) as regenerative or immunosuppressive treatments in the setting of organ transplantation. The aims of the present study were to explore the presence and mobilization of mesenchymal stem cells (MSCs) in adult human liver grafts and to compare their functional capacities to those of BM-MSCs. The culturing of liver graft preservation fluids (perfusates) or end-stage liver disease tissues resulted in the expansion of MSCs. Liver-derived mesenchymal stem cells (L-MSCs) were equivalent to BM-MSCs in adipogenic and osteogenic differentiation and in wingless-type-stimulated proliferative responses. Moreover, the genome-wide gene expression was very similar, with a 2-fold or greater difference found in only 82 of the 32,321 genes (0.25%). L-MSC differentiation into a hepatocyte lineage was demonstrated in immunodeficient mice and in vitro by the ability to support a hepatitis C virus infection. Furthermore, a subset of engrafted MSCs survived over the long term in vivo and maintained stem cell characteristics. Like BM-MSCs, L-MSCs were found to be immunosuppressive; this was shown by significant inhibition of T cell proliferation. In conclusion, the adult human liver contains an MSC population with a regenerative and immunoregulatory capacity that can potentially contribute to tissue repair and immunomodulation after liver transplantation.

Rheumatoid arthritis & Other bones degeneration

Mesenchymal stem cell-based therapies in regenerative medicine: applications in rheumatology


Growing knowledge on the biology of mesenchymal stem cells (MSCs) has provided new insights into their potential clinical applications, particularly for rheumatologic disorders. Historically, their potential to differentiate into cells of the bone and cartilage lineages has led to a variety of experimental strategies to investigate whether MSCs can be used for tissue engineering approaches. Beyond this potential, MSCs also display immunosuppressive properties, which have prompted research on their capacity to suppress local inflammation and tissue damage in a variety of inflammatory autoimmune diseases and, in particular, in rheumatoid arthritis. Currently, an emerging field of research comes from the possibility that these cells, through their trophic/regenerative potential, may also influence the course of chronic degenerative disorders and prevent cartilage degradation in osteoarthritis. This review focuses on these advances, specifically on the biological properties of MSCs, including their immunoregulatory characteristics, differentiation capacity and trophic potential, as well as the relevance of MSC-based therapies for rheumatic diseases.

Intoduction

For several years, mesenchymal stem cells (MSCs; also called mesenchymal stromal cells) have been largely studied and used as a new therapeutic tool for a number of clinical applications, in particular for the treatment of rheumatologic disorders. MSCs indeed have therapeutic potential for bone and joint diseases due to their multipotent differentiation abilities and the secretion of a variety of cytokines and growth factors that confer on them anti-fibrotic, anti-apoptotic, pro-angiogenic and immunosuppressive properties. They are currently being tested in several clinical trials for such diverse applications as osteoarthritis, osteogenesis imperfecta, articular cartilage defects, osteonecrosis and bone fracture. More-over, good manufacturing practices for the production of clinical-grade MSCs at high expansion rates without transformation are now well established. Here, we review the present knowledge on the mechanisms underlying the therapeutic properties of MSCs and their applications in animal models and clinics in the fields of bone and cartilage repair, chronic inflammatory or degenerative disorders, as well as genetic diseases.

Definition of mesenchymal stem cells: location and characterization

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MSCs were first identified in the bone marrow (BM) but are now described to reside in connective tissues and notably in adipose tissue (AT), placenta, umbilical cord, dental pulp, tendon, trabecular bone and synovium. It has also been suggested that MSCs could reside in virtually all post-natal organs and tissues. BM and AT are, however, the two main sources of MSCs for cell therapy due to high expansion potential and reproducible isolation procedures. Historically, the first characterized MSCs derived from BM remain the most intensively studied and are still the reference. AT-derived MSCs (ASCs) are easier to isolate in high numbers. Nevertheless, while they display characteristics similar to BM-MSCs, their transcriptomic and proteomic profiles show specificities particular to the tissue origin. MSCs have also been described to reside in a perivascular location and to express markers specific for pericytes. However, in AT, ASCs are mainly located in the stroma around the adipocytes and only few of them have a perivascular location. Importantly, in the tissue, none or very few ASCs express pericyte markers, even those that are located around the vessels.

MSCs are defined according to three criteria proposed by the International Society for Cellular Therapy. First, MSCs are characterized as a heterogeneous cell population that is isolated by its property of adherence to plastic. In culture, MSCs are able to develop as fibroblast colony forming-units. Second, MSCs are distinguished by their phenotype: MSCs express the cell surface markers CD73, CD90 and CD105 and are negative for CD11b, CD14, CD34, CD45 and human leukocyte antigen (HLA)-DR. More recently, the CD271 marker was used to isolate highly enriched BM-MSC populations [16]. Whereas BM-MSCs are negative for the CD34 marker, native ASCs can be isolated according to CD34 expression, although this rapidly disappears with cell proliferation in vitro. The third criterion to define MSCs, based on a functional standard, is their capacity to differentiate into at least three mesenchymal lineages, namely bone, fat and cartilage.

Functional properties of mesenchymal stem cells

Differentiation capacity and paracrine signaling are both properties relevant for therapeutic applications of MSCs. MSC differentiation contributes by regenerating damaged tissue, whereas MSC paracrine signaling regulates the cellular response to injury.

Differentiation properties

MSCs are an attractive source of cells for bone and cartilage engineering because of their osteogenic and chondrogenic potential. Their differentiation capacity is generally shown in vitro using specific culture conditions but a0lso in vivo in different animal models. Besides this trilineage potential, MSCs can also differentiate into myocytes, tendinocytes, cardiomyocytes, neuronal cells with neuron-like functions  and other cell types. The differentiation potential is dependent on environmental factors, such as growth factors, but also physical parameters, such as oxygen tension, shear and compressive forces, and elasticity of the extracellular three-dimensional environment.

Paracrine properties

MSCs release various soluble factors that influence the microenvironment by either modulating the host immune response or stimulating resident cells.

The immunomodulatory properties of MSCs, characterized by the capacity to inhibit the proliferation and function of all immune cells, have been largely described both in vitro and in vivo. Immunomodulation requires the preliminary activation of MSCs by immune cells through the secretion of the proinflammatory cytokine IFN-γ, together with TNF-α, IL-1α or IL-1β. The induction of MSC immunomodulation is principally mediated by soluble mediators. Among these, indoleamine 2,3-dioxygenase has been shown to be a major player in human MSCs but absent or poorly expressed in murine cells, while nitric oxide is expressed at low levels in human MSCs but at high levels in murine MSCs following IFN-γ stimulation. Transforming growth factor (TGF)-β1, hepatocyte growth factor (HGF), heme oxygenase1, IL6, leukemia inhibitory factor, HLAG5, IL-10 and IL-1 receptor antagonist (IL-1RA) as well as prostaglandin E2 have been proposed as other mediators involved in MSC-mediated immunomodulation. MSCs suppress B- and T-cell proliferation and alter their function, inhibit the proliferation of activated natural killer cells, interfere with the generation of mature dendritic cells from monocytes or CD34+ progenitor cells, and induce an immature dendritic cell phenotype. Finally, MSCs inhibit Th17 cell differentiation and induce fully differentiated Th17 cells to exert a T cell regulatory phenotype.

Although soluble mediators are the main actors in MSC immunosuppression, cell-cell interactions have been shown to be involved in this process. Recently, toll-like receptor (TLR) stimulation has been shown to modulate the action of MSCs on the immune system. Indeed, TLR4-primed MSCs, or MSC1, mostly elaborate pro-inflammatory mediators, while TLR3-primed MSCs, or MSC2, express mostly immunosuppressive ones.

The trophic properties of MSCs are related to the tissue regeneration process through bioactive factors. These factors may act directly, triggering intracellular mechanisms of injured cells, or indirectly, inducing secretion of functionally active mediators by neighboring cells. MSCs are capable of attenuating tissue injury, inhibiting fibrotic remodeling and apoptosis, promoting angiogenesis, stimulating stem cell recruitment and proliferation, and reducing oxidative stress. As an example, in a hamster heart failure model, intramuscularly injected MSCs, or even more importantly MSC-conditioned medium, significantly improve cardiac function. Improvement occurred via soluble mediators acting on proliferation and angiogenesis, resulting in higher numbers of myocytes and capillaries, and on apoptosis and fibrosis, which were significantly reduced. The prominent factors identified in these processes were HGF and vascular endothelial growth factor (VEGF). The authors demonstrate the activation of the JAK-STAT3 axis in myocytes, which increases the expression of the target genes HGF and VEGF. Activation of the STAT3 pathway is crucial since its inhibition by TLR4 activation inhibits MSC-mediated cardioprotection. Secretion of VEGF by MSCs also attenuates renal fibrosis through immune modulation and remodeling properties in different models of kidney injury. The other mediators that are important actors during tissue remodeling and fibrosis formation are matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs). MSC-secreted TIMPs are capable of playing important roles both under physiological conditions in their niche and in pathological situations.

Chemotactic properties

Injured tissues express specific receptors or ligands that are believed to trigger the mobilization of MSCs into the circulation, facilitating trafficking, adhesion and infiltration of MSCs to the damaged or pathological tissues, in a mechanism similar to the recruitment of leukocytes to sites of inflammation. In the damaged tissues, MSCs are believed to secrete a broad spectrum of paracrine factors that participate in the regenerative microenvironment and regulate immune infiltration. Administration of MSCs, either systemically or locally, has been reported to contribute to tissue repair, suggesting the need to enhance the pool of endogenous MSCs with exogenously administered MSCs for efficient repair. A better under-standing of MSC trafficking and homing mechanisms should help in designing novel therapeutic options to compensate for a deficiency in the number or function of MSCs that may occur in injured tissues.

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Therapeutic applications of MSCs in rheumatology

MSCs for bone and cartilage repair

Interest in using MSCs for tissue engineering has been validated in numerous pre-clinical models and is under evaluation in clinics. At least 16 clinical trials are recruiting for the therapeutic application of MSCs for cartilage defects, osteoporosis, bone fracture, or osteonecrosis. For successful tissue engineering approaches, implantation of MSCs will require the use of growth and differentiation factors that will allow the induction of the specific differentiation pathways and the maintenance of the bone or chondrocyte phenotype together with an appropriate scaffold to provide a three-dimensional environment. Defining the optimal combination of stem cells, growth factors and scaffolds is thus essential to provide functional bone and cartilage.

Bone engineering strategies are warranted in cases of large bone defects or non-union fractures, which remain a serious problem as the associated loss of function considerably impairs the quality of life of affected patients. A vast variety of bone graft substitutes is already commercially available or under intense pre-clinical investigation to evaluate their appropriateness to serve as biomaterials for tissue engineering strategies. Briefly, bone substitutes are assigned to the group of either inorganic (mostly calcium phosphate- or calcium sulphate-based materials, or bioactive glasses) or organic matrices (natural processed bone graft or synthetic polymers). Moreover, it has to be stressed that the success of bone graft substitutes needs a functional vascular network to obtain high quality osseous tissue. Enhanced vascularisation is generally achieved by the provision of angiogenic growth factors that have been shown to increase bone healing. To date, corticocancellous bone grafts remain the most frequently used way of reconstructing large bone segments. Despite promising reports on the potential of bone engineering, particularly for oral and maxillofacial surgeries, these innovative therapeutic strategies have so far been too sporadic, and with low numbers of patients, to give interpretable results. Further efforts are needed to state more precisely the indications in which tissue engineered constructs could replace conventional therapies and improve clinical outcome of patients.

After traumatic or pathological injury, the capacity of adult articular cartilage to regenerate is limited. The current proposed surgeries (microfracture, osteochondral auto- or allografts, or cell-based therapies using chondrocytes) may lead to fibrocartilage and not restore hyaline articular cartilage in the long term. Several kinds of combined scaffolds have been evaluated for cartilage engineering using MSCs. More recently, micron-sized fibers, produced by the electrospinning technique, were shown to provide a structure and properties comparable to the cartilage extracellular matrix and to enhance chondrogenesis. Efforts are being made to improve scaffolds by combining several biomaterials (poly(lactic-co-glycolic acid) sponge and fibrin gel) with an inducing factor (TGF-β1) with satisfactory results. Recently, our group has shown that MSC-coated pharmacologically active microcarriers releasing TGF-β3 implanted in severe combined immunodeficiency (SCID) mice resulted in the formation of cartilage, suggesting that they could represent a promising injectable biomedical device for cartilage engineering. An alternative way to avoid direct transplantation of MSCs for tissue engineering is to recruit endogenous progenitor cells. Indeed, the replacement of the proximal condyle in a rabbit by a TGF-β3-infused bioscaffold resulted, 4 months later, in a scaffold fully covered with avascular hyaline cartilage in the articular surface. The scaffold was also integrated within the regenerated subchondral bone, suggesting that the regeneration was probably due to homing of endogenous cells. Although much progress has been made in the manipulation of cells and constructs for cartilage engineering, the generation of functional repaired tissue remains to be optimized.

MSCs for treatment of genetic diseases

Recent advances in stem cell research have prompted the development of cell-based therapies to replace cells that are deficient in genetic diseases. Osteogenesis imperfecta is a rare genetic disorder due to abnormal collagen type I production by osteoblasts, resulting in osteopenia, multiple fractures, severe bone deformities and considerably shortened stature. To replace defective osteoblasts, the infusion of allogeneic whole BM or isolated BM-MSCs producing normal collagen type I was evaluated in two studies. Although linear growth rate, total body bone mineral content, and fracture rate improved in some patients, the relatively short-term follow-up prevented the authors from drawing firm conclusions about the efficacy of MSC therapy. In a subsequent study with infusions of labelled BM-MSCs, Horwitz and colleagues reported that engraftment was evident in one or more sites, including bone, skin, and marrow stroma, in five out of six patients. These five patients had an acceleration of growth velocity during the first 6 months after infusion. Moreover, the trans-plantation of allogeneic foetal liver-derived MSCs in a foetus with severe osteogenesis imperfecta led to 0.3% of cell engraftment and differentiation of the donor cells into osteocytes until more than 9 months after transplant.

Hypophosphatasia, another metabolic bone disease, is a rare, heritable disease due to deficient activity of tissue nonspecific alkaline phosphatase, often causing death in the first year of life due to respiratory complications. In a young girl, transplantation of 5/6 HLA-matched T-cell-depleted BM resulted in clinical and radiographic improvement without correction of the biochemical features of hypophosphatasia during the first 6 months. However, skeletal demineralization occurred 13 months after transplantation and the decision was therefore taken to infuse BM cells that had been expanded ex vivo. Six months later, considerable, lasting clinical and radiographic improvement ensued, still without correction of her biochemical abnormalities. Despite the small number of studies, patients with metabolic bone diseases have benefited from allogeneic MSC therapy.

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MSCs for the treatment of inflammatory disorders

Due to their immunosuppressive properties, MSCs may be of interest in the treatment of inflammatory disorders such as rheumatoid arthritis, which is the most prominent inflammatory rheumatic disease. To date, conflicting results have been reported using the collagen-induced arthritis (CIA) experimental mouse model. In several studies, the injection of MSCs derived from BM or AT in the CIA mouse model after the establishment of the disease improved the clinical score. These effects were associated with a decrease in Th1-driven inflammation and TNF-α or IFN-γ serum levels as well as induction of a regulatory T cell phenotype. More recently, our group has shown that IL-6-dependent prostaglandin E2 secretion by MSCs inhibits local inflammation in experimental arthritis. However, this beneficial effect of MSCs in rheumatoid arthritis is still controversial since different studies have shown that the injection of the C3H10T1/2 MSC line, Flk-1(+) MSCs, or MSCs derived from DBA/1 mice did not exert a positive effect on CIA or even aggravate the symptoms. This discrepancy in the effect of MSCs may be caused by the different sources of MSCs, but we have reported that altering the course of the disease depends on precise timing of MSC administration. This therapeutic window is likely to be associated with the immune status of the mice since it has been recently reported that MSCs are polarized towards an inflammatory MSC1 or immunosuppressive MSC2 phenotype depending on the type of TLR activation.

MSCs for treatment of chronic degenerative disorders

Osteoarthritis is the most frequent rheumatic disease and is characterized by degeneration of articular cartilage, mainly due to changes in the activity of chondrocytes in favor of catabolic activity. However, recent data now suggest that osteoarthritis also involves other joint tissues, with alterations of the meniscus, sclerosis and edema in the underlying subchondral bone as well as intermittent inflammation of synovium. MSC-based therapy may act via two ways, either preventing cartilage degradation through the secretion of bioactive factors, or by differentiating into chondrocytes and contributing to cartilage repair. The different options to deliver MSCs to the osteoarthritis joint have been summarized recently [56]. Indeed, the co-culture of human MSCs with primary osteoarthritis chondrocytes allowed the differentiation of MSCs towards chondrocytes even in the absence of growth factors. This effect was dependant on cell-cell communication for secretion of morphogen by chondrocytes, suggesting that MSCs injected in a joint might differentiate into chondrocytes. Secretion of bioactive mediators by MSCs may prevent loss of chondrocyte anabolic activity or stimulate progenitors present in the cartilage. As an example, the delivery of autologous MSCs to caprine joints subjected to total meniscectomy and resection of the anterior cruciate ligament resulted in regeneration of meniscal tissue and significant chondroprotection. In an experimental rabbit model of osteoarthritis, transplantation of a hyaluronan-based scaffold seeded with BM-MSCs statistically improved the quality of the regenerated tissue compared to the animal control. Loss of proteoglycans and osteophyte formation were less in the animals treated with MSCs. In humans, eight clinical trials are currently recruiting patients to test the efficacy of MSC injection for treatment of osteoarthritis. Indeed, a phase I/II trial is currently evaluating the effect of MSC injection with hyaluronan (in the form of Chondrogen™) to prevent subsequent OA in patients undergoing meniscectomy. The mechanisms of MSC-based therapy remain unknown, but it has been speculated that secreted biofactors might reduce fibrocartilage formation or decrease degradation by inhibiting proteinases. More-over, although osteoarthritis is not considered an inflammatory disease, secretion of cytokines, namely IL-1β and TNF-α, and immune responses may also be suppressed thanks to the immunomodulatory effects of MSCs. The various reports therefore argue for a therapeutic efficacy of MSCs in preventing or limiting osteoarthritis lesions in patients.

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Conclusion

Stem cell therapies represent an innovative approach for the treatment of diseases for which currently available treatments are limited. Because MSCs could operate through many different mechanisms, MSCbased therapies are undergoing rapid development and have generated great expectations. Their therapeutic potential is currently being explored in a number of phase I/II trials, and three phase III trials have been concluded for the treatment of graft-versus-host-disease, Crohns disease (Prochymal®, Osiris Therapeutics) and perianal fistula (Ontaril®, Cellerix). While the data from numerous clinical trials are encouraging, future studies are obviously needed to confirm the phase I/II studies. They have nevertheless paved the way for the establishment of feasibility and administration protocols as well as the safety of the procedures. This should encourage initiating further clinical studies in non life-threatening pathologies such as rheumatic diseases.





Muscular Dystrophy

Stem cell based therapies to treat muscular dystrophy


Muscular dystrophies comprise a heterogeneous group of neuromuscular disorders, characterized by progressive muscle wasting, for which no satisfactory treatment exists. Multiple stem cell populations, both of adult or embryonic origin, display myogenic potential and have been assayed for their ability to correct the dystrophic phenotype. To date, many of these described methods have failed, underlying the need to identify the mechanisms controlling myogenic potential, homing of donor populations to the musculature, and avoidance of the immune response. Recent results focus on the fresh isolation of satellite cells and the use of multiple growth factors to promote mesangioblast migration, both of which promote muscle regeneration. Throughout this chapter, various stem cell based therapies will be introduced and evaluated based on their potential to treat muscular dystrophy in an effective and efficient manner.

Intoduction

Numerous types of muscular dystrophy exist and differ depending on their degree of severity and the muscle types affected. Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is an X-linked genetic disorder that occurs at a rate of approximately 1 in 3500 male births. DMD arises due to either spontaneous mutations or inherited nonsense point mutations in the dystrophin gene, the result of which is progressive muscle wasting and weakness attributed to the loss of a functional dystrophin protein. Dystrophin, an important cytoskeletal protein, and a major component of the dystrophin–glycoprotein complex (DGC), is responsible for the maintenance of cell integrity, mediation of cytoplasmic signaling and muscle cell function. Without dystrophin, muscle cells cannot form the DGC and degenerate as a result of mechanical stress during contraction.
To test prospective therapeutic treatments for DMD numerous large and small animal models have been created; the most common being the mdx mouse, which parallels DMD defects seen in diaphragm muscle as a result of a genetic mutation causing premature termination of the dystrophin transcript. Although the mdx mouse lacks a functional dystrophin protein, it only displays a mild dystrophic phenotype, which is attributed to a greater degree of fiber regeneration and a reduction in endomysial fibrosis compared to DMD. More recent mouse models include the utrophin/dystrophin null mouse,  and the dystrophin/α7-integrin double mutant mouse, both of which more closely resemble human DMD. Feline, zebrafish, and the canine X-linked model of muscular dystrophy, complement the mouse models and provide researchers with additional tools to study this disease.

The function of stem cells in development and tissue homeostasis

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Stem cells are defined by certain characteristics, primarily an ability for long term self renewal and the capacity to differentiate into multiple cell lineages. Stem cells are responsible for the development and maintenance of tissues and organs and self-renew or differentiate in response to a combination of biochemical signals and biomechanical stimuli provided by the stem cell niche. Stem cells can be isolated from either adult or embryonic tissue, and depending on a hierarchical state differ in their ability to give rise to multiple cell lineages. This hierarchy progresses from a state of totipotency through to unipotency; whereby, at each level the ability to differentiate into multiple cell types is progressively diminished (Fig. 1). Stem cell division can be either symmetric or asymmetric. An asymmetric division results in the formation of two non-identical daughter cells; one commits to a specialized fate while the other remains quiescent to maintain the stem cell pool. Conversely, differentiating daughter cells undergo symmetric divisions giving rise to a reservoir of precursor cells that contribute to tissue regeneration.

Small quantities of adult stem cells exist in most tissues throughout the body where they remain quiescent for long periods of time prior to being activated in response to disease or tissue injury. Adult stem cells can be isolated from cells of the hematopoietic, neural, dermal, muscle, and hepatic systems. It is traditionally thought that adult stem cells give rise to the specialized cell types of the tissue from which they originated. However, some recent reports have indicated that adult stem cells can differentiate into lineages other then their tissue of origin, for example transplanted bone marrow or enriched hematopoietic stem cells (HSCs) are reported to give rise to cells of the mesoderm, endoderm and ectoderm. Future experiments elaborating upon the origins and characteristics of adult stem cells are necessary in order to fully distinguish their potential from embryonic stem cells.

The embryonic stem cell (ESC) is defined by its origin—the inner cell mass of the blastocyst. ESCs traditionally differ from adult stem cells in that they are deemed pluripotent; meaning they can give rise to cells derived from all three germ layers. Gene expression patterns observed during the in vitro differentiation of ESCs mimic that seen in vivo; and these cells can give rise to numerous cell types in vitro including neurons, bone, pancreatic islets, and skeletal muscle.

In the past multiple stem cell populations have been assayed for their ability to treat muscular dystrophy, the majority of which have met with limited success. In order to correct the dystrophic phenotype, transplanted cells must fuse to existing, or form new, myotubes. Upon fusion, the contribution of genetically normal myonuclei to the muscle myofiber should result in the production of a functional dystrophin protein. Stem cell based therapies for the treatment of muscular dystrophy can progress via two strategies. The first involves cells from a patient afflicted with DMD and is termed autologous stem cell transfer. In this process cells from the patient are genetically altered in vitro to restore dystrophin expression and subsequently re-implanted. In the second strategy, allogenic stem cell transfer, cells are isolated from an individual with functional dystrophin and subsequently transplanted into a dystrophic patient. Both of these strategies have advantages and disadvantages. Autologous cells are advantageous in that they are derived from the patient and therefore unlikely to elicit an immune response. However, the process of genetic alteration has in the past led to undesirable effects including transformation of donor cells and even death. Allogenic cells, on the other hand, are not subject to genetic modification, making them ideal for functional muscle regeneration. However, the patient is at risk for immune rejection, raising the issues of donor compatibility and appropriate immunosuppressive regimes. In this chapter, multiple sources of stem cells with myogenic potential will be identified and their candidacy as cell sources to treat muscular dystrophy will be assessed. The identification of a stem cell population that provides efficient and effective muscle regeneration is critical for the progression of stem cell based therapies to treat muscular dystrophy.

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Skeletal muscle regeneration

Adult skeletal muscle is capable of a remarkable degree of regeneration, suggesting the presence of a stem cell population either resident within muscle or capable of migrating to muscle. The major component of adult skeletal muscle is the myofiber; a giant syncytial cell containing hundreds of myonuclei within a continuous cytoplasm. Under physiological conditions the ability of adult muscle to undergo regeneration is largely attributed to a distinct subpopulation of myogenic cells, termed satellite cells, located between the basal lamina and sarcolemma of mature skeletal muscle fibers. Despite the fact that satellite cells are multipotent in that they can give rise to osteogenic, chondrogenic and adipogenic cells under appropriate conditions, they are a distinct lineage of myogenic stem cells that remain mitotically quiescent under normal physiological conditions. Upon muscle damage or in a state of disease, satellite cells activate and proliferate giving rise to a population of cells that contribute to muscle regeneration via a process of differentiation and fusion. Satellite cells can be characterized by a panel of cell surface markers including: M-cadherin, c-Met, Syndecan 3 and 4, CD-34, and nuclear markers Pax7, MNF, and Myf5. Patients afflicted with DMD rapidly exhaust their satellite cell reserves due to continuous cycles of muscle injury and regeneration. and as such lose their ability to regenerate, resulting in compromised muscle function and degeneration.

 Applications of typical muscle stem cells

Transplantation of satellite cell derived myoblasts

Satellite cells are present at low quantities in adult muscle and account for 2–5% of sublaminar nuclei associated with myofibers. Due to their scarcity and the difficulties in isolating pure populations, freshly isolated satellite cells have been largely neglected as a source for cell therapy. The progeny of muscle satellite cells, upon culture and expansion in vitro, are termed primary myoblasts, these cells are highly proliferative and can be maintained in an undifferentiated state. Historically primary myoblasts have been the principal source of muscle progenitors for cell-based therapies aimed at treating muscular dystrophy. Myoblast transplantation (MT) involves the delivery of primary unmodified skeletal myoblasts to muscle typically via an intramuscular injection. This method is advantageous in that muscle biopsies are easily conducted on limb musculature, techniques for genetic modification of myoblasts are efficient, and large quantities of in vitro expanded myoblasts are easily achieved.

The potential of MT originates from initial experiments performed in mice which demonstrated the capacity of donor myogenic cells to regenerate recipient muscle. Experiments conducted by Partridge et al. using the immortal C2C12 mouse myoblast cell line validated the ability of exogenous myoblasts to induce synthesis of dystrophin in dystrophin-deficient mdx muscle fibers. Subsequent experiments confirmed and elaborated upon these results by using myoblasts from newborn or adult mice in addition to human myoblasts as donor cells for transplant into mdx mice. These experiments demonstrated the ability to track the transplanted cells in the host through the use of LacZ staining, or in the case of human myoblast transfer antibodies specific for human dystrophin. MT was later tried in non-human primates in order to assess the regenerative capacity and immune response involved. Primate derived myoblasts successfully integrated into allogenic hosts when injected 1 mm apart and in combination with the immunosuppressive FK506. These experiments indicate that primate derived myoblasts could integrate into regenerating muscle and survive after 1 year, however none of these experiments provide any evidence as to whether the transplanted cells provide any physiological correction of the dystrophic phenotype.

On the basis of research conducted in mice and nonhuman primates, human clinical trials involving the transplantation of myoblasts were initiated in the early 1990s. Initial trials involved repetitive intramuscular injections of large quantities of myoblasts (> 106 cells) distributed over multiple sites. Although reported as successful, functional evidence was elusive and plagued with false positives resulting from revertant fibers, which arise from a second mutation and occur due to either a somatic deletion or through splicing of further exons in the dystrophin gene. These events lead to the restoration of the reading frame allowing for the production of a truncated, yet partially functional dystrophin molecule. Later clinical trials involved techniques to distinguish dystrophin-positive fibers derived from donor DNA from host revertant fibers. These techniques eliminate confusion concerning the contribution of donor cells to muscle regeneration and allow for a more confident assessment of physiological benefit post transplantation.

The majority of past experiments involving myoblast transfer to treat DMD failed to show substantial physiological correction of the dystrophic phenotype. Although, recent clinical attempts show improvement in the areas of cell survival, migration, and evasion of the immune response, these issues remain at the forefront of myoblast transplantation. Since grafted myoblasts have limited migration, repeated local injections are required to treat a significant portion of the myofibers in any given muscle. Considering DMD patients succumb to heart and diaphragm failure, repeated injections 1 to 2 mm apart would be required in these muscles to ensure patient survival, a technique that is currently beyond our grasp. In addition, transplanted myoblasts do not participate in long term muscle regeneration making them less than ideal for the treatment of DMD. In conclusion, while myoblast transfer provides transient delivery of dystrophin and improves the strength of injected dystrophic muscle it is considered an interim solution to ease the suffering of patients with muscular dystrophy. In order to be considered a viable widespread treatment option for DMD, myoblasts must contribute to multiple rounds of regeneration and be conducive to widespread distribution throughout the musculature.

Satellite cell transplantation

The ability to directly isolate a pure population of satellite cells from diaphragm muscle, by using a Pax3-GFP knock-in mouse [88], was recently accomplished. This Pax3-GFP mouse incorporates the green fluorescent protein (GFP) under the control of the Pax3 promoter allowing faithful recapitulation of Pax3 expression. The use of fluorescent activated cell sorting (FACS) permits the purification of a GFP positive population of Pax3+/CD34+/Pax7+ cells. Based on gene expression, these results suggest the isolation of a predominantly quiescent population of satellite cells. When injected into dystrophic muscle, this population of cells is capable of restoring dystrophin expression 3 weeks post-transplantation. Importantly, the yield of dystrophin expressing muscle obtained when small numbers of isolated satellite cells were transplanted into irradiated muscle was significant. Freshly isolated satellite cells not only restored dystrophin expression in mdx mice but also formed roughly 17% of the satellite cell pool expressing both Pax7 and Pax3-GFP; an indication that donor cells were capable of contributing to the muscle satellite cell compartment. Moreover, approximately 25 fold more cells are needed to obtain similar levels of regeneration from donor cells isolated by enzymatic dissociation of whole adult muscles, as opposed to grafting Pax3-GFP sorted cells.

However, the full potential of this approach is affected by several limitations. First, the cultivation of freshly isolated satellite cells in vitro significantly reduces their in vivo myogenic potential; therefore, whether or not sufficient numbers of donor satellite cells can be obtained is a key issue. The isolation of sufficient quantities of Pax3-GFP satellite cells is difficult because these cells can only be isolated from the diaphragm and body trunk muscles but not from limb muscles. In fact, the current absence of appropriate cell surface markers to identify a Pax3+/CD34+/Pax7+ population of satellite cells makes this isolation technique impossible in humans. Given that genetic manipulations generally require short-term cultivation in vitro, and in vitro culture decreases the regenerative potential of Pax3-GFP populations, then genetic correction of autologous sorted satellite cells does not appear to be a viable option. This is particularly important from a clinical standpoint since cell transplantation of autologous genetically corrected satellite cells to DMD patients is theoretically the ideal approach to minimize host immune rejection of donor cells. A clinically relevant approach to using fresh satellite cells would involve their isolation from the peripheral musculature, based on a panel of cell surface markers, subsequent culture in vitro under conditions that promote the maintenance of their stem cell state, followed by gene therapy prior to transplantation. In the absence of cell surface markers to isolate quiescent satellite cells from the musculature this alternative is currently not an option; therefore, research into the identification of a feasible isolation strategy is of the utmost importance.

Single muscle fiber

Experiments conducted in the early 1980s involving the transplant of whole muscle indicated that resident satellite cells are capable of initiating regeneratio. While, enzymatic dissociation and the subsequent transplantation of satellite cells from myofibers results in marginal muscle regeneration, the transplantation of satellite cells still associated with a single muscle fiber (containing as few as seven satellite cells) can generate in the range of 100 myofibers with thousands of corresponding myonuclei. Interestingly, the satellite cells resident upon a transplanted myofiber will contribute to the host satellite cell compartment and be available for multiple rounds of regeneration. Transplanted satellite cells appear to migrate throughout the muscle in which the myofibers were implanted; however, no direct quantification of the migratory potential of donor satellite cells exists. The notion that single muscle fibers will be used to treat muscular dystrophy does not in itself present a realistic therapeutic approach. Questions regarding the procurement of donor muscle fibers is somewhat belied by the large regenerative potential of individual satellite cells. However, no evidence suggests donor satellite cells are able to populate neighboring muscles; indicating that this method of cell transplant would involve multiple transplantations.

In general the data presented in this section provides evidence that quiescent satellite cells maintained in their niche retain a large degree of regenerative potential. Once it is possible to simulate the satellite cell niche in vitro the real potential of satellite cells could be harnessed for therapeutic purposes. Experiments conducted with fresh satellite cells as well as intact muscle fibers allude to the necessity of identifying the molecular mechanisms responsible for satellite cell self renewal and differentiation. The drastic increase in regenerative potential from either freshly isolated satellite cells or intact myofibers suggests a link between the maintenance of the satellite cell niche and the efficiency of muscle regeneration. Future experiments to identify the components of the satellite cell niche that are responsible for the activation or maintenance of satellite cells in a quiescent state will be of great importance for the validation of satellite cell based therapies.

 Applications of atypical muscle stem cells


Muscle side population cells

Within muscle, in addition to satellite cells, there exists a population of stem cells that possess myogenic potential, termed side population (SP) cells. This stem cell population, isolated by FACS based on its exclusion of the Hoechst 33,342 dye (via the ABC transporter Bcrp1/ABCG2), can be isolated from many adult tissues. SP cells isolated from bone marrow (bmSP) or muscle (mSP) on their own are unable to undergo myogenic differentiation in vitro, yet upon intramuscular transplantation can give rise to both myocytes and satellite cells. The mSP population when isolated from a Pax7−/− background, where satellite cells are absent, and co-cultured with myoblasts or forced to express MyoD undergo muscle specification. These data suggest mSP cells and satellite cells constitute distinct populations that progress along different myogenic pathways. Studies directly comparing muscle regeneration after intravenous injection of bone marrow side population bmSP and mSP cells indicated a reduced ability of the mSP fraction to reconstitute the hematopoietic compartment in lethally irradiated mice; however, both populations regenerate muscle to a similar degree. 

In contrast to satellite cells or primary myoblasts, mSP cells are able to migrate from the blood stream into muscle, a desirable feature for widespread distribution of a therapeutic cell type. Intravenous transplantation of mSP cells typically yields at most a 1% engraftment rate, however upon delivery into noninjured, nonirradiated mdx mice via femoral artery catheterization mSP cells engraftment into muscle at rates approaching 5–8% in select muscles. These results provide evidence that, under physiological conditions, the mSP population can provide dystrophin to diseased muscle via arterial transplantation.

One aspect of mSP transplantation is puzzling, if mSP cells can take up the satellite cell position, and this is reported in numerous articles, why do they not appear to contribute to long term muscle regeneration? Perhaps mSP give rise to a committed myogenic satellite cell expressing Pax7 rather then a satellite cell with stem cell-like properties? Further advances in the field of mSP transplantation must address the following issues: low levels of integration following arterial or intramuscular transplantation, an inability to partake in long term regeneration, and achieving physiological improvements to dystrophic muscle. Prior to these issues being resolved mSP cells currently do not constitute a viable cell source to treat DMD.

Bone marrow cells
Bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT) involves the transplantation of hematopietic stem cell (HSC) in order to produce new blood cells and repopulate the bone marrow. Evidence of a population of circulating cells with myogenic potential present in the bone marrow was identified in the late 1960s. Ferrari et al. (1998) later confirmed that BM-derived cells can, at very low levels, undergo myogenic differentiation and participate in muscle repair after injury. This research presented the idea of delivering donor cells via the circulation to take part in skeletal muscle regeneration; a potentially powerful development considering the daunting task of injecting donor cells into individual muscle masses. The following year studies involving the transplantation of BM-derived cells conducted in the mdx mouse partially restored dystrophin expression. Bone marrow derived cells persist in the musculature for long periods of time and maintain their dystrophin expression, however quantitatively the amount of muscle generated after a BM transplant does not comprise a therapeutically relevant amount when only 0.5% of regenerating fibers contain donor cells.
A clear mechanism detailing the process by which cells in the bone marrow contribute to muscle regeneration remains elusive. Experiments conducted by LaBarge et al. attempted to elaborate on the process by which bone marrow cells contribute to muscle regeneration by the transplantation of bone marrow derived cells (BMDCs) into irradiated SCID mice. BMDCs appear to contribute following muscle irradiation to the satellite cell niche and further exercise induced damage led to the incorporation of BMDCs into multinucleated myofibers at a frequency approaching 3.5%. These initial experiments have been elaborated upon using exercise as opposed to muscle irradiation leading to the conclusion that BMDC can incorporate into muscle under physiological conditions. However, the question remains whether integration of cells from the bone marrow into muscle is a physiologically relevant process. Experiments by Camargo et al. and Corbel et al. (2003) analyzed the ability of bone marrow derived HSCs to participate in muscle regeneration; and while both studies found HSC progeny could incorporate into muscle, this ability is more likely attributed to fusion rather then the existence of a myogenic HSC [101], [102]. Other hematopoietic stem cell populations exist including the CD45+/Sca-1+ population, which following muscle injury undergoes a 30 fold expansion in regenerating muscle and readily undergoes myogenic differentiation in vitro. These experiments further concluded that Wnt signaling molecules play a role in augmenting the myogenic specification of CD45+/Sca1+ cells. Although, later experiments would lead to the conclusion that under physiological conditions bone marrow, and the HSCs contained within, play a minor role in muscle regeneration [104] this technique in combination with appropriate growth factors and suitable methods for transplantation may eventually serve as a method to treat DMD.
Although the major stem cell component of bone marrow is that of the HSC, and the contribution of bone marrow derived cells to the physiological process of muscle regeneration is considered by some to be trivial, the contribution of cells in the bone marrow may differ significantly between mice and humans. AC133, a human cell surface marker for the hematopoietic/endothelial lineages was recently used to isolate a population of cells from human blood that can, upon in vitro co-culture with myogenic cells or exposure to Wnt-producing cells, undergo a degree of myogenic conversion. This finding has reopened the debate on a blood born population with myogenic potential. These AC133+ cells, when co-cultured or exposed to Wnts, display an mRNA pattern reminiscent of that found in satellite cells including: M-cadherin, Pax7, CD34 and Myf5. Whether AC133+ cells constitute a true myogenic progenitor is difficult to determine considering these cells do not undergo myogenic differentiation spontaneously when myogenesis was induced with low serum levels. Nevertheless, limited myogenic conversion and dystrophin expression is observed upon intra-arterial injection or intramuscular injection of AC133+ cells. This method offers certain advantages over other HSC populations including: engraftment of donor cells under physiological conditions exceeds that shown previously for bone marrow, HSCs or mSP cells, and functional tests of injected muscles revealed a substantial recovery of force after treatment. Considering these qualities this method to treat DMD warrants further analysis regarding the process by which the AC133+ population contributes to myogenic regeneration, the localization of AC133+ cells within the circulatory system, and the ability to expand ex vivo these cells prior to transplantation.

Mesenchymal stem cells

Multipotent mesenchymal stem cells (MSCs), first derived from bone-forming progenitor cells resident in the bone marrow, are capable of producing skeletal muscle in addition to osteoblasts, chondroblasts, and adipocytes [106], [107], [108]. Typical methods for MSC isolation involve percoll fractionation and subsequent culture with varying growth factors [109]. The debate surrounding the pluripotent nature of MSCs continues; indeed reports suggest a population of cells co-purified with MSCs can, in vitro, differentiate into visceral mesoderm, neuroectoderm and endoderm [110]. These pluripotent stem cells are termed multipotent adult progenitor cells or MAPCs. MAPCs appear to reside in the brain, skeletal muscle, and bone marrow of human and mouse tissues [111]. Being adult derived; MAPCs avoid many ethical and immunological hurdles associated with embryonic stem cells facilitating their therapeutic application. Continuing research must focus on the origins of MAPCs and the molecular mechanisms that govern their development as well as the ability to induce myogenesis from these cells prior to them being a potential therapeutic source to treat DMD.
Other MSC populations exist and can be isolated via enzymatic digestion and serial passaging of cells from adult human synovial membrane (hSM-MSCs) [112]. These hSM-MSCs possess multilineage potential in vitro and recapitulate the temporal gene expression typical of embryonic myogenesis when directly injected into injured TA muscles of immunosuppressed mdx mice [113]. hSM-MSCs can engraft into regenerating muscle, express dystrophin, and give rise to putative satellite cells that persist for 6 months after transplantation. However, the gene expression profile of hSM-MSC derived satellite cells must be clarified considering neither M-cadherin, Pax7, or CD34 are shown to be expressed in these cells. A method to resolve this issue would be to conduct single cell clonal analysis to confirm the ability of hSM-MSC derived satellite cells to proliferate and give rise to multi-nucleate myotubes. At this point in time additional research into the physiological benefits of hSM-MSCs post transplantation, as well as the confirmation of satellite cell characteristics are necessary in order to progress therapeutically with this technique.
In addition to the MSC types stated above there exists a method to convert MSCs to the skeletal muscle lineage [114] via infection with activated Notch in the presence of various cytokines. Notch is a transmembrane protein, which upon binding with its ligands Delta or Jagged, undergoes cleavage to release an intracellular domain (NICD) in order to effect downstream signaling. The Notch signaling pathway is involved in embryonic tissue morphogenesis [115], adult cell fate selection [116], and has been linked to satellite cell activation and muscle differentiation, making it a candidate molecule for the myogenic induction of MSCs [117]. Experiments conducted by Dezawa et al. (2005) demonstrate the ability to generate skeletal muscle progenitors from human and rat MSCs. Single cell clonal analysis confirms the ability of isolated muscle-MSCs (M-MSCs) to form multi-nucleate myotubes following treatment with the NICD and culture in low concentrations of horse serum to induce fusion. Intravenous injection of M-MSCs in immunosuppressed rats, pretreated with cardiotoxin in the gastrocnemius muscle, resulted in their incorporation into newly formed myofibers, and not bone, heart, liver, kidney or undamaged muscle. M-MSCs localize to the sublaminar portion of myofibers, express Pax7 and c-Met via immunofluorescence of separate cellular fields and upon multiple rounds of regeneration contribute to muscle repair.
Questions remain regarding the contribution of M-MSCs under physiological conditions to the functional amelioration of the dystrophic phenotype. Interestingly, M-MSCs express myogenin (a terminal marker of muscle differentiation) at high levels along with MyoD, early markers of muscle formation Six1, and Six4 along with Pax7 a satellite cell marker. Considering their gene expression profile M-MSCs appear to be a heterogeneous population of undifferentiated and partially differentiated myogenic progenitors. Further characterization of M-MSCs and the in vivo derived satellite cells is required to answer these questions. In addition, the use of Notch to induce myogenic specification is a unique approach more commonly associated with the maintenance of quiescent satellite cells or the prevention of terminal muscle differentiation. This is not to say the use of Notch to promote myogenic specification is improper, however without a clear molecular mechanism governing the role of Notch in the myogenic commitment of MSCs nor a basis for converting MSCs to the myogenic lineage without over-expressing the NICD this current route is not practical for the treatment of patients with DMD. This method offers the ability to derive myogenic cells that can be easily obtained and expanded from patients, genetically modified in vitro, and re-introduced via the circulation all beneficial therapeutic characteristics.
In summary, multiple different types of MSCs can be isolated and serve as potential cell types for therapy, however in the absence of a defined group of cell surface markers a reproducible system to isolate cells with myogenic potential from MSCs becomes difficult. The reproducible isolation of pure MSC populations and their downstream differentiation into the muscle lineage holds tremendous potential for the treatment of neuromuscular disorders. Given the rapid progress in the areas of cell purification and characterization therapeutic relevancy may not be far off.

Muscle Derived Stem Cells (MDSCs)
Muscle derived stem cells (MDSCs) are a distinct population of stem cells resident in adult skeletal muscle. MDSCs are thought to reside upstream of satellite cells in the terms of their potency, and are not restricted to either the myogenic or mesenchymal lineages [118], [119]. Numerous muscle derived stem cell populations have been shown to contain hematopoietic potential [93], [120], [121] the most prominent being that derived via the preplate technique [22]. These MDSCs represent a heterogeneous population of cells in terms of their high expression of either Sca1 or CD34. While the physiological location of MDSCs remains unknown they often express MNF and the myogenic regulatory factor MyoD and share certain characteristics with a population of hematopoietic stem cells expressing CD34 and Sca1 [122]. When injected into limb muscle or into the circulation of mdx mice MDSCs contribute to muscle regeneration and express dystrophin [123], [124]. In comparison to transplantations conducted with primary myoblasts, MDSCs show a 10 fold increase in dystrophin expression and incorporate into vessels and surrounding nerves [22].
Although certain characteristics of MDSCs including their ease of proliferation in vitro, ability to migrate through the vasculature, and their multipotentiality are amenable to therapeutic applications, a lack of physiological improvement to the dystrophic condition coupled with long term self renewal resulting in their transformation mar the use of MDSCs to treat DMD [125], [126]. In an attempt to determine the physiological location of MDSCs, some reports claim they originate in the bone marrow and reside in the musculature [120] while others claim they are muscle derived [126], [127]. Further research into the origins of MDSCs is required prior to the clinical use of these cells.

Vessel associated stem cell populations

Cells with endothelial and myogenic properties exist and can be isolated at embryonic, fetal [128], and postnatal stages [129] (3 weeks) of development yet the identification of a bona fide adult vessel derived stem cell remains elusive. In 1999 De Angelis et al. discovered a stem cell population resident in the embryonic dorsal aorta having both endothelial and myogenic markers [130]. Although, explant cultures of the dorsal aorta do not initially express any myogenic markers; upon differentiation in culture they gain both endothelial and myogenic markers known to be present on adult muscle satellite cells. Surprisingly, the quantity of satellite cells produced in vitro from the dorsal aorta is in the range of 50 fold more than that derived from somitic explants [130]. Dorsal aorta derived myogenic cells can in vitro fuse with primary myoblasts during differentiation and when transplanted into TA muscles contribute their nuclei to muscle regeneration [130].
Further work on vessel associated stem cells isolated from the dorsal aorta demonstrated their multi-potentiality leading to their classification as mesangioblasts[131]. Studies conducted by Sampolesi et al. using immunocompetent α-sarcoglycan null mice which serve as a model system to study limb-girdle muscular dystrophy indicated that the intra-arterial injection of mesangioblasts results in their migration throughout the vasculature, giving rise to both morphological and functional correction of the dystrophic phenotype, a quality absent in other myogenic stem cell populations [132]. This method was further improved by the treatment of mesangioblasts with either stromal-derived factor (SDF) 1 or tumor necrosis factor (TNF) α which resulted in enhanced transmigration in vitro and migration into dystrophic muscle in vivo [133]. The combination of pretreatment with TNFα and SDF-1 and the expression of α-4 integrin lead to remarkable (∼50%) incorporation of arterially transplanted mesangionblasts into α-sarcoglycan deficient muscle. Long term survival of pre-treated mesangioblasts is observed in muscle masses after 4 months, with α-sarcoglycan mice expressing ∼60% of the α-sarcoglycan detected in a wild type mouse. Although these reports are extremely promising, confirmation of the human equivalent of mesangioblasts that can be isolated from adult tissue is of great importance.
Pre-treated mesangioblast can migrate through the vasculature, engraft at therapeutic levels in muscle, persist and contribute to long term regeneration, and upon genetic modification of autologous cells do not elicit an immune response. Due to these qualities mesangioblasts constitute a potential therapeutic cell type to treat DMD. Prior to their use in clinical trials effective methods to isolate mesangioblasts from adult human tissue must be established, information regarding their long term contribution to muscle function, and the effects of unwanted penetration into non desired cell types must be resolved.
Research supporting the vascular origins of myogenic stem cell populations provides therapeutic potential for the systematic delivery of myogenic progenitors. Whether or not these populations (embryonic, fetal or post natal) of vessel associated stem cells are unique, or derived from an upstream stem cell remains to be determined. Considering these cells are associated with the vasculature it is not a stretch to imagine stem cell populations with myogenic potential residing in multiple adult tissues. While the exact characteristics of vessel associated stem cells remains a mystery their ability to migrate throughout the vasculature coupled with their myogenic potential make them an attractive source for use in cell therapy.

Embryonic stem cells

Small quantities of skeletal muscle were first derived from murine embryonic stem (ES) cells nearly 20 years ago [40], however since then few advances have been made to improve the efficiency of the process. First reports regarding gene expression patterns, functional properties, and morphology of ES derived skeletal muscle parallel observations in vivo. Gene targeting in ES cells is often used to assess gene function, and as a result numerous ES cell lines with modifications to genes important in myogenesis exist. Typically, created to study embryonic development in the mouse these ES cell lines were analyzed during in vitro differentiation to study the molecular mechanisms involved in embryonic myogenesis. Over time limited inhibitors and activators of skeletal muscle formation from ES cells were identified along with strategies to harness their potential for regenerative medicine. 

One study conducted by Barberi et al. and published in 2005 shows therapeutic potential and involves the derivation of skeletal muscle by way of hES derived mesenchymal stem cells. This method offers the ability to derive unlimited numbers of pure MSCs from hESCs, and their subsequent differentiation into the skeletal muscle lineage. In order to obtain skeletal muscle from hES derived MSCs (hESMPCs) either co-culture or conditioned medium from the murine myoblast cell line C2C12 or the addition of 5-AzaC as a demethylating agent is required. Currently these experiments do not address the ability of hESMPCs to give rise to Pax7 positive cells nor their ability to contribute to muscle regeneration in vivo.

ES cells to date have not had a significant impact on the development of cell-based therapies to treat muscular dystrophy. Currently one study exists involving differentiated ES cells (3 days) co-cultured with dissociated skeletal muscle fibers for 4 days prior to transplantation into mdx muscle. After 2 weeks dystrophin expression was detected in select regions over and above that which would occur naturally from revertant fibers. These experiments represent the preliminary steps for the use of ES cells to treat DMD and currently do not demonstrate any indication of the long-term regenerative capacity of transplanted cells. Questions remain as to the mechanism by which ES cells, co-cultured in the presence of adult skeletal muscle, contribute to the process of muscle regeneration.

Currently no methods exist to generate large quantities of skeletal muscle from ES cells and for this reason the ES cell model system remains better suited as a tool to study embryonic myogenesis. Potential benefits of using ES cells to treat muscular dystrophy focus on the isolation of large quantities of myogenic stem cells, their ease of genetic modification in vitro, and the ability to derive immune matched cell lineages for transplant. Although the creation of patient specific hES cell lines remains mired in controversy, the principle retains a high degree of feasibility and in the interm progress on the mechanisms involved in myogenic induction and differentiation continue.

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Synergistic methods to improve stem cell based therapies

In this chapter we have presented numerous stem cell types with varying degrees of myogenic potential. Irrespective of their source, these stem cell populations share certain hurdles to overcome prior to their therapeutic use. Survival and the subsequent migration from the site of injection remains suboptimal for many of the cell populations outlined previously and from a clinical standpoint the immune response generated upon introduction of a foreign cell type, or in the case of DMD a foreign protein, is a concern. As the mechanisms surrounding the survival and proliferation of myogenic cells post transplant are unraveled, and appropriate growth factors are identified the success of cell based therapies to treat muscular dystrophy will improve. Mechanisms involved in the migration of donor cells to skeletal muscle and the satellite cell niche are still poorly understood. Although some stem cell types, namely mesangioblasts, mSP cells, M-MSCs, have the ability to migrate through the vasculature, most do not. Potential future avenues to increase the migratory ability of stem cell populations include the identification of cell surface markers (e.g., l-selectin+ cells) and appropriate growth factors. As is observed in the case of mesangioblasts, pre-treatment with the growth factors TNFα and SDF-1 led to a substantial improvement in their migratory abilities.

Cell transplantation often elicits an immune response, and in addition to immunosuppressive drugs, there exist methods to overcome immune rejection. One such method involves the establishment of central immune tolerance through a process of mixed hematopoietic chimerism. The mechanism surrounding mixed hematopoietic chimerism is not fully understood yet the procedure is well established in animal models and pursued in the clinic. Matching the genetic background of the various stem cell derived myogenic precursors with hematopoietic stem cells could potentially allow the tolerisation of patients to myogenic transplants.

Irrespective of the stem cell population chosen to treat muscular dystrophy the above-mentioned characteristics (survival, localization, and immunogenicity) remain. In order for the chosen cell type to be successful it must be optimized to deal with these issues. The identification of suitable growth factors, appropriate surface markers, and methods to escape immune detection will be of great importance for the progression of this field. 

Conclusions and future perspectives

Stem cell therapy is an attractive method to treat muscular dystrophy because in theory only a small number of cells, together with a stimulatory signal for expansion, are required to elicit a therapeutic effect. In order to achieve clinical relevance candidate stem cell populations must be easily obtained, upon isolation remain capable of efficient myogenic conversion, and when transplanted must integrate into the musculature leading to the functional correction of the dystrophic phenotype

Stem cell populations with myogenic potential can be derived from multiple regions of the body at various stages of development (Fig. 2). Many questions linger regarding the mechanisms by which atypical, or non satellite cell derived precursors, participate in muscle regeneration. A general consensus in the field identifies satellite cells as the primary, if not only, physiologically relevant population that contributes significantly to muscle regeneration. However, because satellite cells are not currently amenable for distribution throughout the vasculature they do not constitute a viable option to treat DMD. A superficial comparison of the atypical myogenic stem cell populations reveals a common theme whereby a cell often linked to the circulatory system is able to migrate to regenerating muscle and contribute in a limited way to this process. The localization of adult mSP, MDSCs, Mesangioblasts, Bone Marrow derived HSCs and HSC populations expressing the cell surface marker AC133 remains unknown. The idea that myogenic potential resides in cells associated with the vasculature is not novel, in fact pericytes which line the capillaries appear to possess qualities of multipotent mesenchymal progenitors If indeed pericytes take part in myogenic regeneration this could explain the widespread distribution of atypical stem cell populations with myogenic potential.

In conclusion, upon comparing the prospective stem cell populations with myogenic potential, the cell type that fulfills the most criteria for use in the treatment of DMD is the mesangioblast. Mesangioblasts serve as a paradigm for widespread distribution, and upon growth factor pre-treatment are able to correct significantly the dystrophic phenotype. Perhaps the application of growth factor pre-treatment to other myogenic stem cell populations may improve their ability to treat muscular dystrophy, however for now mesangioblasts serve as a beacon of hope for patients suffering from various muscular dystrophies.

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Wound Healing


Chronic or non-healing skin wounds present an ongoing challenge in advanced wound care, particularly as the number of patients increases while technology aimed at stimulating wound healing in these cases remains inefficient. Mesenchymal stem cells (MSCs) have proved to be an attractive cell type for various cell therapies due to their ability to differentiate into various cell lineages, multiple donor tissue types, and relative resilience in ex-vivo expansion, as well as immunomodulatory effects during transplants. More recently, these cells have been targeted for use in strategies to improve chronic wound healing in patients with diabetic ulcers or other stasis wounds. Here, we outline several mechanisms by which MSCs can improve healing outcomes in these cases, including reducing tissue inflammation, inducing angiogenesis in the wound bed, and reducing scarring following the repair process. Approaches to extend MSC life span in implant sites are also examined.

Introduction

Wound healing is a complex multi-stage process that orchestrates the reconstitution of the dermal and epidermal layers of the skin. In many pathological circumstances such as diabetes or severe burns, the normal wound healing process fails to adequately restore function to the skin, leading to potentially severe complications from ulcers or resulting infections. As the incidence of obesity and resulting diabetes continues to increase in the western world, the prevalence of chronic wounds related to these conditions continues to be a major focus of wound care research. In fact, non-healing wounds from these conditions have produced a multi-billion dollar advanced wound care market for technologies aimed at stimulating wound healing in patients that suffer from dysfunctional wound repair, with large projected growth in the near future. Most current biological technologies for advanced wound care aim to provide antimicrobial support to the open wound and a matrix scaffold (collagen-based in many cases) for invading cells to reestablish the skin, with some focus on growth factor support of the healing process. However, patient outcomes in this area remain marginal and novel bioengineered approaches to chronic wound repair remain a topic of high interest.

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Chronic wound healing technologies

Mesenchymal stem cells (MSCs) are important cells in orchestrating the three main phases of normal wound healing (inflammatory/proliferative/remodeling), directing inflammation and antimicrobial activity and promoting cell migration during epithelial remodeling. However, recently due to advances in understanding of MSC immunosuppression and secretion of pro-angiogenic factors, MSC-based cell therapy in combination with matrix scaffold approaches to improve wound healing outcomes has become a potential strategy in treatment of non-healing wounds.

Traditionally, MSCs have long been identified for their ability to migrate to sites of injury in the body and differentiate into a variety of cell lineages such as bone, fat, and cartilage,making them attractive candidates for a variety of cell therapies in recent studies. A variety of easy means of isolating and expanding these cells ex-vivo (bone marrow, adipose tissue, placenta, peripheral blood,and others) also makes MSCs useful cells for therapeutic approaches to supplementing tissue regeneration. Additionally, these cells have been shown to have notable immunomodulatory effects on the surrounding environment following transplantation and can support native cells with the secretion of a variety of pro-survival and pro-migratory cytokines and growth factors. As a major problem in chronic wounding is unmitigated inflammation, this characteristic of MSCs has made them good candidates for approaches to cell therapy for chronic wounds in particular.

Clinical sources of MSCs

In this review, we examine current trends in MSC therapy for chronic wound healing, including several major areas of MSC benefit to the wound repair process. Additionally, potential further MSC applications in wound healing and novel technologies are discussed.

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Chronic Skin Wounds

The normal wound healing process is characterized by three main phases that lead to efficient reconstitution of a functional dermis/epidermis and revascularized tissue. Briefly, the inflammatory phase immediately follows wounding, serving to stop bleeding in the wound bed via platelet aggregation and fibrin clot formation. This is followed by invasion of neutrophils and mast cells that follow a chemotactic gradient to clear the wound of dead cells, debris, and residual ECM. The proliferative phase then proceeds, including fibroblast migration into the wound bed and deposition of new ECM (collagen). VEGF and B-FGF also stimulate de novo angiogenesis in the skin. Finally, the remodeling process resolves the wound by organizing collagen fibers that formed during fibroblast proliferation in parallel with further removal of fibronectin to increase the strength of the new skin.

A chronic or non-healing wound is essentially a wound that does not progress normally through the wound healing process, resulting in an open laceration of varying degrees of severity. These conditions can be cause by a number of various pathophysiological conditions (diabetes, venous stasis ulcer progression, nd others), though all causes generally lead to a hyper-inflammatory environment, particularly evidenced by the characteristic presence of neutrophils/high MMP activity that leads to high breakdown of new collagen during the wound healing process and inhibition of pro-healing factors (PDGF, TGF-B, and others). This excessive inflammation phenotype leads to wounds that cannot resolve under normal circumstances, especially until the inflammation in the wound bed is controlled to a normal level and fibroblasts are able to effectively migrate into the wound space and synthesize new matrix.

Clinically, these wounds present a large problem for wound care specialists globally, with approximately 1–2% prevalence and a greater than 50% recurrence rate for diabetic patients. This need has generated a large interest in new treatments for improving patient outcomes in chronic wound therapies. Mesenchymal stem cells, given their immunomodulatory and angiogenic properties, have therefore been studied extensively with regards to cell therapy to supplement wound dressings. With over 350 listed clinical trials for MSC therapies (clinicaltrials.gov), many include studies utilizing MSCs for healing ischemic/diabetic foot ulcers and similar wounds.

Clinical trials for MSCs and chronic wounds


OUltimately, this interest in MSCs for cell therapies in wound healing revolves around several key aspects, including immunosuppression, angiogenesis stimulation, and scar reduction. As MSCs play a normal role in the wound healing process, they are an obvious candidate for study in this context as opposed to embryonic stem cells or other regenerative sources. Recent studies have outlined some successful approaches to promoting wound healing with MSCs, including autologous bone marrow-derived MSCs in fibrin matrix or, more recently, intramuscular injection of autologous MSCs to improve diabetic wound closure. While some trials have been aimed primarily at safety of MSC use for wound healing, several clinical trials have shown the potential benefit of MSCs for inclusion in wound healing devices, including improved average rate of wound healing and general limb perfusion after treatment and also improved acute wound healing correlating to the number of injected cells.Despite any effects on healing, there was some doubt as to any reduction in limb amputation rate or relative pain levels among groups, a major consideration for effective therapy in chronic wounds. In general, the consensus from completed trials has been an overall improvement in chronic wound closure with application of mesenchymal stem cells, particularly as a part of a matrix delivery system (wound gel, etc.).

MSC Immunomodulation In Wound Beds



Chronic hyperinflammation in the wound bed is the most substantial barrier to treatment in non-healing wounds, as outlined previously. Mesenchymal stem cells have recently been shown to hold a variety of immunomodulatory effects on host immune cells in both wound healing and transplant biology contexts. These characteristics are potentially what make MSCs the most attractive cell type for cell therapy in chronic wounds, as they exert pleiotropic effects on the inflammatory mechanisms to move the wound past static inflammation and fibrosis.
It has been known for some time that donor MSCs are able to suppress host T cell proliferation, a key activity in reducing wound bed inflammation. More recently, this was demonstrated to be dependent on MSC induction of IL-10 in native T cells and macrophages, as well as TGF-β activity. Additionally MSCs have been shown to be capable of modulating host TNF-α production to mediate excessive inflammatory effects, and reduce NK cell function in the inflammatory phase, lowering IFN-γ activity in the process. Conversely, in the later stages of inflammation, active TGF-α is able to stimulate implanted MSCs to produce a variety of pro-healing growth factors and cytokines, including VEGF to stimulate angiogenesis in the wound bed. n 2008 Ren et al. showed a dependence on pro-inflammatory factors for these processes to be effective, suggesting a potential time window for application of allogeneic MSCs to be efficient in reducing inflammation. 

Importantly, recent research into the immune response to allogeneic MSCs has shown that in most systems, the donor MSCs are ‘immunoprivileged’ and do not induce a significant response in the host, suggesting that allogeneic cell sources may be possible for chronic wound therapies, where diabetic patients may have already-defective endogenous MSC populations making autologous therapy less than optimal. This characteristic of allogeneic MSCs is crucial in this particular wound environment, where excessive inflammation already drives the chronic phenotype and additional immune response from cell implantation must be as low as possible. However, several studies have shown that this immunoprivileged characteristic is lost as the MSCs differentiate, leading to a gradual host response to the implanted cells. Thus, approaches to keeping MSCs undifferentiated may be key in future chronic wound therapies.
MSCs have also recently been identified as having antimicrobial effects, a significant advantage in reducing excess inflammation from any contaminants in the wound during injury and treatment.This was identified in 2008 by Krasnodembskaya and colleagues  as a mechanism based on secretion of LL-37, a peptide with a wide array of antimicrobial properties including broad spectrum microbial defense via disruption of bacterial cell membranes and directly limiting bacterial macrophage activity via upregulation of chemokine receptors, all while ignoring pro-inflammatory cytokine activation. In terms of cell therapeutics, the concern for reducing infection is great and many products attempt to seal wounds with silicone barriers to dressings. Silver nanoparticles have also been examined for antimicrobial properties, which can be conveniently included in wound healing gels and allowed to leach into the wound locally. Combined with MSC antimicrobial activity, this would help to reduce any additional inflammation seen during the healing process.
As a whole, all of the effects produced by MSCs here help to solve the problem of chronic hyperinflammation in the wound bed and advance wounds such as diabetic ulcers into the next stages of wound healing. Allogeneic application of MSCs in gel-based products for wound healing holds promise for combatting these issues, as has been done in various applications for MSC therapy using fibrin-based gel systems and related mimetics.


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Stimulation of Angiogenesis

Revascularization of the wound bed is a crucial stage of the normal wound healing process, where new vessels form as granulation tissue develops to supply blood to the wound area, which is in need of oxygen and nutrients. Endothelial cells therefore need to be able to break through the dermis of the wound and form tubes in the newly developing tissue, a process that is balanced by the growth factor production cascade during wound healing. Mesenchymal stem cells play a normal role in this process as they are recruited to the wound bed following mobilization from endogenous sources. The ability of mesenchymal stem cells to promote angiogenesis in vivo is not necessarily unique, as several other cell types have been shown to been integral in stimulating angiogenesis via cell therapy, such as hematopoietic stem cells73 Takakura N, Watanabe T, Suenobu S, Yamada Y, Noda T, Ito Y, Satake M, Suda T. A role for hematopoietic stem cells or resident cardiac progenitor cells.However, the unique role of MSCs during normal wound repair and additional effects of MSCs discussed in this review make the application of MSCs to stimulate vessel growth in chronic wounds particularly interesting for future studies in clinical MSC application for wound therapy.

There is evidence that MSCs can differentiate into a variety of skin cell types, contributing to repopulation of the wound bed with normal dermal structure, as well as endothelial cells to yield new vessels. Recently several groups have focused on differentiation of MSCs into endothelial cells, an approach that has potential to be useful in direct transplant into anti-angiogenic environments such as ischemic wound beds. Results have shown endothelial-like cell populations derived from human MSCs in vitro with varying degrees of donor variation, while Bago et al. showed similar results for amnion-derived MSCs in glioma tumors. Furthermore, pericytes that stabilize vessel walls and promote vessel maturation during angiogenesis have been shown to be derived from bone marrow populations following injury. Recent evidence suggests that these pericytes in fact represent a sub-population of mesenchymal stem cells that contribute to the healing process. These cells all have the potential to support new vessel growth in a chronic wound bed, a critical aspect of overcoming barriers to current therapies.

Perhaps more important than differentiation, secreted factors also play a substantial role in MSC regulation of angiogenesis in the wound bed. Chronic wounds are often subject to anti-angiogenic conditions, including reduced growth factor production as a result of increased MMP production in the wound bed, as outlined by Krisp et al. recently in a global secretome analysis of wound exudates.MSCs naturally produce a variety of pro-angiogenic factors following recruitment to the wound bed that stimulate endothelial cell proliferation and tube formation in the wound bed, most notably VEGF, a potent stimulator for angiogenesis that is regulated by IL-6 and TGF-α in the wound bed. Though it has been shown that exogenous VEGF application to wounds can stimulate angiogenesis, MSCs used in cell therapeutics also have been shown to stimulate EC recruitment and wound healing via VEGF secretion or via pre-differentiation into angiogenic precursors. Ultimately, MSCs are able to stimulate de novo angiogenesis in wound beds upon transplantation, a crucial factor in stimulating healing in chronic wounds that lack this normally due to the hyperinflammatory environment.

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Reduction in Scar Formation

Another consideration in repair of wounds under all circumstances is the formation of scars, caused by deposition of excess ECM by fibroblasts in the wound bed. These structures carry a variety of undesirable consequences, including unsightly appearance on the skin and, more critically, scars lack many of the normal makeup of the skin such as follicles and nerve endings and also do not retain the normal tensile strength of undamaged skin. While scar reduction research has been a field in of itself for quite some time, it is a notable consideration for patients with large non-healing ulcers.

As discussed previously, anti-inflammatory mechanisms of MSCs have several effects on fibrotic phenotypes in the wound, and thus play a major role in reducing scar formation following wound healing. Most notably, MSC production of PGE2 drives a variety of changes in the scarring phenotype. PGE2 from MSCs has been shown to increase secretion of IL-10 by T cells and macrophages, an important anti-inflammatory cytokine in the wound environment. PGE2 secreted by MSCs in response to the inflammatory wound bed plays a crucial role in the healing process, reducing T cell migration and NK cell proliferation during the inflammatory phase. The upregulation of IL-10 in the wound by MSCs also has a multitude of effects on general scar formation, including downregulation of IL-6 and IL-8 to reduce collagen production in the wound and inhibition of neutrophil invasion and macrophage activity to suppress ROS generation, all leading to support of regenerative healing in recent experimental scar formation models. ROS generation is also affected by nitric oxide secreted by MSCs, acting as a scavenger to prevent the fibrotic activity of the oxygen radicals. Though these anti-inflammatory mechanisms are part of normal MSC function following homing to acute wound sites, the hyperinflammatory environment of a chronic wound makes the MSC ability to modulate excessive inflammation and reduce excessive scarring critical. Ultimately, reduced scar formation is not an outcome desired specifically for chronic wounds, but nevertheless is a significant potential benefit of utilizing MSCs to promote closure of such non-healing wounds. Experimentally, recent experimental evidence has shown that MSCs can indeed reduce a fibrotic phenotype in a mouse model, showing promise for reduced scar formation in future MSC therapeutics.

Mesenchymal stem cells also produce a variety of anti-fibrotic factors throughout the wound healing process. Aside from IL-10, HGF is a major contributor to reduced fibrosis, which has been shown to be effective in advancing clean wound healing in a variety of tissues such as liver and various skin contexts. HGF has also been attributed to chronic wounds, with differential regulation of HGF production and presence of c-Met in chronic wound dermis. Specifically in relation to fibrosis, HGF has been demonstrated to reduce TGF-β and collagen production in fibroblasts, and also have a multitude of effects on cell recruitment to the wound bed, including endothelial cells and promotion of keratinocyte migration. Ultimately, HGF production by transplanted MSCs would yield a more normal state of cell migration and matrix production than what is normally seen in chronic wound beds.

Together in concert with the other immunomodulatory mechanisms of MSC function in wound repair, addition of MSCs to chronic wounds may prove to be an effective means of promoting cleaner healing on a smaller time scale than traditional treatments.

Promotion of MSC Survival

In typical cutaneous wound healing, MSCs are mobilized from host sources and home to the site of injury, persisting to support immunomodulation and improved angiogenesis in the wound bed as the skin repairs itself. These host MSCs are able to perform these normal functions despite somewhat challenging conditions in the wound site, such as hypoxia or lack of nutrients. However, in the case of chronic wounds, the normally-ischemic wound environment becomes even harsher, with excessive inflammation and an environment not conducive to angiogenesis compared with normal wounds. Therefore, a significant barrier to successful use of MSCs in any potential cell therapy has been post-implant cell survival in a variety of ischemic injury models. Past studies have shown marginal MSC preservation in various models including cardiac infarct or cerebral injury, but still MSC use in any of these models is limited by MSC death due to the harsh wound environment. As all of the benefits of MSC therapy for any type of wound healing are dependent on cell survival in the wound, strategies to improve survival following implantation are of interest in future research efforts. Recent studies have examined the effectiveness of preconditioning human MSCs with varying oxygen concentrations or pan-caspase inhibitors to improve the MSC survival response immediately following implantation. Saini et al. showed hyperoxic and pan-caspase pre-treatment of the cells substantially decreased MSC apoptosis in a cardiac infarct model, a scenario that produces an ischemic environment for implanted MSCs. Conversely, Chang et al. recently demonstrated the advantages of preconditioning MSCs in hypoxia, which was shown to improve the secretory capabilities of the cells (VEGF, HGF, and others), a main benefit of MSC therapy.  Gene therapy in MSCs has also received some attention, as Wang et al. recently showed that adenoviral upregulation of protein kinase G1α improved MSC survival following implantation into a similar cardiac infarct model.

Additional recent studies have examined the possibility of exploiting endogenous signaling pathways in promoting MSC survival in a variety of wound healing contexts. The possibility of activating pro-survival pathways via matrikine moieties is a relatively novel concept that has been demonstrated to affect MSC signaling during wound healing normally. This research led to the idea that EGFR could be activated by EGF molecules tethered to growth scaffolds, which was shown to improve MSC survival in vitro during cell death assays. This system of activating EGFR artificially to promote survival signaling has been applied to several tissue engineered surfaces, and provides the MSCs with a variety of survival advantages that could be used to combat the ischemic wound environment. More recently, Rodrigues et al. showed that the matrix protein Tenascin C could produce similar effects in vitro. Tenascin is easily incorporated into collagen-based scaffolds, and could potentially be combined with current therapeutic gels to modulate MSC survival. This could be beneficial in scaffold design for MSC delivery to chronic wound beds, as biomaterials used in scaffold design can at times produce a more robust artificial inflammatory response.

Limitations of MSC Use in Chronic Wound Repair


As discussed in this article, one potential limitation to use of MSCs for treating chronic wounds is varying degrees of cell survival following implantation that might curtail any therapeutic effect in the long-term. However, there are other more fundamental hurdles to use of MSCs. One clear limitation to using MSCs as a standard therapy in any context is a general functional heterogeneity that makes a “standardized” MSC for manufacturing and quality control purposes a serious challenge. Changes in cell proliferation rate and differentiative capacity between donor sources have been reported for many years, as well as functional differences among subpopulations of MSCs from a single source based on variation in RNA production. Additionally, use of MSCs as therapeutic agents requires ex vivo expansion that, while somewhat more accessible than other stem cell types, can remain problematic due to the aforementioned heterogeneity as well as a very limited natural in vitro lifespan. This all ultimately leads to issues in commercialization, where there remains no real set of guidelines for MSC expansion and general use in therapeutics for companies using the cells, despite their relatively common use in clinical trials and some marketed products.

Finally, the source of MSCs used can create a great deal of variation among therapeutic cells and also have an effect on harvesting methods. Many sources of MSCs have been discovered over time, including those mentioned in this article. There are complex and sometimes vast differences between MSCs obtained from bone marrow, adipose tissue, and other sources, ranging from cell surface marker expression to differentiation limitations and, importantly, immunomodulatory capacity (recently reviewed by Hass et al.  In terms of therapeutic strategy, this makes selection of a cell source an important consideration and/or limitation for any given therapy. For cutaneous wound healing, bone marrow-derived MSCs are the most often-used source, as evidenced by their inclusion in most of the clinical trials cited in this article However, adipose-derived cells and umbilical cord-derived cells have also been used for treating diabetic ulcers in clinical trials, suggesting continued disparity among researchers as to the optimal source.

Conclusions and Future Directions

Cell therapies for improving wound healing have become a topic of great interest, particularly for non-healing wounds such as diabetic or venous stasis ulcers. Patients with these wounds continue to be subject to inefficient wound care technologies that do nothing to stimulate the wound environment itself, instead providing secondary support via antimicrobial action or space filling matrix. Despite some potential limitations, we have here outlined several reasons why mesenchymal stem cells provide unique and effective support for stimulating the wound healing process in a chronic wound bed. Ultimately, these cells have the ability to suppress excessive inflammation and reduce scarring while stimulating de novo angiogenesis in the wound bed, all leading to promising outcomes in chronic wound repair.

Future directions for research in this field might focus on optimization of MSC function in chronic wound contexts, both in delivery systems and scaffold designs as well as improving cell survival via independent technologies or in combination with these delivery systems. The most promising current technologies, as outlined in several clinical trials cited in this review, include basic fibrin mesh scaffolds in gel form for seeding of MSCs; however, technologies for improving MSC efficiency in wound healing continue to emerge, such as recent studies for microsphere delivery in wound gel. MSC therapy also holds promise in improving wound healing outcomes in other wound care settings, such as surgical wounds or burns. Ongoing studies into MSC immunomodulation and wound support in other wound models (myocardial infarction, brain, long bone defects, and others) may continue support advanced wound care research with insights into novel biomaterials and beneficial properties of MSCs in cell therapy.

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Neurological disorder


Several studies have reported that mesenchymal stromal/stem cells (MSCs) restore neurological damage through their secretion of paracrine factors or their differentiation to neuronal cells. Based on these studies, many clinical trials have been conducted using MSCs for neurological disorders, and their safety and efficacy have been reported. In this review, we provide a brief introduction to MSCs, especially umbilical cord derived-MSCs (UC-MSCs), in terms of characteristics, isolation, and cryopreservation, and discuss the recent progress in regenerative therapies using MSCs for various neurological disorders.


Introduction

Recently, mesenchymal stromal cells (MSCs) have been attracting much attention for their potential to treat neurological disorders.
The concept of MSCs introduced by Caplan in 1991, can be traced to investigation of the inherent osteogenic potential associated with bone marrow (BM). MSCs have now been reported to be isolated from several sources, including BM, umbilical cord blood (UCB), adipose tissue (AD), and the umbilical cord (UC) . Among various sources of MSCs, we focused on the UC because of  their abundance and ease of collection, non-invasive process of collection, little ethical controversy, low immunogenicity with significant immunosuppressive ability, and  migration ability towards injured sites.
Several studies using neurological disorder models have reported improvements after MSCs administration, and clinical studies using MSCs to treat brain injuries have been already conducted.
In this review, we characterize UC-MSCs, in terms of characteristics, isolation, and cryopreservation, and discuss the recent progress of regenerative therapies using MSCs in various neurological disorders.

Methods to isolate UC-MSCs

There are several protocols used for isolating and culturing UC-MSCs. We review two major methods: explant method and enzymatic digestion method.

Improved explant method

Collected UCs are manually minced into approximately 1–2 mm3 fragments. These fragments are aligned and seeded regularly on a tissue culture dish. After the tissue fragments attach to the bottom of the dish, culture media is added, slowly and gently to prevent detachment of the fragments [16], [17], [18]. When the fibroblast-like adherent cells growing from the tissues reach 80%–90% confluence in 2–3 weeks, the cells and tissue fragments are detached using trypsin. The culture is then filtered to remove the tissue fragments.

When using the explant method, it is critical that the UC tissue fragments tightly adhere to the dish to obtain MSCs consistently and efficiently. This is because MSCs can only migrate from the adherent UC tissue fragments and not from floating fragments. In fact, we demonstrated that only the adherent part of the cells in UC tissues showed positive CD105 expression. To prevent the floating of tissue fragments from the bottom of the culture dish, we improved the explant method by using a stainless-steel mesh (Cellamigo®; Tsubakimoto Chain Co.). In addition, the incubation time required to reach 80–90% confluence is reduced upon inclusion of the mesh.

Enzymatic digestion method

UCs are minced into small pieces and immersed in media containing enzymes such as collagenase, or a combination of collagenase and hyaluronidase with or without trypsin. Tissues are then incubated with shaking for 2–4 h, washed with media, and then seeded on a tissue culture dish. MSCs are then obtained as described above.

Cryopreservation


Long-term cryopreservation of UCs and UC-MSCs is desirable, because the same donor sample may be required multiple times in the future, and because the cells may be further investigated in the future with techniques yet to be devised. Long-term cryopreservation extends the usability of UC-MSCs. The main technique used to prevent damage is a well-studied combination of slow freezing at a controlled rate, and addition of cryoprotectants.
As a cryoprotectant, 5–10% dimethyl sulfoxide (DMSO) with animal or human serum is typically used. Ten percent DMSO and various amounts of fetal bovine serum (FBS) with or without culture medium is the common standard cocktail for the cryopreservation of cells in research facilities. There are several reports of cryopreservation of the UC tissue and MSCs, using serum-free and xenogeneic animal free (xeno-free) cryoprotectants.

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Characteristics of MSCs

MSCs and characteristics of MSCs are defined by criteria that form the basis for their use as therapeutic agents

Criteria for MSCs; biomarkers and differentiation potentials

The International Society for Cellular Therapy proposed minimal criteria for defining human MSCs. Firstly, MSCs must be plastic-adherent when maintained in standard culture conditions. Secondly, MSC must express CD105, CD73, and CD90, but not CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules. Thirdly, MSCs must differentiate into adipocytes, chondroblasts, and osteoblasts in vitro. UC-MSCs as well as MSCs derived from other sources meet these criteria.

Immunosuppressive properties

Immunosuppressive and immunomodulatory effects have now become the most popular property of MSCs for their clinical use. Defect of HLA-class II expression in UC-MSCs can theoretically rescue them from immune recognition by CD4+ T cells. Moreover, MSCs don't express co-stimulatory surface antigens, CD40, CD80, and CD86, which activate T-cells. Thus, MSCs can escape activated T cells and exert immunomodulation. The immunomodulation may be resulted from soluble factors such as indoleamine 2,3-dioxygenase (IDO), PGE2, galectin-1, and HLA-G5. On the other hand, several studies have reported that UC-MSCs may display immunosuppressive properties only after exposure to inflammatory cytokines and/or activated T-cells, a process called licensing or priming. With these anti-inflammatory actions, MSCs could be good therapeutic candidates for neurological disorders accompanying inflammation.

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Migration ability

MSCs are reported to exert migratory action similar to those of leukocytes with respect to cytokine responsiveness and the ability for transendothelial migration, and this migration capacity towards injured sites is one of the important factors in MSCs-based transplant therapy. In addition, Teo GS et al. reported that, BM-MSCs preferentially adhere to and migrate across tumor necrosis factor-α-activated endothelium in a vascular cell adhesion molecule-1 (VCAM-1) and G-protein-coupled receptor signaling-dependent manner and transmigrate into inflammatory lesion like leukocytes. We actually reported the migration ability of UC-MSCs towards injured neural cells with glucose depletion in vitro. Considering the treatment of neurological diseases, the passage of the MSCs through the blood–brain barrier (BBB) is a critical issue. Lin MN et al. reported phosphatidylinositol 3-kinase (PI3K)/Akt and Rho/ROCK (Rho kinase) pathways are involved in MSCs migration through human brain microvascular endothelial cell monolayers. In addition, Matsushita T et al. revealed MSCs transmigrate across the brain microvascular endothelial cells (BMECs) monolayers through transiently formed intercellular gaps between the BMECs. The migration ability of MSCs and the elucidation of mechanisms broaden the possibility of cellular therapy of neurological diseases.

Tissue repair properties

Neurorestorative and neuroprotective effects as a tissue repair property of MSCs can be mainly characterized by two mechanisms of action: neurogenic differentiation and cell replacement, and secretion of neurotrophic factors. Regarding the former, it has been reported transplantation of UC-MSCs can significantly alleviate ischemic injury, and the rescue arises from differentiation of transplanted cells into neurons and astrocytes. On the other hand, concerning the latter point, it has been reported the paracrine effects of UC-MSCs on nerve regeneration, showing that UC-MSCs secret neurotrophic factors and that UC-MSCs-conditioned medium enhances Schwann cell viability and proliferation via increases in nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression. We also found that UC-MSCs which secrete neurotrophic factors such as BDNF and hepatocyte growth factor (HGF), but not nerve growth factor (NGF), attenuate brain injury. BDNF has been reported to improve hypomyelination via Erk phosphorylation or TrkB signaling, and HGF has been reported to influence the development and growth of oligodendrocytes, as well as the proliferation of myelin-forming Schwann cells, and also reduces gliosis by suppressing MCP-1 induction. These BDNF and HGF are reported to activate the phosphatidylinositol 3-kinase/Akt and MAP-kinase pathways which lead to neurorestorative, anti-apoptotic and neurogenic effects. In addition to the anti-inflammatory effect mentioned above, these neurogenic differentiation ability and the paracrine effects of UC-MSCs are expected to contribute toward their use as therapeutics for neurological injuries.

Article Title with Solid Background

Most of these clinical studies were performed in adults, and trials focusing on cerebral palsy were performed in children. With regard to the source of MSCs, most of the BM and AD are autologous, whereas the UC are allogeneic. Autologous transplantation is desirable considering the possibility of transplant rejection, but it depends on the sources and administrative timing. Regarding sources, it is difficult to isolate autologous MSCs derived from BM and AD in infants and children compared to the autologous MSCs from UC and UCB, because sampling of BM and AD is an invasive procedure. As for administrative timing, as it takes 3–6 months to mass culture MSCs and to confirm that they pass quality tests such as infection tests and chromosome tests, it is impossible to administer autologous MSCs in the acute phase of neurological injuries. On the other hand, allogeneic MSCs can be ordered as a preparation and administered in the acute to subacute phase.
As for the administration route of MSCs, intravenous injection is usual for graft versus host disease, liver disease, and heart disease, whereas local injection is used for arthrosis, and so on, but in clinical studies targeting neurological disorders, most studies were performed using intrathecal injection.

  1. Spinal cord injury - A recent study reported the safety of intrathecal injection of autologous BM-MSCs in nine patients with spinal cord injury [50]. In this study, patient received two or three injections with a median of 1.2 × 106 cells/kg and no treatment-related adverse event was observed. Vaquero J et al. reported that variable improvement was found in the patients, and that mean values of BDNF, glial-derived neurotrophic factor, ciliary neurotrophic factor, and neurotrophin 3 and 4 showed slight increases compared with basal levels after subarachnoid administrations of BM-MSCs. In a study using UC-MSCs study, it was proved that transplantation of allogeneic UC-MSCs has advantages in neurofunctional recovery in comparison with rehabilitation therapy and self-healing alone. Other reports using BM, AD-MSCs showed the feasibility of MSCs administration in spinal cord injury patients, and subjects displayed variable improvements especially in motor functional recovery.
  2. Amyotrophic lateral sclerosis (ALS) - Oh K et al. reported two repeated intrathecal injections of autologous BM-MSCs were safe and feasible for ALS patients throughout the duration of the 12-month follow-up period. In addition, there is a report showing intrathecal and intramuscular administration of BM-MSCs secreting neurotrophic factors (MSC-NTF) in patients with ALS is safe and indicates possible clinical benefits. On the other hand, Staff NP et al. reported the safety of intrathecal autologous AD-MSCs treatment for ALS patients, but the results didn't directly address the efficacy of MSCs therapy. It has been reported that therapeutic effectiveness of intrathecal administration of MSCs was related with the level of neurotrophic factor and anti-inflammatory cytokines in ALS patients and that the potential therapeutic effect of MSCs would not be long-lasting because MSCs gradually disappear over time in cerebrospinal fluid, therefore multiple administration of MSCs would be needed to sustain therapeutic effects.
  3. Spinocerebellar ataxia (SCA) - There are two clinical trials to assess the feasibility and efficacy of allogeneic MSCs therapy in patients with SCA. Tsai YA et al. reported the safety and possible efficacy (scale for assessment and rating of ataxia and sensory organization testing scores, metabolite ratios on the brain magnetic resonance spectroscopy, and brain glucose metabolism) during the 1-year follow-up after intravenous administration of allogeneic AD-MSCs. On the other hand, Jin J.L et al. reported intravenous and intrathecal infusion of allogeneic UC-MSCs was performed with no serious transplant-related adverse events and the majority of patients showed improved Berg Balance Scale (BBS) and International Cooperative Ataxia Rating Scale (ICARS) scores continuing for at least 6 months.
  4. Cerebral palsy - Recently, stem cells are emerging as a new treatment and possible cure for cerebral palsy. Liu X et al. investigated whether BM-MSCs and BM-mononuclear cells (MNC) have any difference of curative effect for the treatment of spastic cerebral palsy. The results indicated that BM-MSCs transplantation for the treatment of cerebral palsy is safe and feasible, and can improve gross and fine motor function significantly compared with the results of BM-MNC treatment. In addition, both of UC-MSCs and UCB-MSCs transplantation in children with cerebral palsy were reported to be safe and effective in improving gross motor function. These reports suggest transplantation of MSCs in subjects with cerebral palsy is safe and may promote neurological improvements.

Conclusion

MSCs for neurological disorders are expected as a new cell therapy to be effective by combining with rehabilitation and other medication therapy suggested by recent clinical trials.

Considering the suppression of inflammation, administration of allogeneic third-party MSCs in the acute to subacute period is considered desirable, whereas administration of autologous MSCs is sufficiently effective in the chronic phase possibly resulting from neurotrophic and neurorestorative effects rather than immunosuppressive effects in recent clinical trials. Therefore, it is important to investigate the appropriate administration protocol of MSCs considering the sources and timing, and also there are many problems to be solved including the number of MSCs to be administered, the kinds of medium used for culturing MSCs, the number of culture passages, and the preservation state until use. Further studies and clinical use in the near future will extend our knowledge of MSCs.





Urological disorder


Voiding dysfunction encompasses a wide range of urologic disorders including stress urinary incontinence and overactive bladder that have a detrimental impact on the quality of life of millions of men and women worldwide. In recent years, we have greatly expanded our understanding of the pathophysiology of these clinical conditions. However, current gold standard therapies often provide symptomatic relief without targeting the underlying etiology of disease development. Recently, the use of stem cells to halt disease progression and reverse underlying pathology has emerged as a promising method to restore normal voiding function. Stem cells are classically thought to aid in tissue repair via their ability for multilineage differentiation and self-renewal. They may also exert a therapeutic effect via the secretion of bioactive factors that direct other stem and progenitor cells to the area of injury, and that also possess antiapoptotic, antiscarring, neovascularization, and immunomodulatory properties. Local injections of mesenchymal, muscle-derived, and adipose-derived stem cells have all yielded successful outcomes in animal models of mechanical, nerve, or external urethral sphincter injury in stress urinary incontinence. Similarly, direct injection of mesenchymal and adipose-derived stem cells into the bladder in animal models of bladder overactivity have demonstrated efficacy. Early clinical trials using stem cells for the treatment of stress urinary incontinence in both male and female patients have also achieved promising functional results with minimal adverse effects. Although many challenges remain to be addressed prior to the clinical implementation of this technology, novel stem-cell-based therapies are an exciting potential therapy for voiding dysfunction.

introduction

Voiding dysfunction represents a diverse spectrum of urologic conditions including stress urinary incontinence (SUI) and overactive bladder (OAB): two of the most common and challenging conditions faced by urologists today. Several of these disorders are precipitated by an acute injury such as vaginal delivery in female SUI, or radical prostatectomy in male SUI. The underlying pathophysiology comprises a complex interplay of injuries or altered function at the tissue, nerve, and vascular level that is then exacerbated by underlying comorbidities, including obesity, increasing age, and diabetes mellitus.

Despite significant strides in our understanding of the underlying pathophysiology of these clinical conditions, current gold standard therapies remain lacking in their ability to target these fundamental mechanisms of injury or etiology of disease. To date, none of the standard therapies focus on halting disease progression or reversing existing injury. Regenerative medicine concepts have emerged as an exciting means of fulfilling this therapeutic void by restoring and maintaining normal function via direct effects on injured or dysfunctional tissues. Over the past decade, the use of stem cells has shown promise for a host of urologic disorders including applications in lower urinary tract dysfunction, ureteral and bladder trauma, erectile dysfunction, and renal disease 

Stem cells are classically thought to improve tissue repair via multilineage differentiation and self-renewal. Stem cells may also exert a therapeutic effect via the secretion of bioactive factors that have antiapoptotic, antiscarring, neovascularization, and immunomodulatory effects on innate tissues and can direct innate stem and progenitor cells to the area of injury. Multiple treatment avenues using stem cells for voiding dysfunction, especially SUI, have been evaluated with preclinical animal models and clinical trials demonstrating their potential to restore function via direct effects on the underlying mechanisms that lead to incontinence or voiding dysfunction Nonetheless, many challenges remain to translate these promising results to clinical practice.

In this review, we provide a brief overview of some of the most prevalent clinical conditions that constitute voiding dysfunction and urinary incontinence. We review stem cell sources and their potential mechanisms of action in aiding tissue repair. We then discuss the key preclinical and clinical trials using stem cell therapy for SUI and OAB, and, finally, highlight some of the challenges in translating this promising research from the bench to the bedside as well as future avenues for development.

The clinical problems

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SUI in women

SUI, the involuntary leakage of urine during events that result in increased abdominal pressure in the absence of a bladder contraction, is a prevalent condition in women that results from failure of the urethral sphincter, pelvic floor muscles, and fascial support tissues to provide sufficient closure to prevent leakage . SUI occurs when intra-abdominal pressure exceeds urethral pressure, resulting in leakage. The incidence of incontinence increases with increasing age and, while daily leakage is less common in young women, up to one third of middle-aged females report leakage at least weekly with 10% reporting daily or severe leakage In females, urinary continence relies on an intact urethral sphincteric mechanism. Multiple factors contribute to urethral pressure including bladder neck position, urethral sphincter musculature, sphincter innervation, and surrounding vascular supply and tissue support. Pregnancy and childbirth are well-recognized risk factors for SUI and four related major mechanisms of injury have been identified: injury to connective tissue support during vaginal delivery, vascular damage due to fetal compression of surrounding pelvic structures, traumatic injury to pelvic nerves and musculature and direct injury to the lower urinary tract during childbirth.

Patients with SUI can benefit from initial conservative measures including pelvic floor physiotherapy, biofeedback, electrical stimulation, and, in some countries, pharmacotherapy; however, surgical options remain the mainstay for cases nonresponsive to conservative measures. Urethral slings and suspensions aim to correct SUI by correcting hypermobility and augmenting intrinsic sphincter deficiency by allowing urethral compression during periods of increased intra-abdominal pressure without causing obstruction during voiding. At 48 months postoperatively, current surgical techniques for SUI have success rates of 30% for collagen injection, 73% for urethral suspensions, and 82–96% for urethral slings. A number of complications can result from sling implantation, including erosion rates in up to 23% of cases, permanent retention in up to 5% of cases, as well as wound complications, bladder perforation, and persistent groin/suprapubic pain and dyspareunia in up to 15% of cases. Although SUI is the culmination of diverse injuries to a host of pelvic structures, the current gold standard therapy only indirectly addresses one such aspect of injury by providing mechanical support to a weakened pelvic floor.

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SUI in men

SUI in men is most commonly precipitated by radical prostatectomy with a historical incidence between 0.8% and 87% . Contemporary series report improved continence rates following prostatectomy although wide variation continues to exist due to discrepancies in data collection methodology, and patient- versus surgeon-reported outcomes. Nonetheless, while treatment options have improved in recent years, the burden of disease remains high due to the increasing numbers of radical prostatectomies performed annually . Post-prostatectomy incontinence results from a failure to store urine secondary to inadequate resistance of the outlet sphincter. Surgical damage to the urethral sphincter occurs due to direct injury to the sphincter itself, as well as to surrounding nerves and supportive tissue. As in females, surgical therapies are the mainstay of therapy and include transurethral bulking agents, bulbar urethral slings, and artificial urinary sphincters (AUS). These treatments aim to augment a deficient urinary sphincter by allowing urethral coaptation during periods of increased abdominal pressure without causing outlet obstruction during voiding. However, 5 years post-AUS implantation, up to 24% of men still report severe incontinence, and 28% will have undergone an AUS revision.


OAB

OAB affects up to 12% of men and women. While overall prevalence of OAB is similar between genders, the severity of symptoms and phenotype differs. OAB without urgency incontinence is more prevalent in men, and increases in prevalence with increasing age from 0.3% to 8.9% with a marked increase by 64 years of age. In contrast, OAB with urgency incontinence is more prevalent in women and increases in prevalence with age from 2.0% to 19.0% with a significant increase after 44 years of age. OAB is characterized by urgency, with or without urgency urinary incontinence, usually accompanied by frequency and nocturia in the absence of infection or other pathology, and may or may not be associated with detrusor overactivity (DO) on urodynamic evaluation. Normal lower urinary tract function comprises a complex integration of detrusor muscle, urethral, and pelvic floor muscle with hierarchical neural control. In OAB, DO may result from increased cellular excitability of the detrusor muscle and/or abnormal neural propagation locally as well as altered peripheral afferent nerve and central nervous system function. Many treatment options are available for patients with OAB, including pharmacologic interventions such as anticholinergics and beta-3 adrenergics, and nonpharmacologic options such as behavioral/dietary modification, biofeedback, and sacral neuromodulation. However, not all patients obtain adequate relief with current therapies. In addition, many suffer complications or side effects requiring discontinuation of therapy. In a retrospective study by Kelleher and colleagues of women with DO who were treated with immediate release oxybutynin, 59.5% of women reported symptom cure or improvement at 6 months; however, 40% had discontinued medication due to side effects. Open-label extension studies of five commonly used OAB medications generally show higher levels of medication adherence, although discontinuation of therapy due to adverse events was reported to be as high as 24% of study participants 
In all of these clinical conditions, current therapies do not repair the original cause of the pathology and may be associated with significant side effects or the need for further invasive treatment. Regenerative medicine represents one promising avenue to restore normal voiding and continence function.

Stem cells: an overview

The field of stem cell research began with the discovery of mouse embryonic stem cells (ESCs) in the early 1970s and with the description of human ESCs in 1998. Stem cells comprise a unique population of cells with three defining characteristics:  the ability to self-renew, multipotent differentiation potential, or the ability to differentiate into a number of different cell types,  and clonogenicity, or the ability to form clonal cell populations derived from a single stem cell. It is these unique abilities for differentiation and self-renewal that give these cells the potential for restoration of function in multiple tissue types.

Stem cell procurement

The procurement and isolation of stem cells for therapeutic use typically occurs in three stages. The patient initially undergoes a procedure to harvest the cells. Following procurement, the tissue specimen is then transported to a regulated facility for isolation from other cell types. Here, the cells are grown via a process of ex vivo stem cell expansion involving multiple cycles of differentiation and senescence until adequate numbers are reached. Finally, cell sorting may or may not be used to isolate stem cells prior to therapeutic application, as this process is often labor-intensive and expensive. Thus, the cells that are ultimately administered to the patient may comprise a heterogeneous combination of stem cells and differentiated cells. Although still investigational, it is feasible during this process to manipulate the cells for a variety of therapeutic applications. For example, cells can be predifferentiated toward a specific cell lineage, pre-exposed to an environment similar to the in vivo post-transplant environment, or transfected to produce specific cytokines or growth factors.


Sources of stem cells for therapy

Currently, four broad categories describe the diversity of stem cells being investigated in regenerative medicine: ESCs, stem cells derived from placenta or amniotic fluid (AFPS), induced pluripotent stem cells (IPSC), and adult stem cells (ASC) [Martin, 1981; Thomson et al. 1998; Goldman et al. 2012; Vaegler et al. 2012; Kim et al. 2013]. ESCs are stem cells isolated from an early stage embryo. They represent a pluripotent cell source and can differentiate into all adult cell types. These cells have great therapeutic potential; however, their use is limited by ethical factors. In addition, their clinical application is limited because they represent an allogeneic cell source with the potential to provoke an immune response and because of concerns regarding potential tumorigenicity. Nonetheless, ESCs are currently being investigated for application to type 1 diabetes mellitus and cardiomyopathy. However, for the reasons stated above, ESCs are not being investigated as a treatment for voiding dysfunction or urinary incontinence.
AFPSs are a new class of stem cells with properties intermediate to those of ESCs and ASCs. They represent a heterogeneous stem cell population derived from the amniotic fluid and placental membrane of the developing fetus. Cells found in these tissues include mesenchymal stem cells (MSCs) as well as multipotent AFPS cells that possess extensive self-renewal capacity. In addition, AFPS cells can be induced to differentiate into cells of all three germ cell layers including cells of adipogenic, osteogenic, myogenic, endothelial, neural, and hepatic lineages. AFPSs are currently being investigated for a variety of applications including treatment of acute tubular necrosis, cardiac valve regeneration for the early correction of congenital cardiac malformations, and as a source of immunomodulatory cells for a variety of immunotherapies, but are not being investigated for application in urology.
IPSCs are a unique class of stem cells recently discovered by Takahashi and Yamanaka who demonstrated that specific transcription factors could be used to reprogram differentiated cells to a pluripotent state. Like ESCs, these cells have been shown to possess capacity for multipotent differentiation and self-renewal. In addition, they can be used autologously and their derivation from adult cells obviates concerns regarding ethical issues with ESCs. A disadvantage of IPSCs, however, is the time involved in resetting the cells to a pluripotent state followed by the additional time required to induce the cells to differentiate into the desired lineage. Furthermore, there remain concerns that full transition to this new desired lineage may not occur. To date, IPSCs have not yet been used for urologic applications.
ASCs are the most well-studied and well-understood cell type in the field of stem cell therapy and, thus far, are the only stem cell type that have been investigated for urologic applications. Over the past decade, ASCs have been identified throughout the body and are thought to act as tissue-specific progenitors that repair damage and restore function locally. MSCs, also known as multipotent adult progenitor cells, are a unique subset of ASCs first described by Friedenstein and colleagues in the 1970s. Classically, MSCs were isolated from bone marrow stroma although more contemporary studies have demonstrated that they may also be found in other well-vascularized tissues including adipose, muscle, endometrium, and kidney. Unlike tissue-specific progenitor cells, they can be induced to differentiate into multiple cell lineages including bone, neuronal, adipose, muscle, liver, lungs, spleen and gastrointestinal tissues.
At the present time, MSCs are the primary source of stem cells tested for therapeutic benefit in urologic applications. However, this cell population is relatively rare in bone marrow (approximately 1 per 10,000 cells) and traditional bone marrow procurement is painful, requires general or spinal anesthesia, and potentially produces low yield [Krause et al. 2001]. Alternative cell sources that have been investigated for urologic application include both muscle-derived stem cells (MDSCs) and adipose-derived stem cells (ADSCs) which can be obtained via less-invasive biopsies and in larger quantities under local anesthesia. Investigators continue to identify novel, less-invasive cell sources for urologic application including cells derived from hair follicles, menstrual fluid, and urine.
Urine-derived stem cells (USCs) are of particular interest for potential urologic applications . In recent studies, Zhang and colleagues have reported the successful isolation and expansion of stem cells from voided human urine. These cells, thought to be MSCs or pericytes from the kidney, can be obtained noninvasively from human urine specimens and demonstrate stem-cell-like features including clonogenicity, self-renewal, and multipotent differentiation capacity. Single clones of USCs have been shown to have the capacity to expand to yield a large population. Comprehensive characterization studies of these USCs demonstrate their ability to differentiate into multiple cell lineages at the gene/protein expression, cellular and tissue levels. In vitro studies show that USCs derived from a single clone were, following induction, able to differentiate into smooth muscle and urothelial lineages as evidenced by histologic examination and gene/protein marker assays. USCs can also be induced to differentiate into osteogenic, adipogenic, and chondrogenic lineages using different differentiation protocols. In vivo studies using BMP-2 and -9 transduced USCs also demonstrated their ability to form bone, fat, and cartilage tissue in immunodeficient female mice. USCs hold several advantages as a cell source for incontinence and voiding dysfunction. They are easily harvested and do not require invasive surgical techniques to obtain. Furthermore, they are easily isolated with a significant cost advantage at US$50 to obtain cells from urine compared with approximately US$5000 to obtain cells using a biopsy procedure. Finally, they can be used autologously, obviating potential ethical issues or the potential of adverse immune reactions.


Stem cell mechanisms of action

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Stem cell homing

The process of innate systemic stem cell delivery to the site of injury is termed “homing” and can be taken advantage of in delivering cells systemically rather than locally. In contrast to tissue-specific progenitor cells, MSCs derived from the bone marrow traverse the circulatory system with access to all tissues in the body but then migrate to specific locations such as areas of acute injury following chemokine gradients where they can engraft and facilitate healing and regeneration. Much of the insight regarding stem cell homing derives from literature regarding leukocyte migration into injured tissue, metastatic cancer cells, and hematopoietic stem cells. Similar to leukocytes, MSCs express cell surface receptors and adhesion molecules responsible for directing cellular migration and homing to particular tissues, including the chemokine receptor CXCR4 and its binding partner CXCL12 as well as the chemokine ligands: CCR1, CCR4, CCR7, CCR10, CCR9, CXCR5, and CXCR6. MSCs are hypothesized to migrate to target tissues via a process similar to that of leukocyte migration: initial localization by means of chemoattraction, adhesion to vascular endothelial cells at the target site, and, finally, transmigration across the endothelium to the site of injury. Integrins and selectins are other classes of cell surface molecules that direct migration and adhesion of a variety of cells, including MSCs. While the role of these molecules in leukocyte-endothelial adhesion is well established, their exact role in facilitating MSC interaction with endothelium is less well characterized. Ruster and colleagues found that binding and rolling of MSCs was mediated by P-selectin while migration involved binding of the integrin VLA-4 on MSCs with VCAM-1 on endothelial cells.

A large body of literature utilizing animal models has demonstrated the ability of MSCs to home to injured tissues in several disease models including cardiac injury, renal failure, and skin wounds. In addition, recent work investigating MSCs for SUI has shown that chemokine ligand 7 (CCL7), a homing factor for MSCs, is upregulated in both the urethra and vagina after vaginal distension (VD), suggesting that intravenously administered MSCs could have the potential to home to sites of injury in SUI. Subsequent literature examining intravenous injection of MSCs in a VD rat model of simulated childbirth injury showed that MSCs home to the urethra and vagina and facilitate recovery of continence as measured by leak point pressure (LPP). Recent work by Lenis and colleagues used a rat model of childbirth injury with both virgin rats that had undergone VD and postpartum rats to further investigate the expression of chemokines and receptors involved in stem cell homing and tissue repair. They showed that VD in virgin and postpartum rats resulted in upregulation of urethral CCL7 expression. In addition, pregnancy and delivery was found to upregulate the chemokine receptor CD191 but to decrease the expression of hypoxia inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF). Similarly, in a mouse model of OAB via bladder outlet obstruction, Woo and colleagues showed that intravenously injected MSCs following bladder injury were associated with increased chemokine ligand 2 (CCL2) expression in the affected tissue.

In therapeutic applications, stem cell homing is affected by a variety of factors including age and passage number of the cells, culture conditions, and the delivery method. Rombouts and Ploemacher demonstrated that with increasing age and passage number, the efficiency of MSC engraftment decreases, possibly due to rapid aging of the cells with increased in vitro multiplication. Reproduction of the innate MSC microenvironment in vitro is challenging and plays a significant role in stem cell homing potential. For example, in vitro expression of matrix metalloproteinases (MMPs), key factors to stem cell migration, is affected by cell culture factors such as hypoxia and culture confluence and may be upregulated with the addition of certain inflammatory cytokines, such as transforming growth factor beta 1 (TGFβ1), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α [De Becker et al. 2007]. Despite the appeal of delivering cells systemically rather than locally, systemically infused MSCs often suffer from a first-pass effect whereby these larger cells become trapped in capillary beds of various tissues, especially the lungs, liver, and spleen, decreasing their therapeutic bioavailability and functionality [Fischer et al. 2009]. To overcome this, recent investigations have utilized an intraperitoneal delivery route for MSCs and have obtained higher yield in target tissues.

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Stem cell differentiation

The mechanism of action of stem cells was initially thought to primarily derive from their ability to differentiate into multiple cell types and regenerate damaged tissues. To date, treatment avenues for voiding dysfunction and urinary incontinence have focused on this potential to restore function via differentiation to replace injured or diseased tissues such as smooth or striated muscle for urethral sphincter regeneration and urothelial tissue for bladder, urethral, and upper urinary tract reconstruction. It is thought that MSCs restore function in SUI primarily by their ability to differentiate into multiple cell lineages, with animal studies demonstrating increases in urethral muscle, nerves, and connective tissue following MSC injection. MSCs have also been induced to differentiate into a smooth muscle phenotype when exposed to conditioned media from smooth muscle cell cultures or when induced with specific myogenic growth factors for application to bladder reconstruction. More recent work, however, suggests a more complex role of stem cells in functional recovery, with some studies demonstrating that paracrine secretions of MSCs play an important role in the regeneration process.

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Bioactive effects of stem cells

Recent research has demonstrated that, in addition to differentiating into target tissue types, stem cells likely exert a therapeutic effect via the secretion of bioactive factors since few MSCs engraft and remain long term in target tissues, in contrast to their large therapeutic effect. MSCs also activate and direct endogenous stem and progenitor cells to areas of injury via the secretion of cytokines and chemokines. In addition, their secretions have antiapoptotic, antiscarring, and neovascularization effects, as well as immunomodulatory properties. The investigation of this protein milieu or “secretome” is a subject of growing interest with the increasing recognition of the paracrine/autocrine role of cell secretions in the regulation of many physiological processes and their potential for therapeutic application. The investigation of these cell-specific proteins often begins in cell culture. While in vitro studies cannot fully capture and test the totality of MSC secretions in the in vivo microenvironment, researchers seek to replicate the effects of the MSC secretome via the use of media conditioned by the MSCs and containing their secretions.

MSCs also possess immunomodulatory and immunological tolerance inducing characteristics. These cells typically express MHC-I but lack expression of MHC-II, CD40, CD80, and CD86. Owing to the lack of co-stimulatory cell surface molecules, MSCs fail to induce an immune response by the transplant host. MSCs have also been shown to play a role in suppressing immune responses by modulation of T-cell activation and proliferation through both direct cell–cell interaction and via the action of soluble factors. These immunomodulating properties are currently being investigated in myriad applications including prevention of graft-versus-host disease following allogeneic transplantation and Crohn’s disease. Clinical trials in cardiology have taken advantage of these properties of MSCs to investigate the efficacy of nontype-matched allogeneic MSC transplantation. In a study by Hare and colleagues 1 year after intravenous administration of allogeneic human MSCs in reperfused myocardial infarction patients, recovery, as measured by global symptom score, ejection fraction, ambulatory electrocardiogram monitoring, and pulmonary function testing, was significantly improved in treated patients compared with those that received a placebo [Hare et al. 2009]. In addition, no signs of rejection were observed and adverse event rates were comparable between treated and placebo arms.

A recent study by Dissaranan and colleagues demonstrates that the secretome of MSCs can facilitate recovery of continence as measured by LPP following VD in a rat model; moreover, rats treated locally with MSC secretome, as contained in conditioned media, exhibited an increase in elastin fibers and urethral smooth muscle, which may have contributed to the restoration of continence. A study of hamsters with heart failure suggests that MSCs act systemically as well as locally. In this study MSCs injected into the hamstrings of affected animals were unable to migrate from the injection site; however, the authors found that treated animals still benefited from stem cell injection based on histologic and functional analysis of the myocardium. More recent work by Timmers and colleagues utilizes human MSC secretions collected as conditioned medium in a pig model of myocardial infarction. Pigs underwent left circumflex coronary artery and were then administered intravenous conditioned media for 7 days. At 3 weeks following initial cardiac injury and treatment, pigs treated with MSC conditioned media were found not only to have increased myocardial vascular density but also reduced infarction size and more preserved cardiac function.

These findings have been borne out in other urologic investigations as well. Lin and colleagues investigated rats subjected to VD and ovariectomy that were subsequently treated with intraurethral injection of ADSCs; despite limited cell engraftment on histologic analysis, these rats demonstrated significant functional recovery. Similarly, a study by Song and colleagues utilizing bladder wall injections of MSCs demonstrated functional recovery in a rat model of OAB from partial bladder outlet obstruction (PBOO) despite limited engraftment 4 weeks post-treatment. Further investigations into the use of stem cell bioactive factors could someday obviate the need for cellular injections in future stem cell therapies.

Stem cell therapy for voiding dysfunction


Stem cells for stress urinary incontinence

Various animal models of simulated childbirth have been used to mimic the injuries that can produce SUI and enable preclinical testing of potential therapies for this prevalent condition. The first animal model was introduced by Lin and colleagues in 1998 and utilized VD in female rats to simulate the trauma of childbirth. VD results in damage to surrounding muscles and nerves responsible for continence as evidenced by increased levator muscle edema and smooth muscle disruption, as well as hypoxic injury. Since this initial study, multiple models have been developed to investigate other putative mechanisms of injury to the continence mechanism, including nerve injury, direct urethral injury, and pelvic ligament injury. These include:  simulation of nerve injury via pudendal nerve crush (PNC), simulation of anatomic support damage with urethrolysis or pubourethral ligament injury,  simulation of intrinsic urethral defects with periurethral cauterization, urethral sphincterectomy, or pudendal nerve transection,  and combination models such as those with both VD and PNC. Most research testing novel cell-based therapies for SUI has focused on direct stem cell injection into the urethra with the hope of repairing or regenerating damaged rhabdosphincter tissue. Local injections of MSCs, MDSCs, and ADSCs have all demonstrated efficacy in animal models of either mechanical, nerve, or external urethral sphincter injury, as demonstrated by both anatomic and functional outcomes.

MDSCs have been shown to improve sphincter function in several animal models of SUI. Chermansky and colleagues investigated MDSCs in an animal model of urethral injury by midurethral cauterization. MDSC injection 1 week after injury led to significant tissue recovery as evidenced by increased LPP 2, 4, and 6 weeks afterwards compared with controls. Tissue staining confirmed integration of MDSCs into the striated muscle layer of the urethra. Similarly, Kim and colleagues injected MDSCs into the urethra of rats that had previously undergone pudendal nerve dissection 2 weeks prior. By 4 weeks after MDSC injection, LPP and urethral closure pressure in animals that had undergone stem cell administration and nerve injury were restored to values comparable to rats that had undergone only a sham operation. Furthermore, the injected MDSCs stained positive for muscle-specific markers, suggesting the potential for MDSCs to differentiate into muscle lineage cells that may contribute to the repair of damaged muscle tissue.
ADSCs have also been investigated for treatment of SUI. A pilot study by Jack and colleagues investigated the potential for ADSCs to survive within bladder and urethral smooth muscle. Human ADSCs derived from lipoaspirate of female patients undergoing liposuction were directly injected into the bladder and urethra of athymic rats and mice immediately following laparotomy. Histologic analysis of the bladder and urethra of treated animals was performed 2, 4, 8, and 12 weeks afterwards and demonstrated ADSC viability and incorporation into the recipient smooth muscle. By 8 weeks, ADSCs also demonstrated in vivo expression of α-smooth muscle actin, an early marker of smooth muscle differentiation. In a more recent publication by Fu and colleagues ADSCs that had been induced into myoblasts were injected periurethrally in rats that had undergone VD. Both maximal bladder capacity and LPP were shown to be significantly increased 1 and 3 months post-injury compared with control rats that had been implanted with untreated ADSCs. In addition, histologic analysis showed increased thickness of the inferior muscularis in urethral mucosa which may have contributed to improved sphincter contractility and decreased SUI in injured rats. A study by Lin and colleagues investigated the use of periurethrally injected ADSCs in postpartum rats that then immediately underwent VD to simulate prolonged delivery. One week following injury, rats were subjected to ADSC transplantation. Urinary function and histological analysis was then performed 4 weeks later. Cystometric analysis demonstrated that ADSC-treated animals had a significantly lower incidence of abnormal voiding compared with untreated animals. Histologic analysis demonstrated that treated animals, particularly treated animals that then achieved normal voiding function, had higher urethral elastin and smooth muscle content.
Recent studies have focused on elucidating the mechanism of action of cell treatment in SUI models: specifically, the potential contribution of paracrine/autocrine factors to tissue recovery following childbirth injury. A study by Cruz and colleagues demonstrated that intravenous injection of MSCs following childbirth injury may represent an effective route for cell-based therapy. In their work, green fluorescent protein (GFP)-labeled MSCs from bone marrow were injected into rats 1 hour after VD or sham VD. Imaging 4 and 10 days post-treatment demonstrated that significantly more MSCs homed to the urethra and surrounding tissues in animals that had undergone VD. Their work suggests that, following injury, tissues release factors that promote mobilization and attraction of MSCs to the injured site. A subsequent study by Dissaranan and colleagues further investigated MSC homing as well as the potential role of the MSC secretome in facilitation of functional recovery after VD. MSCs or media conditioned by the MSCs and then concentrated, or concentrated conditioned media (CCM), was administered intravenously and periurethrally, respectively, to rats 1 hour after VD or sham VD. Urethral histology and function were assessed one week following treatment. As with the study by Cruz and colleagues, significantly more MSCs were found in the urethra and surrounding tissues in animals that had undergone VD versus sham VD. Interestingly, continence, as assessed by LPP, was found to not only be improved in injured animals that had received intravenous MSCs but also in those that had received a periurethral injection of CCM. The recovery of function utilizing only the secretions of MSCs is a fascinating discovery that suggests the possibility of an acellular stem cell-derived therapy.
Carr and colleagues performed the first North American MDSC therapy trials for SUI. A follow-up expanded study by the same group has demonstrated promising results [Carr et al. 2013]. A total of 38 women with SUI that had not improved with a trial of 12 or more months of conservative therapy received intrasphincteric injection of MDSCs derived from autologous lateral thigh muscle biopsies. Subjects also had the option of electing a second treatment of the same dose of MDSCs at 3-month follow up. Assessments were then made 1, 3, 6, and 12 months following the last treatment. Of the 33 subjects who completed the study, nearly 90% in the high-dose group experienced a 50% or greater reduction in pad weight, and nearly 80% had a 50% or greater reduction in diary-reported stress leaks. Adverse side effects from the treatment were limited to minor events such as pain or bruising at the injection or biopsy sites.
Gotoh and colleagues recently investigated the efficacy and safety of periurethral injection of ADSCs in 11 men who had persistent SUI more than 2 years following prostate surgery (radical prostatectomy or holmium laser enucleation of the prostate). One-year outcomes demonstrated 59.8% decrease in leakage volume on a 24-hour pad test, decreased frequency and improved quality of life. In addition, mean maximum urethral closure pressure and urethral functional profile length were increased compared with pretreatment values, and magnetic resonance imaging demonstrated the sustained presence of injected adipose tissue. No adverse events occurred during ADSC procurement and administration nor were any severe side effects or changes in serum prostate specific antigen levels reported during the follow-up period. As with the study by Carr and colleagues, however, these feasibility studies are limited by their lack of a control group and small sample size. Nonetheless, these initial open label safety trials are necessary prior to moving on to controlled double-blinded clinical trials.
The use of stem cells in conjunction with existing or emerging therapies is another nascent area of research. For instance, Zou and colleagues have combined stem cell therapy with slings in an effort to augment the long-term efficacy of slings and reduce rates of sling erosion or extrusion . In this study, female rats underwent bilateral proximal sciatic nerve transection and developed SUI confirmed by LPP measurement 4 weeks following injury. At this time, rats were treated with either no sling, a silk sling, or a tissue-engineered sling composed of a silk scaffold that had been seeded with MSCs. Histology and LPP testing 4 and 12 weeks later, and collagen and mechanical testing 12 weeks after implantation, showed that animals treated with the tissue-engineered sling had nearly normal LPP, higher collagen content, and higher failure force compared with untreated animals. Liu and colleagues tested genetically modified urine stem cells that overexpress VEGF. Collagen hydrogel scaffolds containing VEGF-overexpressing stem cells were subcutaneously implanted in mice. This cell subset was found to enhance angiogenesis and improve cell engraftment in addition to promoting myogenic differentiation and innervation by USCs.

Stem cells for OAB

The reliance on urgency as a defining symptom in OAB has been a complicating factor for its investigation because of the difficulty of assessing this symptom in animal models. At present, PBOO is the most well-known and well-established model for DO. However, PBOO only comprises one possible etiology of OAB symptoms. Other models of voiding dysfunction including those involving bladder ischemia, diabetes, spinal cord injury, and cryo-injury have also been utilized to test cell-based therapies for voiding dysfunction. In a recent study by Lee and colleagues human MSCs labeled with nanoparticles were injected into the bladder wall of rats 2 weeks following induction of PBOO. Follow up studies, including magnetic resonance imaging (MRI), histology, and functional studies, were performed 4 weeks after treatment. Following induction of PBOO, collagen and TGF-β protein levels increased, consistent with the hypothesis that increased collagen deposition in older men with PBOO leads to lower urinary tract dysfunction. Following MSC transplantation, however, the expression of both proteins returned to pre-injury levels along with maximal voiding pressures and residual volume. MRI confirmed engraftment of MSCs in the bladder wall while maximal voiding pressure and residual urine volume were found to be recovered after MSC transplantation. In a different experiment mice received MSCs intravenously 3 days after induction of PBOO. Urodynamic and histological evaluation were performed 4 weeks post-treatment and MSCs were found in the detrusor muscle and were correlated to decreased levels of tissue hypoxia, hypertrophy, and fibrosis as well as improved bladder compliance. PCR studies revealed increased CCL2 expression in the affected tissue, suggesting the CCL2 overexpression may contribute to MSC recruitment following PBOO

As in SUI, growth factors also play an important role both in normal bladder development and in bladder remodeling following injury. In addition to direct effects via stem cell implantation and differentiation, the paracrine effects of MSCs have been studied in the repair of PBOO-induced DO. In a study by Song et al. injection of human MSCs into the bladder wall of rats was performed 4 weeks after onset of PBOO. MSC therapy led to normalization of OAB outcomes, including maximal voiding pressure, residual urine volume, and intercontraction interval within 4 weeks. Notably, histologic analysis demonstrated that the transplanted MSCs did not engraft well into the damaged bladders despite enhancing the expression of several genes responsible for stem cell trafficking [Song et al. 2012]. These changes in gene expression suggest that paracrine mechanisms of tissue repair may be the dominant mechanism of action of MSC therapy.

Stem cell therapy using other models of DO have also shown promise. Huang and colleagues report the use of autologous ADSCs to ameliorate DO in a hyperlipidemia-associated rat model of DO. Rats were administered a high-fat diet for 5 months. At this time, all rats underwent fat harvest for procurement of ADSCs. After culture and purification, ADSCs were delivered by direct bladder injection as well as intravenously. Functional and histologic studies were then performed 1 month after treatment. ADSC-treated rats were found to have longer micturition intervals compared with untreated rats. In addition, nerve and blood vessel density were lower in untreated rats compared with ADSC-treated animals. While more ADSCs collected within the bladder following local injection compared with systemic injection, treatment with ADSCs via both routes improved both angiogenesis and cholinergic nerve innervation. In another study, rats underwent transection of the bladder branch of the pelvic plexus and were immediately injected around the damaged pelvic plexus with rat MDSCs. Four weeks following injury and treatment, the transplantation group demonstrated significantly greater functional recovery as assessed by intravesical pressure compared with control treated groups. Histologic analysis showed that transplanted cells engrafted into the injured area with differentiation into multiple cell lineages.

Bladder dysfunction manifesting as urothelial dysfunction, alteration in smooth muscle, and neuronal damage is a known sequelae of diabetes mellitus. In a study by Zhang and colleagues rats were fed a high-fat diet and treated with low-dose streptozotocin to induce diabetes. A month later, rat ADSCs were either injected intravenously or directly into the detrusor. One month post-treatment, cystometric analysis demonstrated improved bladder function compared with untreated animals. Furthermore, histologic analysis showed that only a small fraction of transplanted ADSCs differentiated into smooth muscle cells, suggesting that cellular differentiation plays a minor role in the therapeutic benefit of ADSCs. They hypothesize that paracrine release of cytokines and growth factors may, in part, account for the beneficial effects of stem cell transplantation.

Conclusion

To date, stem cells have shown promise for the treatment of voiding dysfunction. Applications in SUI and OAB have demonstrated success in both preclinical animal trials and limited clinical trials. However, stem cells have not yet been tested as a potential treatment for interstitial cystitis or painful bladder syndrome, both of which have the potential to be reversed or ameliorated via the immunomodulatory effects of stem cells. Many questions need to be answered before stem cell therapy can be introduced into urologic practice for voiding dysfunction.

For example, there remains much to be elucidated regarding the mechanisms of stem cell action. Previously, the beneficial effects of stem cells were primarily attributed to their ability to differentiate into multiple cell types and to thus directly augment injured or dysfunctional tissues in voiding disorders [Pittenger et al. 1999]. However, emerging concepts regarding the paracrine actions of stem cells and their effects on tissue healing from angiogenesis, antioxidant properties, and immunomodulation, have opened up new avenues for investigation. The successful use of stem cell bioactive factors, or secretome, in nonurologic and early urologic applications could eventually pave the way for cell-based but cell-free therapies in the future. 

Practical considerations in translating stem cell technology from the bench to the bedside comprise another critical hurdle to overcome. SUI and many of the causes of OAB syndrome are chronic in nature. However, the preclinical models used to date only produce and address acute disease symptomatology. Assessing the efficacy of stem cell therapy for chronic voiding dysfunction will need to be validated further in clinical trials. In addition, work still needs to be performed to identify the best delivery method for stem cells including how to overcome first pass effects and subsequent loss of bioactive potential, as well as to identify the best sources of stem cells for urologic applications. In this vein, recent work by Zhang and colleagues utilizing USCs represents a promising alternate autologous cell source that could be easily harvested and isolated. However, to date, the potential of USCs in animal models of voiding dysfunction remains to be investigated.

Immune considerations regarding stem cell therapies represent another important avenue of necessary investigation. Future stem cell therapies may not always rely on the constraint of using autologous cell sources since autologous cell sources from older individuals, those with genetic disorders, and those who have received systemic treatments such as radiation may not be as efficacious as stem cells derived from younger healthy individuals. While allogeneic stem cells have been tested safely for other diseases, efficacy of allogeneic cell sources for voiding dysfunction have yet to be demonstrated in humans.

In summary, voiding dysfunction and urinary incontinence constitute a variety of common disorders affecting millions of men and women worldwide. Current treatments are limited and do not address the underlying pathophysiology of disorder or disease, nor do they repair damaged or diseased tissue to restore normal function. In contrast to current therapies, stem cells have emerged as an exciting treatment avenue targeting disease progression and potentially correcting pathophysiology. Although many challenges remain to be addressed before clinical implementation of this technology, preclinical animal and early clinical studies show promise in the use of stem cell therapy for voiding dysfunction and urinary incontinence.





Stroke


Stroke represents a public health enemy. Currently, and in spite of multiple clinical trials, thrombolysis remains as the only approved therapy. Most preclinical trials and animal trials employing stem cell-based therapies have shown very promising evidence of benefits. The aim of this review is to provide a landscape of what has been done in human clinical trials, and what are the possible ways that stem cell therapy may enhance functional recovery in stroke patients.

introduction

Acute stroke is the sixth most common cause of death among Mexicans and the third cause in elderly people; just in 2014 it was responsible for 30,000 deaths.1 Though these numbers may even be sub-registered.2 More startling is the fact that stroke is the leading cause of disability in this country and worldwide.3 This is because in spite of the high mortality, 75% of patients survive a stroke with some kind of sequel; this is 258 patients daily.4

The main mid-term and long-term sequels of stroke are dysphagia, fatigue, muscle weakness, paralysis, visual problems, incontinence, chronic pain, seizures, insomnia, depression, dementia, aphasia, amnesia among many others.5

The annual incidence of stroke is 232/100,000 people, with a prevalence of 8/1000.6 With a population of 112 million Mexicans, this means almost 900,000 people survive with some type of disability or sequel. Worse, after a year almost half of them survive with significant sequels that affect their quality of life and their capacity to perform daily life activities (Modified Rankin Scale [mRS] between 2 and 5).7

As if that were not enough, the economic impact is notorious as well. Among hospital expenses, studies, treatments, doctors’ fees, work incapacities, early retirement, rehabilitation programs and more, each patient surviving stroke yearly spends around $25,000 dollars.4 This is of course without taking into consideration the social and emotional expenses.

Stroke prevalence is on the rise worldwide,8 and our country is not the exception.9 In Mexico the median age of a first stroke event is of 68 years (ranges 52–84).7 Epidemiology studies project that by 2050, the group conformed by people aging between 55 and 80 will almost triple, to 28%.10 Therefore, by that year there will be more than 2 million stroke survivors; 1 in every 60 Mexicans.

Even though there have been many attempts to develop new stroke cures, a thrombolytic approach with tissue plasminogen activator for an acute ischemic stroke remains as the only approved therapy.11 It is imperative to find or develop a new treatment to enhance recovery and restore, at least partially, the lost neurological functions.

Physiopathology of stroke

Etiology

Stroke can be divided into ischemic stroke, constituting 85% of cases (atherothrombotic, cardioembolic, small vessel disease), and hemorrhagic stroke (subarachnoid hemorrhage, intraparenchymal hemorrhage) which accounts for the remainder 15%.12 The later one has an overall worse prognosis.13 The primary risk factors for stroke include, but are not limited to, hypertension, diabetes mellitus, smoking, dyslipidemia and atrial fibrillation. 

Cerebral ischemia is graded depending on the cerebral blood flow (CBF; which would normally be of 50–55 ml/100 g/min). Anatomically, stroke lesions can be divided into an ischemic penumbra (CBF of 15 ml/100 g/ml), with functionally impaired neurons, though still reversible with acute stroke therapy, and an ischemic central core (CBF of 6 ml/100 g/ml) with irreversible neuronal death.

Natural history

Lack of energy substrates quickly leads to dysfunction of energy-dependant ion transport pumps and depolarization of neurons and glia.16 This depolarization releases excitatory neurotransmitters, primary glutamate, which amplify the damage by the release of free radicals and interruption of the electron chain transport.17 The following oxidative stress contributes to neuronal death by disruption of the cell membrane.18 Apoptosis also mediates many of the lost neurons, predominantly in the penumbra region if no acute treatment is installed.19

Afterwards, astrocytes concentrate along ischemic lesions, and produce proteoglycans to form a glial scar, which act as both a physical and biochemical barrier to axonal regeneration and sprouting, limiting the reconnection of neural circuits and contributing to many of the long-term sequels of stroke.20, 21

Most important for the matter of this review it is the robust inflammatory reaction following cerebral ischemia. Inflammatory molecules (e.g. interleukin-1 [IL-1], interleukin-6 [IL-6] and tumoral nectrotic factor-alpha [TNF-α]) are predominantly deleterious in the early phase after an ischemic stroke22 and paradoxically promote brain regeneration and neurovascular remodeling in the later or chronic phases.23

Finally, stroke releases many chemotactic molecules (e.g. interleukin 8 [IL-8], monocyte chemoattractant protein-1 [MCP-1]) both for leucocytes and stem cells.24 Particularly stromal-derived factor 1a (SDF1a) is released by activated endothelial cells after hypoxic injuries, and its receptor (CXC chemokine receptor-4 [CXCR4]) is up-regulated as well. They both acts as chemoattractants that mediate neural and bone marrow stem cell migration to injured areas, which is critical for stem cell-based therapies.

Neurorestorative therapies

After neuroprotection (acute) therapies have failed, and scarring, inflammation and edema have installed, the approach must be shifted then to a nuerorestorative therapy rather than on preventing the extension of a damage that has already been well established. This type of therapy focuses on orchestrating through all type of parenchymal cells (i.e. neuroblasts, immune cells, astrocytes, oligodendrocytes and neurons), the enhancement of endogenous neurogenesis, angiogenesis, axonal sprouting and synaptogenesis in affected brain tissue. 

Neurorestorative therapies include, but are not limited to, stem cells. There are also ongoing pharmacological investigations and other type of treatments such as electromagnetic stimulation, device-based strategies, repetitive training and task-oriented strategies. Rehabilitation could exploit the combination of functional reorganization and adaptation after stroke. Of all, currently only constraint-induced therapy has evidenced some type of efficacy.

Stem cells


Types of stem cells

Since their first descriptions in 1963 by Till and McCulloch,33 stem cells have been used as an alternative to many diseases, especially those without any other treatment options. Neurologic conditions have not been the exception. In fact they represent the fifth most common cause for ongoing clinical trials based just on mesenchymal stem cell therapy (after bone, heart, gastrointestinal and autoimmune disorders).
A stem cell is considered as such when two properties are met: capacity for long-term self-renewal without senescence and the ability to differentiate into one or more specialized cell types. Based on their transdifferentiation spectrum stem cells are classified as totipotent if they can form any cell in the body (including placental cells), as pluripotent if they are able to divide into any cell of the three germ-lines of early embryogenesis, and as multipotent if they are already committed to only one germ-line (e.g. most bone marrow stem cells can only differentiate into mesoderm derived cells).
Stem cells are also classified based on their origin as embryonic or adult type. Embryonic stem cells (ESC) are pluripotent cells that are obtained by manipulating embryos before implantation.36 Adult (somatic) stem cells are multipotent (or pluripotent) cells obtained from mature differentiated tissues such as bone marrow, umbilical cord, human olfactory mucosa, fat tissue and brain.
Finally, another type of stem cell is the induced pluripotent stem cells (iPSC). In this technique, differentiated mouse fibroblasts are reprogrammed to an embryonic-like state (pluripotency) by transfer of nuclear contents into oocytes or by fusion with embryonic stem cells.

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Neural stem cell

Contrary to old assumptions, evidence of neurogenesis in the adult human brain has been demonstrated. Neural stem cells (NSC) are a multipotent variant of stem cells present in the brain. These cells are located in the subventricular zone (SVZ) of the third ventricle and in the subgranular zone (SGZ) of dentate gyrus, and respond to brain insults that cause neuronal death such as stroke, Huntington's disease, and Alzheimer's disease. NSC not only proliferate but also migrate to areas of lesion even in elderly patients. NSC can be cultured in vitro for stem cell therapies, and even if administered intravenously, have the capacity to migrate into ischemic areas.

It is been documented that after a stroke NSC expand and mature into well differentiated neurons and integrate functionally into neuronal circuits. After a stroke, the brain's environment holds a rise in many growth factors that induce changes in NSC's mitotic cell cycle such as reduction of G1 phase which boosts mitotic rate up to a 12-fold increase in  as well as activation of phosphatidylinositol 3-kinases-Akt signaling pathway which enhances cell survival, proliferation, differentiation and migration. Stroke also activates many genes involved in neurogenesis during embryonic development, especially those of transforming growth factor-beta [TGF-β] superfamily (bone morphogenic protein 8 [BMP2], bone morphogenetic protein type 1 receptors [BMPR1] and growth differentiation factor 2 [GDF2]). These newly formed neurons differentiate into the phenotype of most of the neurons that were lost during ischemia, in an attempt to regenerate lost circuits and recover lost functions. 

Discouragingly, one of the setbacks is their slim capacity to migrate into areas of the cortex where higher mental functions lie. What is more, after a couple of weeks 80% of these newly formed neurons die and actually just 0.2% of dead tissue is replaced. We hypothesize that if the percentage of incorporated renewed cells could be increased somehow, (e.g. neurotrophic, or angiogenic factors) restoration of neurological functions would be much greater as well.

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Bone marrow stem cells

Bone marrow stem cells (BMSC) are an array of different type of multipotent and pluripotent cells homed in the spongy tissue of almost all bones. Two basic lineages prevail: hematopoietic stem cells (HSC, PBSC if obtained peripherally) and mesenchymal stem cells (MSC). HCS give rise to all the type of blood cells and are typically CD34+, CD133+ and negative for all markers of differentiation or further lineage commitment (CD13−, CD71−, CD19−, CD61−). MSC lie on the stroma of the bone marrow, and contrary to HSC, they can differentiate into a broader variety of cell types, such as osteoblasts, chondrocytes, myocytes and adipocytes and even neurons. MSC are usually CD34- 

BMSC actually have limited cellular differentiation ability in comparison to other type of stem cells; evidence suggests instead that the beneficial properties are due to immunomodulatory mechanisms, as they migrate to sites of inflammation (by the mechanisms explained before)56 and secrete many bioactive molecules. This is supported by the fact that PBSC are also used with efficacy in the autologous therapy of non-hematopoietic tissues like neurons, skeletal muscle and heart. In multiple sclerosis and amniotrophic lateral sclerosis for instance, immunomodulatory effects and improvements were observed just 24 h after intrathecal delivery of MSC, which would be an irrational time frame for differentiation and rather backs up the hypothesis of a bystander effect instead.61 Furthermore, six months later, evidence of integration or even survival of these cells was very poor. In an animal model, CD34+ cells (HSC) were tracked by magnetic resonance, where they prove they migrate to lesion sites but just persisted for about 3 to 4 weeks.

Even though there is a very low rate of transdifferentiation into neurons, there is still clinical recovery, motor evoked potential improvements, as well as reconstruction of the ischemic tissue. As stated before, the benefits of BMSC would be by enhancing endogenous neurogenesis rather than cellular lineage reprogramming. The mechanisms involved appear to be paracrine secretion of bioactive molecules and upgrade regulation of receptors that reinforce and augment the natural recovery processes implemented by the brain; subsequently increasing the number of new functional neurons derived from endogenous neuroblasts.

It has been proved that exogenous administration of brain-derived neurotrophic factor (BDNF) stimulates neuroegenesis,65 therefore, endogenous secretion of BDNF and similar trophic factors by stem cells would aid in such purposes. BMSC increases concentration of SDF1a as well as expression of the SDF-1 receptor, CXCR4 in the perischemic area. There is also promotion of basic fibroblast growth factor (bFGF) and other trophic factor like β-nerve growth factor (β-NGF) which would not only promote proliferation, but will reduce apoptosis as well. 

BMSC increase the number of oligodendrocyte progenitors and increase axonal density around the ischemic lesion, extending and orienting axons parallel to the boundary of the penumbra. They do this by reducing expression of axonal growth inhibitory proteins, such as reticulon and neurocan, enabling axonal and neurite outgrowth.

MSC also share the properties of secreting many trophic factors (BDNF, SDF-1, NGF, bFGF, and VEGF) and promoting neurogenesis with the added benefit of a greater potential than regular HSC to transdifferentiate into neurons themselves,  MSCs carry the benefit of being readily obtained from bone marrow and easily expanded by culture in vitro, though this involves a time frame of 4 to 5 weeks before being delivered back to patients.

MSC are pretty safe. Because of their low major histocompatibility complex proteins they are considered immune privileged and cause no immunogenicity, neither acute or chronic. In a recent meta-analysis, there was no association between MSC and neoplastic potential, infection, embolism or zoonosis; in fact the only side effect was transient low-grade fever (OR: 16.82). 

Angiogenesis also plays a critical role in functional recovery. As in neurogenesis, angiogenesis is induced by several growth factors present in the penumbra 3 to 4 days after a stroke. It is so relevant, that patients who have a high density of blood vessels after stroke survive longer than those who do not. Animal models with denser vascularization have a better functional outcome as well. This density is determined by the presence of vascular growth factors, for there is a correlation between greater concentration gradients of them and increased blood vessel neoformation. Interestingly, neurogenesis actually enhances symbiotically angiogenesis by secreting the same factors.  Given that BMSC up-regulate expression and paracrine secretion of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) as well as angiopoeitins 1 and 2 and their receptor (TIE-1 and TIE-2), it is hypothesized that they would magnify the beneficial properties of neurogenesis and angiogenesis along with improving clinical outcomes and survival rate. 

Deciding which is better among HSC, PBSC or MSC is still unachieved. The only human clinical trial comparing HSC and expanded MSC found out that patients had better clinical outcomes (Barthel Index [BI]) with HSC. 
Other type of stem cells

Besides BMSC and NSC, other type of stem cells may be used for stroke and other diseases. Pluripotent stem cells (e.g. ESC), with a wider transdifferentiation spectrum, have the theoretical advantage over multipotent cells in its use for regenerative medicine; however, the downside is the accompanying increased risk of developing malignancies as well.86 In addition to this ESC bear ethical, technical and legal issues regarding the use of human embryos. 

As promising as they might be, iPCS have not been approved yet to be used in any clinical trial involving humans as concerns involving tumorigenicity abound. iPSC display more genetic and epigenetic abnormalities than other type of stem cells. Actually, their capability of developing pluripotent malignancies, such as teratoma surpasses that of ESC.

Stem cell therapy for stroke

Design of clinical trial
The first attempt to treat stroke using stem cells was more than 15 years ago. Hitherto there is still no optimum model for a clinical trial. With stroke being so diverse and many aspects of stem cell therapy still unexplored, many variables have to be thrown into the equation. We will discuss each of these variables separately.

Selection of patient

Stroke lesion sizes and locations are broadly heterogeneous in addition to normal neuroanatomical variations among individuals. Besides, depending on the etiology, stroke outcomes and prognosis vary hugely as well.9 Therefore, stroke outcomes are extraordinarily diverse among patients; even without intervention, most patients exhibit limited spontaneous recovery, though a subgroup will remain severely impaired. On the other hand, even with effective thrombolysis most patients will still have neurological deficits.

So without a control or a placebo group, it is difficult to distinguish whether improvements are stem cell therapy-based or just the natural history of the disease. Ideally, and specially to address efficacy, inclusion criteria should be the most homogenously possible (in age, etiology and comorbidities) to avoid confounding biases, even if this comes at the cost of shortening the number of patients.

Selecting patients with little to no predicted natural recovery may highlight the benefits of cell therapy, though this represents an obstacle given that most patients do not exhibit explicit recovery until 3–6 months after stroke, a time frame which limit most of the clinical trials that advocate for administration of stem cells much earlier.95 The expected recovery can be anticipated early (within days after stroke) by the use of specialized techniques of neuroimaging (e.g. fiber numbers asymmetry) and neurophysiological assessments (e.g. motor-evoked potentials), which would help us select patients with the worst prognoses to treat them in acute phases; though these are not yet used routinely.

Double blinding enhances statistical power to the clinical trial, but this may not be fitting for the more invasive interventions, such as intrathecal or intracerebral approaches.



Dosage

A consensus regarding dosage has not been met. Nonetheless, there is clear relation between more cells administered and better outcomes.67, 98 Therefore, given the safety profile of autologous stem cells, efforts to recollect the highest number cells possible must be done. This of course would not apply for allogenic stem cells, where the risk of graft versus host disease and rejection are much greater with higher doses.99
Another matter regarding dosage concerns the use of granulocyte colony stimulating factor (GCS-F) either as an attempt to increase number of available stem cells for collection, or even as a mean of treatment itself. GCS-F, as an up-regulator of hematopoiesis has demonstrated to increase exponentially the number of PBSC and could theoretically work as if these have been exogenously administered (i.e. migrate to penumbra and enhance recovery).100 Safety of GCS-F has been established in hyperacute stages of stroke (24–48 h after onset),101 which would carry an enormous advantage over stem cells, given that these would be difficult to have at hand that much early, especially in unstable patients. Although the trend is toward better outcomes,102 efficacy of GCS-F has not been thoroughly proven and is yet to be determined if they could be used as an alternative therapy alone or even as a coadjuvant of stem cell therapy.


Time of intervention

If any natural recovery is going to happen is not seen until around 3–6 months after the onset of stroke, and waiting that much could limit many of the immunomodulatory effects of stem cells. Besides most data points toward better functional outcomes if stem cells are administered much earlier.104 In one animal model, only the rats receiving BMSC intravenously 7 days after artery occlusion exhibited decreased ischemic lesion volume in contrast to those who received them at days 14 and 28. Interestingly tough, all three groups displayed clinically significant better functional outcomes compared to the placebo group, suggesting that 1 month after stroke might be a suitable time-frame.
Nevertheless, patients with much more chronic stroke still exhibit improvements in neurological functions. Considering the importance of inflammatory chemotaxis for stem cell therapy, this hints to the hypothesis that stroke constitutes a state of very chronic state of inflammation. Therefore, independent of the time of administration, stem cells will always migrate to some degree to the areas of lesion or even to the scarring tissue. Evidence suggests that stem cells in early stages work as anti-inflammatory molecules and in chronic stages aid in endogenous recovery and neurorestoration.
Administration time should also be decided depending on the feasibility of the route of administration as patients with hyperacute stroke (<72 h) are usually neurologically unstable and cannot tolerate invasive procedures.

VestaCell

Route of administration

The American Stroke Association in its recommendation for future stem cell research states that the safest and most effective route of cell delivery should be defined using preclinical trials. There are four major possible routes: intravenous (IV), intra-arterial (IA), intrathecal (IT) and intracerebral (IC)

It is clear due to the many clinical trials, that the IV route is the safest and most feasible for administrating stem cells.74, 85, 110, 111 Unfortunately, the most effective route is yet to be determined.

The basis for peripheral and non-invasive approaches (IV and IA) is the phenomenon of the selective permeability of the blood-brain barrier (BBB). After a brain insult, specially a hypoxic one, the tight junctions between the endothelial cells of the capillaries loosen up, therefore increasing its permeability and allowing income of many molecules including inflammatory cells. This breakdown may even persist for weeks or even months after the original insult, justifying stem cell therapies in chronic stages.Despite this, it remains unclear whether systemically infused stem cells are able to cross in a significant extent the BBB under both normal and pathological conditions.

IA administration of stem cells theoretically increments the number of cells delivered to the lesion area in comparison to IV; yet stem cells through these route, especially in high doses, may cause recurrent stroke. On the other hand, another study that displayed safety of IA infusion in a window time of 3–7 days, though this was just made in 4 patients. Even though considered not as an invasive approach as others, safety of IA route must be reevaluated in larger clinical trials.

Another major pitfall of peripheral routes is the substantial loss of cells in other parts of the body. When injected IV, stem cells are distributed all around the body and are homed in other organs like the liver, kidney, lungs and spleen. Nonetheless, perhaps it isn’t strictly necessary for the cells to be homed in the penumbra area for them to perform their anti-inflammatory properties. Some types of stem cells may not even enter the central nervous system and may instead promote stroke recovery by acting on peripheral organs. Whether this contributes significantly to clinical improvements remains an interrogatory.

In one animal model of human umbilical cord blood-derived MSC, it was observed that there was a major migration and concentration of stem cells in the areas of hypoxia after IT administration compared to an IV approach, as well as a longer survival period of these giving a great advantage to this approach. Additionally, IT administration of stem cells for other neurological disorders has been found to be safe and well tolerated; with the main side effects being headache and transient low-grade fever. Nevertheless, some serious adverse effects have been described as well. Case reports of inflammatory hypertrophic cauda equine, demyelinating encephalomyelitis, and spinal myoclonus following intrathecal injection of stem cells (combination of ESC, MSC and HSC) for different diseases have been published. However, these interventions were performed in stem cell therapy clinics, with no more information given.

When implanted IC via stereotaxis, grafted MSC cells are visualized prominently just 24 h after implantation and are homed almost exclusively in the affected site, positioning this route as the most effective in terms of cell concentration. Though as with animal models, evidence of these cells by neuroimaging (hypointensity on T2) became smaller gradually in the following 4 weeks until finally dissapearing. One must weight the risk and benefits when considering this invasive approach, particularly when using BMSC where benefits may just be temporal. Stereotaxis may be more justifiable if using exogenous NSC where there is a reasonable expectation of functional engraftment and a permanent incorporation to neural circuits. One must take into account that IC administration is unfeasible in acute and in unstable patients.


Randomized clinical trials

The first ever randomized clinical trial74 used ex vivo cultured autologous MSC and then infused them IV twice (weeks 4 and 8). All patients from the MSC group (n = 5) had cerebral infarcts that involved the middle cerebral artery territory. They reported significant improvements in BI (3 and 6 months) and mRS (3 months). One year outcomes were not significant. The main limitations of this study are the small treatment group and a short follow-up period. A similar approach was used by the same authors but with a larger population (MSC group n = 16 and control group n = 36) and a longer follow-up (5 years).110 Significant improvement of the mRS were reported in the MSC group (p = 0.046), in contrast with the control group (p = 0.257). The mortality rate in the MSC group was lower than in the control group (Log rank: p = 0.059), and there was no difference in comorbidites during the follow-up period. Notably, there was also a correlation between higher levels of the SDF-1a and clinical improvements, emphasizing the crucial role played by chemoattractants in this type of therapies.
An IC approach has been used twice. Kondziolka et al. implanted stereotactically 5 or 10 million allogenic neuronal cells cultivated from human embryonic carcinoma-derived cells (LBS®) to 14 patients with chronic stroke. They demonstrated safety of the procedure, as no serious adverse effects occurred after a 5 year follow-up. Non-significant improvements were found, especially in those having an ischemic stroke. Regarding neuropsychological testing, marked improvement was seen, as well as improved F-fluorodeoxyglucose (FDG) uptake in hypoxic areas. The other clinical trial also used stereotaxis in 15 chronic stroke patients, implanting 3–8 million CD34+ cells after stimulated (with G-CSF) PBSC where recollected by apheresis. The treated group showed a significant improvement in the National Institute of Health Stroke Scale (NIHSS), European stroke scale (ESS) and ESS motor subscale (EMS). Further, there were reductions in fiber number asymmetry of the damaged corticospinal tracts as well as restoration of motor evoked potentials response, both correlating with better functional outcomes. These changes were not observed in the control group. Safety end-points were acknowledged.
The only IA clinical trial conducted showed functional improvements, though these were not significant. Ten patients were injected with 1.5 × 108 autologous HSC between 5 and 9 days after ischemic stroke. No serious adverse events, stroke recurrence (clinical or by image), nor tumor formation were observed during the follow-up period (6 months). Interestingly, there was a trend toward better clinical outcome when higher numbers of CD34+ cells were injected.
The most recent, and by far, the larger clinical trial performed (60 cases and 60 controls) infused intravenously 2.9 × 106 CD34+ obtained by HSC in subacute stroke patients (median of 18.5 days after stroke).111 Even though safety was met, no changes were observed as to functional improvements. This contrasts with results obtained by the same research group a few years back, where they used either HSC or MSC in chronic patients (n treated = 20), and found statically significant improvements in BI, as well as increased number of cluster activation in motor cortex area, suggesting neuroplasticity.

Follow-up

A biological marker that can assess selectively improvements of neurological functions after an ischemic or hemorrhagic event is in great need,94 but that as it may, there is currently no validated marker for such purposes.128 Therefore, we must rely on other tools such as clinical scales and neuroimaging.

On this matter, more extensive, objective and specific neurological outcomes that measure beyond the classical NIHSS, BI, mRS, ESS, or EMS need to be developed and implemented for restorative treatments.

Magnetic resonance imaging (MRI) is an invaluable resource to gauge more objectively improvements after stem cell therapy. The focus should not be on the reduction of stroke volume size or edema, as these do not translate directly into better functional outcomes, and should remain then as secondary endpoints.28 Rather, restructuring of white matter tracts, neurogenesis and angiogenesis can be better used as to monitor recovery, which can be evaluated through more sensitive MRI techniques such as anisotropy130, 131 and magnetically labeled cells.132

Future & perspective

The feasibility and safety of stem cells in stroke patients have both been roundly confirmed. But in spite of the remarkable improvements observed in animal models, translation to clinical scenarios has not been achieved so far. The overall results of stem cell therapy for stroke have been inconclusive, at best. Yet, the tendency seems to lean toward better functional outcomes. Many unsolved issues remain regarding timing, dosage, type of cell, and route of administration. And until these are not addressed, conclusions concerning efficacy should not be given at all. Therefore, larger double-blind randomized clinical trials with homogenous selection criteria and domain-specific end points are strongly encouraged to clarify this matter. Certainly, a predictive marker of which patients would benefit the most from cell therapy would be of immense aid.

Given the magnitude of the physical, emotional and economic burden that stroke survivors have to endure, and its colossal impact on society as a whole, efforts to find the appropriate stem cell therapy for neurorestoration should not surcease but be encouraged.

Regenerative Medicine


Myths and stories about regenerative medicine have long been present in our history, ever since the search began for the elixir of youth. However, recently all the hoping and searching has been focused on the stem cell because of its abilities to reproduce itself and thus give rise to virtually all the functioning cells and tissues in our body. We are now witnessing new discoveries about stem cells and their ability to repair or regenerate damaged cells and tissues thus alleviating many degenerative diseases.

 Apart from the ongoing research on stem cells, a biomolecular substance i.e. organopeptide or homeopathic suiss organ, has also been proven to induce body repair processes as well. From Germany originally, this medical innovation is being widely used now in almost all European countries.
AH Regenerative Medicine provides effective treatment for osteoporosis, autoimmune disease, degenerative disease of endocrine organs and for eye, kidney, liver, ear, heart, brain, blood vessels, adrenal gland, pituitary gland, nerve, muscle etc.

VESTACELL

Mesenchymal Stem Cell Therapy in Multiple System Atrophy


Randomized clinical trials

The purpose of this study is to determine whether mesenchymal stem cells (MSCs) can be safely delivered to the cerebrospinal fluid (CSF) of patients with multiple system atrophy (MSA). Funding Source - FDA OOPD.
Primary Outcome Measures : Adverse event frequency (by severity, type, attribution, and intervention dose). [ Time Frame: 14 months ] Secondary Outcome Measures : Rate of change of Unified Multiple System Atrophy Rating Scale (UMSARS) I score from baseline to 12 months (or last available date), compared with placebo limb of Rifampicin trial (historical control cohort). [ Time Frame: 12 months ] Rate of change from baseline to 12 months (or last available date) in UMSARS II score. [ Time Frame: 12 months ] Rate of change from baseline to 12 months (or last available date) in UMSARS total score. [ Time Frame: 12 months ] Rate of change in COMPASS-select score from baseline to 12 months. [ Time Frame: 12 months ] Change in CASS score and thermoregulatory sweat test (TST) % from baseline to 12 months. [ Time Frame: 12 months ] MRI morphometric changes using dedicated algorithms to evaluate rate of atrophy of defined areas of brain from baseline to 12 months. [ Time Frame: 12 months ] Change in CSF biomarkers from baseline to 2 months. [ Time Frame: 2 months ]
The most recent, and by far, the larger clinical trial performed (60 cases and 60 controls) infused intravenously 2.9 × 106 CD34+ obtained by HSC in subacute stroke patients (median of 18.5 days after stroke).111 Even though safety was met, no changes were observed as to functional improvements. This contrasts with results obtained by the same research group a few years back, where they used either HSC or MSC in chronic patients (n treated = 20), and found statically significant improvements in BI, as well as increased number of cluster activation in motor cortex area, suggesting neuroplasticity.

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