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Why do stem cells injected into muscle become muscle cells?

Why do stem cells injected into muscle become muscle cells?



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In this article published in Science Daily, researchers injected teratoma-derived stem cells into mice with muscular dystrophy, and the stem cells regenerated 80% of the lost muscle. But what is the mechanism that results in their becoming muscle cells, rather than any other type of cell?

I'd have to assume some sort of cell signaling is responsible for this directed differentiation into muscle cells, so what is it? Can it be reproduced in vitro?


Looking at the paper itself, we find that they prepared the stem cells to have myogenic (muscle-specific) properties. They first injected stem cells into the irradiated (which kills muscle stem cells) muscles of immune-deficient mice. This resulted in tumor growth (teratoma - a stem cell derived tumor, where different kinds of tissues are spontaneously formed. Growing in a muscle environment increases the chance of muscle tissue formation.). The tumor cells, which expressed muscle-specific markers, were isolated from the other tissues and then used for regeneration of an injured muscle.

In their teratomas they checked for cells expressing α7-integrin and vascular cell adhesion molecule-1 (VCAM-1), which are markers expressed in satellite cells (muscle stem cells). These factors were expressed in about 10% of the fraction that did not show any hematopoietic and endothelial markers (meaning: there were a lot of other tissues formed as well). They also showed expression of Pax3 and Pax7, which are muscle-specific transcription factors, important for differentiation of myogenic progenitors. These cells were therefore already muscle-specific cells before they have been transplanted for regeneration.

The researchers did, in fact, do in vitro and in vivo differentiation experiments. For in vitro differentiation they subjected their muscle-marker expressing cells to a myogenic medium. This mimics the muscle environment and enables cell differentiation signaling. Here is an example protocol how this can be done. Then they transplanted their muscle stem cells into a muscle and they also managed to differentiate in vivo and regenerate the injured muscle.

The entire process of pluripotent stem cell to muscle cell differentiation can be performed in vitro, but seems to be tricky (1,2). Medium factors or genetic activation of transcription factors can activate different steps in the differentiation cascade.


‘Stem Cell’ Injections: Emerging Option for Joint Pain Relief?

Are you suffering from chronic joint pain? If so, you may have heard about “stem cell” injections and may want to ask your doctor whether these injections are right for you. If you want to avoid the surgical route of repairing a damaged knee or treating an arthritic shoulder, novel cell-based injections are becoming available and may give you the relief you need. However, this is a dynamic field and multiple questions remain unanswered.

Cleveland Clinic is a non-profit academic medical center. Advertising on our site helps support our mission. We do not endorse non-Cleveland Clinic products or services. Policy

What are stem cells?

Stem cells are special types of cells with the ability to multiply and self-renew. They also have the potential to replicate into specialized cell types forming potentially any tissue in your body. In other words, they can become a cartilage cell, a muscle cell or a nerve cell, says orthopedic surgeon Anthony Miniaci, MD.

“They have a tremendous capacity to differentiate and form different tissues, so that’s the thought behind regenerating cartilage, regenerating nerve cells and healing any injured tissues,” he says.

There are multiple sources of adult stem cells found in your body, including bone marrow or fat, although you can also receive stem cells from donor sources, Dr. Miniaci says.

“However, the term ‘stem cell’ is often widely and inappropriately overused in orthopaedics to include all kinds of treatment with uncharacterized, minimally manipulated cells, and even therapies that do not contain stem cells at all,” explains orthopaedic surgeon Nicolas Piuzzi, MD.

How cell-based treatment works

The treatment team harvests cells from your bone marrow or fat, or uses donor cells. Later on, your treatment team injects the cells precisely into your joint, ligament or tendon.

Theoretically, the cells when delivered will adjust inflammation and/or then divide and duplicate themselves and develop into different types of cells depending on the location into which they have been injected.

However, for patients with a severe loss of cartilage or no cartilage at all, a cell-based injection is unlikely to create a new joint, Dr. Miniaci says.

“Severe loss of cartilage typically leads to bone erosion or bone deformity, so a cell-based injection is highly unlikely to work in terms of reversing those changes,” he says.

It can, however, improve your symptoms of pain and swelling.

“The earlier you can treat someone’s joint pain, the better chance this has of working, making it less painful for the patient, less inflamed, and improve their function,” he says.

Risks

To date, there have not been any major adverse events reported associated with cell-based therapies in orthopaedics.

The main risk from a cell-based injection is probably in harvesting the cells. For example, when taking the cells from your bone marrow, the treatment team inserts a needle into your pelvis and removes some bone marrow cells and blood.

“However, the risk of having a complication during or after bone marrow aspiration in orthopaedics is minimal,” Dr. Piuzzi says.

“If you’re taking cells from fat, you can remove some out from under the skin,” Dr. Miniaci says. “To consider, anytime you make incisions or insert sharp instruments into a patient’s body, they can have problems such as acquiring an infection.”

Treatment is in its infancy

While the use of cell-based injections to treat joint pain holds much promise, Drs. Miniaci and Piuzzi caution that this treatment option is still very new. Researchers need to study its safety and determine if they are effective or not.

“We don’t have proof indicating that cell-based injections actually repair the joint,” Dr. Miniaci says.

He explains that if you have cartilage or bone damage, stem cells could differentiate and produce bone and cartilage and other tissues. So, theoretically, they could heal damaged tissue within a muscle, tendon, bone or cartilage.

“That’s the theory behind it, but this type of treatment and research is just in its infancy,” he says.

“We really don’t know what’s effective, what’s not effective, how many cells are necessary, how many actual injections you need and how often,” he says. “Nobody knows how well or even if it works yet. But we will eventually.”

Anecdotally, Dr. Miniaci finds that some patients can have improvement in their symptoms with cell-based injections. But he has not seen any proof yet that they are regrowing or regenerating a joint.

“Many people think that they’re going to come in with their arthritic joint and leave with a newer version of their knee joint. That doesn’t happen,” he says.

“What does occur is a biological reaction which makes the environment in their joints a little healthier, which probably makes it less inflamed, and as result, gives them less pain.”

Cleveland Clinic is a non-profit academic medical center. Advertising on our site helps support our mission. We do not endorse non-Cleveland Clinic products or services. Policy


Muscular dystrophy: how could stem cells help?

Muscular dystrophy is a muscle wasting disease that has many different forms. About 1 in every 3,500 boys worldwide is born with the most common form of the disease, Duchenne muscular dystrophy. How might stem cell research lead to new treatments?

What do we know? ▼

Muscular dystrophies are a group of genetic diseases causing weakness and progressive decline of heart and skeletal muscles.

People with Duchenne muscular dystrophy (DMD), lack a protein called dystrophin, which makes their muscles easily damaged. Muscle damage may lead to inflammation that causes further damage to muscle tissue.

Normally, muscle stem cells, called ‘satellite cells’, create myoblast cells that repair damaged muscle fibres. However, satellite cells in DMD patients struggle to make enough myoblasts and quickly become depleted.

What are researchers investigating? ▼

Researchers are investigating many details about satellite cells and the causes of muscle damage as well as treatments that help reduce muscle damage, such as anti-inflammatory treatments.

Studies are examining ways to preserve, and possibly restore, muscle function by transplanting dystrophin-producing cells into patients. These cells could be healthy donor cells or a patient’s own cells that have been genetically modified.

Induced pluripotent stem cells (iPSCs) are being studied as an option for making large numbers of cells with healthy dystrophin genes.

What are the challenges? ▼

One major challenge for treatments using donor cell transplants is the potential for transplant rejection by a patient’s immune system. Treating patients with their own cells (either genetically modified cells or iPSCs) can largely overcome transplant rejection but have other risks.

Another major challenge is engraftment: most muscles in a patient are weakened and need treatment. Evenly distributing cells to muscles throughout the body is a big challenge for cell therapy treatment. Currently cell therapies also have a relatively low success rate because of the unavoidably low ratio between healthy (or genetically corrected) cells versus resident diseased cells.

Muscular dystrophies are a group of genetic diseases that affect skeletal muscles and often also heart muscle. The symptoms include muscle weakness and progressive muscle wasting. Duchenne muscular dystrophy (DMD) is the most common and a very severe form of the disease. It is caused by a genetic fault which prevents the production of a protein called dystrophin. Without dystrophin, muscles are fragile and are easily damaged. Over time so much damage builds up that the body can’t repair it and muscles waste away, causing progressive disability in patients.

The majority of a muscle is formed from bundles of muscle fibres, long cells containing many nuclei but muscles also contain many other types of cells, including stem cells. Stem cells are part of the body’s inbuilt repair system. They can generate progenitor cells and also make copies of themselves. Skeletal muscles contain a type of stem cell called satellite cells. When muscle fibres are damaged they send chemical signals to satellite cells telling them to form new muscle fibres or to fuse with existing fibres to repair the damage. At the same time some satellite cells copy themselves to ensure enough stem cells are available to continue to repair and replace muscle fibres in the future.

Scientists believe that because the muscles are constantly damaged in DMD the repair burden placed on satellite cells is so big that they become exhausted and lose their ability to copy themselves. Satellite cells are essential for muscle repair so as the number of these cells decreases, the muscle becomes less and less able to repair itself. Instead damaged muscle fibres are replaced by fat cells and scar tissue, weakening the muscle until it can no longer work effectively.

Currently there is no definitive cure for DMD. Treatments aim to strengthen patients’ muscles and reduce some of the symptoms of the disease. Steroids are routinely used to slow down muscle wasting but they have many side effects, including weakening of bones leading to osteoporosis, hypertension and delayed growth. Physiotherapy may partially help to maintain muscle strength and flexibility. Researchers are hoping that, in the future, they may be able to repair or replace damaged muscle fibres using different strategies, including transplantation of dystrophin-producing cells to restore or at least preserve muscle function.

There are a number of different types of stem cells that scientists think may be used in different ways to develop treatments for muscular dystrophy. The main stem-cell-based approaches currently being investigated are:

  1. Producing healthy muscle fibres: Scientists hope that, if stem cells without the genetic defect that causes DMD can be delivered to patients’ muscles, they may generate working muscle fibres to replace the patient’s damaged ones.
  2. Reducing inflammation: In muscular dystrophy damaged muscles become very inflamed. This inflammation speeds up muscle degeneration. Scientists believe certain types of stem cells may release chemicals that reduce inflammation, slowing the progress of the disease.

Beside stem cells, other therapeutic strategies such as gene therapy or small-molecule drugs for repairing the damaged gene are being tested in patients and in pre-clinical models. Future therapies are likely to use a combination of more than one of these approaches. Scientists are also studying the role of stem cells in the maintenance and repair of healthy muscles in order to understand in more detail what goes wrong in muscular dystrophy and how the problem could be corrected.

Much current research is focussed on developing ways to restore production of the missing protein dystrophin in the muscles of DMD patients.

Myoblasts
Myoblasts are a type of cell formed from satellite cells after birth. Myoblasts fuse together to form muscle fibres. When injected into the muscles of mice with muscle damage similar to that caused by DMD, myoblasts from healthy donor mice fuse with the diseased muscle fibres and partially restore dystrophin production. However, clinical trials have shown that myoblast transplants are not effective to treat very large muscles in humans. Only a few of the transplanted myoblasts survive when injected into dystrophic muscles and, if derived from a donor, are attacked by the body’s immune and inflammatory cells, causing rejection.

There are also practical problems: although myoblasts may eventually be useful as part of treatments for types of muscular dystrophy that affect only a small and specific area of muscle in the body (as recently shown for oculo-pharynegeal muscular dystrohpy, OPMD), DMD affects most of the body’s muscles. This makes it extremely challenging to treat with intra-muscular injections as the cells do not move away from the injections site, thus many thousands of injections would be necessary. Obtaining the large numbers of cells that would be needed for transplantation and then delivering these cells to all the muscles in the body would be extremely challenging. Alternatively, to treat every muscle, the cells would need to be injected into the bloodstream to be carried around the body. Myoblasts cannot be delivered in this way because they cannot pass across blood vessel walls and so cannot travel from the blood to surrounding muscles.

Mesoangioblasts
Mesoangioblasts (MABs) are a type of 'progenitor' cell. MABs are found in the walls of blood vessels. Researchers have shown that healthy mesoangioblast cells can form dystrophin-producing muscle fibres in both dogs and mice with muscular dystrophy. The extent to which this formation of new fibres reversed muscle wasting varied between individual animals. Importantly, MABs can cross blood vessel walls so they can be delivered to all muscles in the body by injecting them into the arterial blood stream.

An early phase 1/2 clinical trial has now been completed with 5 patients in Italy. This trial showed MABs from healthy donors are safe to use as a treatment for children with DMD, under a regime of immune suppression, but had minimal efficiency (this was in part linked to the advanced stage of the condition in the patients involved in the trial). Researchers are now optimising the transplant procedures, moving to using patients' own cells, after genetic correction in the laboratory to produce normal dystrohpin. Results will not be available for several years. In the future, younger patients will be treated (now that the safety of the procedure is established) so that the remaining muscle cells have not yet been exhausted by the progression of the disease

Induced pluripotent stem cells (iPS cells)
iPS cells can be made in the lab from virtually any cell of the body and are pluripotent – this means they can make any cell of the body. Recently researchers discovered how to turn iPS cells, originally grown from patients’ skin cells, into cells that behave like healthy MABs. When these MAB-like cells were injected into mice with muscular dystrophy the mice gained muscle strength, could exercise for longer and produced normal muscle proteins. This was a very early study and a lot of research is still needed to establish whether this type of treatment would be safe and effective in humans. However, this research does suggest that MAB-like cells grown from iPS cells may be promising as a way to treat different types of muscular dystrophy. Since iPS cells can copy themselves indefinitely, an unlimited number of MAB-like cells could potentially be grown from a patient’s own skin cells and delivered in the bloodstream. These cells would be easier to grow in the lab and might be less likely to be rejected by the patient’s immune system than donor cells.

Possible route to cell therapy for muscular dystrophy: Mesoangioblast (MAB)-like cells can be made from iPS cells. The MAB-like cells were transplanted into mice in a recent study, with some positive results. More research is needed to find out whether the strategy could lead to treatments for patients.

In muscular dystrophy, both the damaged muscle cells and immune system cells produce inflammatory chemicals. These chemicals kill muscle cells and make the muscle environment hostile so new muscle cells can’t grow and survive. Scientists think they may be able to slow muscle wasting in patients by decreasing muscle inflammation. Steroids are currently used to achieve this but researchers are looking for alternative solutions. These include:

Drug treatments
Researchers are investigating alternative anti-inflammatory or muscle growth promoting drugs to treat muscular dystrophy. Many clinical trials are currently running with large numbers of molecules but conclusive results are not available at this time. Some new molecules such as PTC124 (that works to repair the gene defect) got market authorization in Europe but, again, results are non-conclusive. A similar situation holds for small molecules (oligonucleotides) that cause the cell machinery to skip the mutation that prevents synthesis of dystrophin (exon skipping).

There are currently no stem-cell-based therapies for muscular dystrophy. Research has provided some exciting avenues for potentially effective future treatments. A lot of work is still needed to determine whether these treatments will be safe and effective in humans. The main challenges scientists still need to address are:


Stem cells: definitions, characteristics and recognition

Adult stem cells are defined by two major functions: self-renewal and multilineage differentiation. Stem cells are functionally responsible for development and regeneration of tissue and organs. Developmental signals, both biochemical and biomechanical, trigger the proliferative action of the stem cells in early development. However, as we attempt to harvest and utilize adult stem cells, it is necessary to understand the specific signals which the cell requires in order to be directed along a desired pathway. Nonetheless, at this point, we are limited to a more descriptive understanding of their behavior.

The hierarchy of multilineage differentiation leads to the terms totipotential, pluripotential, multipotential, progenitor and precursor cells. In the earliest stages of development, the totipotential zygote and early blastocyst cells give rise to a fully differentiated adult organism. Just a few divisions into development, totipotency is lost. At this stage, pluripotential cells are present that give rise to cells of all three germ layers, but are no longer capable of giving rise to an entire organism. Germ layer-specific multipotential cells emerge later in development and are present in the adult tissues to repopulate and regenerate in response to environmental cues. The organ-specific progenitor and precursor cells give rise to mature, tissue-specific, differentiated cells.1

A second defining characteristic within the classical definition of a stem cell is its self-renewal ability. In tissues in which stem cell function is essential, homeostasis is maintained by balancing any tissue loss (due to injury or apoptosis) with repopulating organ-specific cells, while at the same time preserving the ability of the tissue to undergo future rounds of re-population. In this respect, it is thought that asymmetric cellular divisions occur such that the stem cell gives rise to one cell destined for differentiation and one renewed stem cell. An alternative possibility is that individual cells of the stem cell population respond stochastically by either differentiating or self-renewing. With either mechanism a stem cell, or reserve population, is maintained.

Stem cells are also often recognized by their quiescent behavior, though it is certainly not characteristic of stem cells of early development. In general, the stem cells of adults most likely remain quiescent until activated by injury or tissue damage. Indeed, the degree of the injury may dictate the level of stem cell activation and participation in response to the insult. Precursor cells may be readily available for homeostasis, while the more potent stem cell may be kept safely quiescent until serious injury occurs.

Isolation and identification of stem cell populations from various tissues is dependent upon specific markers, usually either exclusive proteins or characteristic profiles of more common surface proteins. Currently, one of the most well-defined stem cell populations is the mouse hematopoietic stem cell, which can be readily identified by a characteristic marker profile (Sca-1-positive, c-kit-positive, and differentiated hematopoietic lineage markers-negative).234 To date, only Sca-1 has been consistently identified on the putative MDSC. It is possible that the more committed the level of the stem cell, the more unique the organ-specific marker. As the level of pluripotency increases, these organ-specific markers may diminish and sets of common markers among stem cells from various organs may become more apparent.


Old Mice Made "Young"—May Lead to Anti-Aging Treatments

Stem cell injections prolonged lives of rapidly aging mice.

Aging mice can be made "young" again, according to findings one scientist initially found unbelievable.

The key is muscle-derived stem cells, which—like other stem cells—are unspecialized cells that can become any type of cell in the body.

When injected with muscle stem cells from young mice, older mice with a condition that causes them to age rapidly saw a threefold increase in their life spans, said study co-author Johnny Huard, a stem-cell expert at the McGowan Institute for Regenerative Medicine in Pittsburgh.

"I've been doing science for the last 20 years," Huard said. What "makes the story so amazing is that in the beginning, I didn't believe the result," he said.

"I bet that we mixed up the animals—you know, scientists are always skeptical."

"Tired" Stem Cells Reenergized

The study mice were genetically engineered to have a condition similar to a rare human syndrome called progeria, in which children age quickly and die young. (Learn more about the human body.)

The fast-aging mice typically die around 21 days after birth, far short of a normal mouse's two-year life span.

When scientists looked at the muscle stem cells of the fast-aging mice, they found what Huard called "tired" stem cells, which don't divide as quickly.

The team then examined mice that had aged normally and found their stem cells were similarly defective.

Curious if these deficient stem cells contribute to aging, Huard and colleagues injected stem cells from young, healthy mice into the fast-aging mice about four days before the older animals were expected to die.

To Huard's astonishment, the treated mice lived an average of 71 days—50 more than expected, and the equivalent of an 80-year-old human living to be 200, he said.

Not only did the animals live longer, they also seemed healthier, the scientists found.

Mysterious Secretions Make Cells Young

The "drastic" results bore out with repeated experiments, leaving the scientists to wonder how exactly the stem cells were working their magic, Huard said.

To find out, the team "tagged" stem cells injected into the fast-aging mice with a genetic marker that tracked where the cells went inside the body. Surprisingly, the team found only a few stem cells in the mouse organs, squashing a theory that the introduced cells were repairing organ tissues.

The scientists went back to the lab to test another idea: that stem cells secrete some kind of mysterious anti-aging substance.

The team put stem cells from the fast-aging mice on one side of a flask and stem cells from normal, young mice on the other side. The two sides were separated by a membrane that prevented the cells from touching.

Within days, the aging stem cells began acting "younger"—in other words, they began dividing more quickly.

"We can conclude that probably normal stem cells secrete something we don't know that seems to improve the defects in those aging stem cells," Huard said.

"If we can identify that, we have found an anti-aging protein that is going to be important" for people, said Huard, whose study appeared January 3 in the journal Nature Communications.

Stem-Cell Research "Intriguing" but Preliminary

But other scientists are cautious about how soon the discovery may help people delay the aging process or treat age-related disease.

"They did a beautiful job of showing that, when they put the muscle stem cells in [the mice], they improved function," said Justin Lathia, an assistant professor of cell biology at the Cleveland Clinic's Lerner Research Institute.

But as far as people go, it's still not clear what exactly stem cells do in the body, as well as what the mysterious stem cell secretion really is, Lathia emphasized.

Jeremy Rich, chair of the department of Stem Cell Biology and Regenerative Medicine at the Cleveland Clinic, also pointed out that the study is limited to muscle stem cells. That means the research can't be generalized to include all stem cell types, which are often very different from each other.

Paul Frenette, a stem cell and aging expert at the Albert Einstein College of Medicine in New York, called the research "intriguing," but said one of the messages for "patients is not to get too excited."

"You see all these clinics that are popping up all over the world—even in New York—where they're injecting stem cells" into people to treat disease, even though such therapies have not been proven.

"I don't think people should run to the clinic right now to have injections of stem cells to live longer."

Stem Cell Therapy to Help People "Age Well"?

Indeed, study co-author Huard noted that before any human anti-aging trials can begin, scientists need to repeat the experiment in normally aging mice to show whether these mice also live longer.

If that turns out to be true, Huard could imagine a scenario in which some of a person's stem cells are harvested at about age 20 and then injected back into his or her body at around age 50 or 55.

Stem cell therapies do already exist for conditions such as incontinence and heart problems, so he thinks "we're not that far [from applying] this approach clinically down the road."

But Huard warned that such a treatment would not mean a 55-year-old will suddenly look and feel 25 again.

"The goal of doing this research is not to [be like a] movie star with a ton of money [who wants to] look great for the rest of their lives," he said.

"The goal is, if you delay aging, maybe you can delay Alzheimer's or cardiovascular problems."

In other words, he said, such stem cell treatments would help people "age well."


Conclusions

Parabiotic studies have demonstrated extrinsic components, whereas transplantation assays have confirmed intrinsic components that cause age-dependent muscle stem cell decline. At first glance, the results based on satellite cell transplantation appear to be in conflict with those from parabiosis. The main purpose of this review has been to highlight the potential causes of discrepancy among the different papers and discuss how these differences teach us new facets about muscle stem cell biology and aging. We believe that the procedure chosen to assess the capacity of the young environment to rejuvenate aged satellite cells greatly accounts for the differences. We propose that the isolation of satellite cells and their subsequent transplantation may exacerbate intrinsic changes that importantly alter their functions. Thus, stem cell potency is greatly influenced by their capacity to overcome the stress of engraftment. From this viewpoint, old satellite cells would be less resistant to this stress. Importantly, strategies that modulate stress pathways in old satellite cells constitute a robust method for functional rejuvenation. Noteworthy, acute inflammation and stress, induced by the surgical joining of two animals in parabiosis experiments, might cause systemic perturbations that could affect satellite cell functions in yet unpredictable ways. In addition, the choice of genotype and age of the recipient mice for engraftment assays might also influence the regenerative outcome.

Another issue that may contribute to the apparently discrepant results between parabiosis and transplantation experiments relies on the molecular and functional diversity within the satellite cell population. Rejuvenation of aged satellite cells, in the context of transplantation, might derive from the augmented expansion of a small subpopulation of the fitter and more stress-resistant stem cells. Whether distinct satellite cell subsets are mobilized preferentially in the context of repair and transplantation or are differentially sensitive to intrinsic and extrinsic stressors warrants testing. A greater comprehension on the causative factors that drive satellite cell dysfunction during aging will lead to strategies that promote tissue rejuvenation and a beneficial impact on the quality of life of the elderly.


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Part III: How to Prepare for Stem Cell Therapy

Part III: How to Prepare for Stem Cell Therapy?

When it comes to stem cell preparation, there are a few things you could do that could actually help us maximize the therapy results.

However, it’s important to note that proper recovery (discussed in Part IV) is much more important for the final results of your treatment.

Stem cells need a healthy environment to develop and grow.

Nevertheless, here’s what you can do before the procedure to make the whole process smoother and more effective:

To-do #1: Cut Down Calories

Reducing the amount of calories taken—even for a short period of time—has been shown to positively affect the number of stem cells [43] in your body circulation.

The more stem cells you have, naturally, the easier it is to harvest a needed amount of them for the treatment, which will increase your chances of a successful recovery.

Another study has shown that short-term calorie reduction enhances skeletal muscle stem cell function [44] , too. The study also concluded that short-term calorie reduction improves muscle regeneration and “enhances stem cell transplant efficiency.”

In 2014, a group of scientist also found that calorie restriction is strongly linked to slower aging [45] of our stem cells, “prolonging the capacity of stem cells to self-renew, proliferate, differentiate, and replace cells in several adult tissues.”

After all, there’s a reason why low-calorie Japanese communities live the longest, healthiest lives [46] .

To-do #2: Cut Sugar

Related to the previous point, sugar restriction deserves a separate point of its own.

The most important reason why we suggest cutting down on sugar is because there is evidence that high sugar levels may cause several adverse effects [47] on the therapy, which basically means that your procedure will be less effective, or the change will take longer to manifest.

In general, cutting sugar is always a good idea, as it stimulates a more healthy lifestyle, which is the cornerstone of a successful stem cell treatment—both before and after the actual procedure.

To-do #3: Exercise

Exercise is actually both scientifically and practically proven to increase stem cell activity, which is a big advantage going into the therapy.

This 2017 study [48] found that “for skeletal muscle, the effect of physical exercise is becoming more and more clear”, which could be summarized in simple terms as stem cell activation. Which means that stem cells function better and multiply faster, given physical exercise stimulation.

Another 2009 study has shown that strenuous exercise has significantly increased [49] the number of stem cells in circulation.

It has even been shown in professional athletes that intense exercise can help cells migrate [50] better from bone marrow into the circulation, improving their quantity and function.

What is the proper exercise to activate your stem cells?

To put it briefly, it doesn’t matter what you do exactly as long as you’re taking your muscles to exhaustion. Whether it’s high repetitions with low weights, or few repetitions with heavy weights, the important thing is to exhaust the muscle. (As long as you’re doing everything with correct form, of course.)

Other (Non) Tips

You’ll find many tips online that may or may not be true. Some of them will be too vague to prove useful, while others will be specific but incorrect.

The three tips we’ve outlined so far—we’ve witnessed their effects ourselves. However, there are very few peer-reviewed pre-procedural protocols that have been properly researched.

So, the next time you come across tips like these, use a healthy dose of skepticism:

  • Go on a high-altitude vacation. We’ve seen this quoted on several occasions on the internet. The argument is that lower oxygen levels improve the function of stem cells. We would advise not to follow this point blindly, because there are other mechanics at play that could “cancel out” the effects of lower oxygen.
  • Stop taking over-the-counter medications. The other thing we see often is a definite list of all the supplements and medications that “all” patients should avoid taking. In reality, each case is different, and which medications and supplements could interact with the treatment depend on a number of factors, including the patient’s age, nature of the injury, type of stem cells used, and a number of other criteria. Always consult with your physician before making a decision to start/stop taking any supplements or medications.

Part IV: How To Maximize Stem Cell Results After the Treatment (Recovery and Guidelines)

Part IV: How To Maximize Stem Cell Results After the Treatment (Recovery and Guidelines)

Stem cells multiply and differentiate within your body naturally.

Therefore, in order to achieve best possible results, we need to create a healthy environment for the stem cells to thrive. We can do that by:

  • Cutting down on harmful habits
  • Exercising regularly and staying active
  • Eating a healthy diet

Here is your approximate activity guideline and recovery timeline for the next few months:

Week 1-2: Light Exercise and Mild Activity

During the first two weeks, moderation is key. You do not want to overload your body as it can cause another injury while your body is experiencing during this vulnerable period of inner transformation.

At this time, the stem cells are just starting to form and differentiate within your body, therefore overstretching and overloading your joints can be dangerous and reverse the effects of the therapy, if not make them outright worse.

Be extremely cautious to put pressure on the joints near the treatment area.

Here are a few important guidelines for weeks 1 and 2:

  • Avoid lifting more than 10 pounds of weight at once
  • Avoid walking up long flights of stairs
  • Avoid intense stretching
  • Avoid intense cardio exercise and active sports

However, it is absolutely crucial that you do not start off your recovery in a fully sedentary manner. Your body needs the oxygen and active stimulation to continue to grow and develop the stem cells.

Even though you might feel some mild pain and discomfort in the treatment area, do not give in to the natural instinct of laying in your bed for two weeks.

Staying active is extremely important during the recovery phase to achieve optimal results. In fact, prolonged inactivity after your stem cell treatment can dramatically reduce results.

Recommended exercises during this period:

  • Light yoga, stretching, pilates
  • Lifting weights (20-30% of your normal weight load)
  • No running or outdoor sports

Week 3-6: Walking, Swimming, Light Lifting, but No Running

Weeks 3 and 4 are typically when patients stop feeling any pain after the procedure.

For that reason, it is also the period when most patients injure themselves by overstretching or putting too much pressure on the musculoskeletal systems.

During this period, everything applies that was said about weeks 1 and 2, except:

  • You can now increase your stretch intensity a little
  • Weight lifting can now be safe up to 50% of your normal weight load
  • Active walking and swimming can now be resumed
  • Running is still strongly not recommended

Week 7-8: Slowly Progressing Back to Normal Exercise

“Active but cautious” is still the name of the game during weeks 7-8.

However, as your body continues to get stronger, you can slowly start raising your workout intensity to your normal levels. Always listen to your body as you increase the workload.

Month 2-6: Regular Exercise Without Sudden Changes

This period of a few months is crucial for the success of your stem cell therapy.

The most important thing during this period is to stay active and exercise regularly, but at the same time to not increase your workload or change your exercise plan suddenly.

For example, some bodybuilding enthusiasts and high-intensity athletes tend to often switch things up in their workout routine, in order to “keep their muscles guessing.” This way, hypertrophy and other favorable body reactions are stimulated.

While this might make sense for an entirely healthy individual for a period of time, it’s best to keep things simple and consistent during your recovery phase.

Part V: Questions People Ask, Answered

Part V: Questions People Ask, Answered

1. Are stem cell procedures covered by insurance? Are they covered by Medicare?

Currently in the US and many other westernized countries, only stem cell therapies designed to treat blood and bone marrow diseases—such as leukemia and lymphoma—can be fully or partially reimbursed by insurance and Medicare.

Therapies aimed to help patients avoid surgery after an injury or when suffering from a chronic condition are still mostly considered “experimental” and are usually not covered by insurance or Medicare however, exceptions do happen on a case-by-case basis.

2. Is stem cell therapy legal?

The usage of adult (bone marrow and adipose tissue) and umbilical cord stem cells to treat various conditions and diseases is perfectly legal [10] .

The only stem cells that are prohibited in therapy and are highly regulated in research are embryonic stem cells.

3. Is stem cell therapy FDA-approved?

FDA allows for “minimal manipulation” [12] to happen during the transplantation phase. Here is what “minimal manipulation” means:

  • The processing of the tissue does not alter its original characteristics
  • The processing does not alter any biological characteristics of cells and tissue

4. Is stem cell therapy ethical?

As we’ve mentioned previously, there is a lot of controversy surrounding stem cell therapy.

However, the controversy is mainly sparked by embryonic stem cells, which are not used in practical therapy. It is not legal to use embryonic stem cells in therapy within the US.

Umbilical cord stem cells are donated by informed consent by a newborn’s parents for research and medical purposes. The transfer of these stem cells to therapy clinics is a highly controlled process.

Other sources—your own bone marrow or adipose (fat) tissue stem cells—shouldn’t raise any ethical questions as they are part of your body.

5. Can stem cell therapy cure diabetes?

The potential of stem cells is as powerful as it is exciting. There is important ongoing research in the possible application of stem cell therapy in curing a number of previously unchallenged diseases, including diabetes [51] . As we do not provide this type of treatment, we will refrain from further comment.

6. Are stem cell injections painful?

This is a question we get quite a lot. The injection itself is no more painful than your typical shot at the doctor’s office. For a couple of weeks after the procedure, however, you might feel mild pain in the area, which is completely normal and shouldn’t interrupt your daily activities or light exercise.

7. How long does it take for stem cell therapy to work?

It takes a few weeks to a few months for stem cells to start actively multiplying and differentiating within the patient’s body. Most of our patients feel significant results within 2-6 months period.

Want to activate your body’s natural ability to heal itself?


Muscles May Preserve A Shortcut To Restore Lost Strength

Skeletal muscle cells from a rabbit were stained with fluorescent markers to highlight cell nuclei (blue) and proteins in the cytoskeleton (red and green). Daniel Schroen, Cell Applications Inc./Science Source hide caption

Skeletal muscle cells from a rabbit were stained with fluorescent markers to highlight cell nuclei (blue) and proteins in the cytoskeleton (red and green).

Can muscles remember their younger, fitter selves?

Muscle physiology lore has long held that it is easier to regain muscle mass in once-fit muscles than build it anew, especially as we age. But scientists haven't been able to pin down how that would actually work.

A growing body of research reviewed Friday in the journal Frontiers in Physiology suggests that muscle nuclei — the factories that power new muscle growth — may be the answer. Rather than dying as muscles lose mass, nuclei added during muscle growth persist and could give older muscles an edge in regaining fitness later on, new research suggests.

This work could affect public health policy and anti-doping efforts in sports, says Lawrence Schwartz, a biologist at the University of Massachusetts, Amherst who wrote the review. But some scientists caution against extrapolating too far from these studies into humans while conflicting evidence exists.

One thing is for sure: Muscles need to be versatile to meet animals' needs to move. Muscle cells can be sculpted into many forms and can stretch to volumes 100,000 times larger than a normal cell. Muscle cells gain this flexibility by breaking the biological norm of one nucleus to a cell some muscle cells house thousands of nuclei.

In mammals, these extra nuclei come from stem cells called satellite cells that surround the muscle. When demands on the muscle increase, these satellite cells fuse with muscle cells, combining their nuclei and paving the way for more muscle.

"To build muscle mass you need to make more of the contractile proteins that create that force," says Kristian Gundersen, a muscle biologist from the University of Oslo. Nuclei power the building of more muscle, making them "a bit like factories," says Gundersen. The more nuclei, the bigger and stronger the muscle.

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But what happens to those extra nuclei as we slide into a more sedentary, less fit lifestyle?

Physiologists had thought that a single nucleus supported a certain volume of cell. As a muscle cell grew, it needed more nuclei to support that extra volume. But as a muscle shrinks from lack of use, it gets rid of those unnecessary extra nuclei.

"The idea was you exercise a muscle and it changes," says Gundersen. "Stop exercising and it goes back to how it was." Better to raze unneeded factories than expend energy to keep them running.

This view found support in studies that found nuclei were scrapped as muscles atrophied. But Gundersen and Schwartz say those experiments overlooked what was really happening.

Take a cross section of muscle tissue and you'll find a sort of marbled mishmash of muscle cells surrounded by numerous other cell types, such as satellite cells and fibroblasts. "It can be very difficult to distinguish between muscle nuclei from other nuclei," says Gundersen.

Researchers could have been measuring the death of cells that support muscle and incorrectly inferred that muscle cells lose their nuclei, according to Gundersen and Schwartz.

Gundersen and colleagues developed another method that zoomed in on individual muscle cells. The researchers injected a stain into muscle cells that mice use to flex their toes.

The stain spreads throughout the muscle cells, illuminating their nuclei. Gundersen could then track the nuclei over time as he induced muscle growth by giving the mice testosterone, a steroid hormone. Later, after stopping the testosterone, he could watch what happened as those muscles atrophied.

Unsurprisingly, testosterone boosted nuclei number. But those extra nuclei stuck around, even as the muscle shrank by half.

Gundersen thinks the results contradict the conventional wisdom that nuclei disappear when muscles atrophy. "Nuclei are lost by cell death," he says, "just not the actual muscle nuclei that confer strength." What's more, he says these retained extra nuclei might explain how a muscle remembers its past fitness.

To test this idea, his lab gave some mice testosterone, and left others untreated. The doped mice got a boost in muscle mass and muscle nuclei. Then the scientists left both groups to atrophy for three months, about 15 percent of a mouse's life span.

Muscle size decreased, but the extra nuclei in the treated mice were still there.

The researchers then put the mice through an intense fitness regimen. After six days, the nuclei-rich muscles exposed to testosterone grew 36 percent, while the untreated muscles grew only 6 percent.

Gundersen has an analogy to explain what happened: "If you have to build the factories anew, it probably takes more time and is more difficult, but if the factories are already there you just need to start them up."

University of Massachusetts' Schwartz believes this phenomenon can probably be generalized to most muscle types across the tree of life. He points to his own work in moths, where he also found that nuclei remain as muscles atrophy.

If it's true in mice and moths, he thinks, then it could be generally true that once a muscle gains nuclei, it keeps them. And recent evidence suggests muscle cells can stick around for decades.

Schwartz and Gundersen say their research could have major implications for public health and anti-doping rules in sport.

Muscle weakness is a major cause of injury in the elderly, and as we age it becomes harder to grow new muscle. "Of course we need further confirmation in humans, but the idea that is you exercise, you get more nuclei and you have them forever," says Gundersen.

Schwartz adds, "If we can bank muscle nuclei early in life, when it's easier to build muscle, we could then draw on these later in life to slow the effects of aging." He thinks early physical education classes, which are often on the chopping block when schools tighten budgets, gain added importance in light of this research.

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This work may touch the world of sport as well. Athletes who dope could reap benefits, in the form of banked muscle nuclei, long after they stop taking any drugs. Currently, the World Anti-Doping Association has a maximum first-time ban of four years. "If the mechanisms are similar in mice and men, then I think that time is far too short," says Gundersen.

Olivier Rabin, WADA's science director, says the association is aware of Gundersen's work but will wait for further confirmation in humans before suggesting any changes to current ban rules. "If we want to start banning athletes for six years, 10 years, or life, we'd better be very sure the science is solid."

Muscle biologist Charlotte Peterson, a professor at the University of Kentucky, also thinks it is too soon to translate this science into any kind of policy. "To say that muscle nuclei do not undergo atrophy, period, is absolutely not true," she said. Results are mixed, she said, citing a study of humans and bed rest that showed muscle nuclei are lost.

Peterson praised Gundersen's imaging work and was convinced that in the muscle cells he observed, nuclei are not lost during atrophy. But she argues that assuming this experiment represents the norm would be a mistake.

Other muscles with different demands, like postural muscles, or muscles more directly involved in functional movement, could behave differently. Timing may also play a role. "Just wait long enough depending on the function of the muscle and you will lose muscle nuclei with atrophy," says Peterson.

As for muscle memory, Peterson thinks that changes in DNA expression in response to exercise may play a greater role than muscle cell nuclei, as was suggested in a recent human study.

"We still have a lot to learn," says Peterson. "We do not know enough at present to translate to humans, and we certainly don't know nearly enough to influence any kind of policy."

Jonathan Lambert is an intern on NPR's Science Desk. You can follow him on Twitter: @evolambert


The Institute for Creation Research

Embryonic stem cells are the basic building blocks for some 260 types of cells in the body and can become anything: heart, muscle, brain, skin, blood. Researchers hope that by guiding stem cells in the laboratory into specific cell types, they can be used to treat diabetes, Parkinson's disease, heart disease, or other disorders. The primary clinical source is the aborted fetus and unused embryos currently housed in frozen storage at IVF facilities. A developed stem cell line comes from a single embryo, becoming a colony of cells that reproduces indefinitely. Consider now the following ten problems with Embryonic Stem Cell Research (ESCR).

1. The issue of who or what

As the nation sits embroiled over the battle of where to draw the line on ESCR, the real issue that truly divides us is whether embryonic stems represent a who or a what. In other words, are we talking about people or property?

Since Roe v. Wade we have not been willing or able as a nation to address the issue. As a result, those who oppose ESCR and those who support it will never reach an acceptable point of compromise. Still, in the midst of the flurry of all this biotechnology and all the problems it presents, there is some very good news that has been overlooked by almost everyone. Ready? Cloning proves scientifically that life begins at conception&mdasha position to which the author and most Christians philosophically already adhere.

Additionally, the insights provided by cloning technology destroy the scientific and legal basis of distinguishing a preembryo from an embryo, the popular distinction made at 14 days after conception. This is significant because this distinction determines the handling and treatment of human life less than 14 days old, which is so basic to all ESCR.

In short, our understanding of embryonic development as provided by cloning technology could force not only those who participate in ESCR specifically, but also those who participate in in-vitro fertilization (IVF) procedures generally, to recognize there is no real preembryo&mdashembryo distinction and that all human life begins at conception. Therefore, as a nation, we should rightly adjust the moral and legal treatment and status of all embryos to people not property from the point of conception.

2. The deliberate misuse of terminology in defining stem cells

Proponents of ESCR often use the term pluripotent. This word intends to imply that the ESC cannot make or reform the outer layer of the embryo called the trophoblast. The trophoblast is required for implantation of the embryo into the uterus. This is a distinction used by proponents of ESCR to imply a fully formed implantable embryo cannot and does not reform after the original embryo is sacrificed. This is significant because to isolate the stem cells, scientists peel away the trophoblast or skin of the embryo much like the peel of an orange. They then discharge the contents of the embryo into a petri dish.

At this stage of development, the stem cells that comprise almost the entire inner body of the early embryo look and function very similar to one another. Once put into the petri dish, the un-programmed cells can be manipulated to multiply and divide endlessly into specific cell types. The question regarding use of the term pluripotent is whether stem cells emptied into the petri dish can reform the trophoblast creating an implantable embryo of the originally sacrificed embryo?

The uncomfortable truth is, James Thomson, who led the effort that first isolated and grew embryonic stem cells in the laboratory says the trophoblast can reform under certain circumstances. That means even after months of continuous proliferation of the cells, implantable cloned human beings of the original embryo might be forming as the stem cells are grown in petri dishes. Therefore, use of the term pluripotent is scientifically inaccurate and deliberately misleading.

3. ESCR is related to human cloning

Understanding how ESCR and human cloning relate requires delineation between the two forms of human cloning: reproductive and therapeutic.

Reproductive cloning creates a later born twin from a single cell of another person by transplanting the DNA of the adult cell into a human egg whose nucleus has been removed. This process is somatic cell nuclear transfer. In this procedure, the resulting embryo is implanted in a woman and carried to birth. Proponents say that reproductive cloning is a logical extension of infertility treatments, hence the intimate link to IVF procedures.

By contrast, therapeutic cloning occurs when an adult undergoes a cloning procedure to duplicate his own cells in order to stave off personal disease, illness or the effects from sudden and serious injury. This procedure also begins by creating a clone of the adult through somatic cell transfer. In therapeutic cloning however, the embryos are allowed to live up to 14 days, at which time their trophoblasts are removed, as in standard ESCR, to harvest the highly prized stem cells for the donor's treatment.

In summary, therapeutic cloning begins with the same procedure as reproductive cloning. The goal of reproductive cloning is to produce a baby. The goal of therapeutic cloning is to produce embryonic stem cells for research and or treatment.

Additionally, whenever embryonic stem cell research results in the spontaneous reformation of the trophoblast around other stem cells, a fully implantable cloned life of the originally sacrificed embryo is created, however temporarily.

4. The current status of ESCR in the U.S. is unsettled at best

President Bush announced on August 9, 2001, that federal funds would not be used for ESCR that result in the future destruction of embryos. They can, however, be used to conduct research on the 64 stem cell lines that currently exist because "the life-and-death decision has already been made." However, scientists who work with some of these cells say many of the 64 lines are not yet developed and some may never pan out. Some researchers are uncertain about the quality of the cells and wonder if the limited number is enough. Proponents of this research are now focused on gaining more ground by passing legislation in Congress.

5. There is law that could apply to ESCR

Originally attached to the 1995 Health and Human Services (HHS) appropriations bill, the "Dickey Amendment" has prohibited federal funding of "any research in which a human embryo or embryos are destroyed, discarded or knowingly subjected to risk of injury or death." Unfortunately, there are no laws to protect preembryos (embryos under 14 days old) or that prohibit private individuals, research firms, or pharmaceutical companies from forming, manipulating, or destroying stem cells, human clones, or embryos.

6. Polls show that the American people do not approve using public money to destroy human embryos in medical research

7. ESCR puts us on the road to growing humans for body parts

The un-programmed cells of an early embryo are derailed from their natural course of development and coaxed through chemical manipulation to become very specific tissue types that will be used to treat the unhealthy or diseased tissue of those already born. Opponents of funding ESCR have argued vehemently against this stark utilitarian treatment of human life, unfortunately with little effect.

Regarding the justification that the embryos "left over" in IVF clinics (reportedly >300,000 in the US alone) will simply be discarded anyway, reflects a chilling absence of moral conscience. We do not consider it appropriate to take organs from dying patients or prisoners on death row before they have died in order to increase someone else's chances for healing or cure. Neither, then, should we consider any embryos "spare" so that we may destroy them for their stem cells.

How far down this road have we already come? Consider the story of Adam and Molly Nash. Molly was diagnosed with Fanconi anemiaa hereditary and always fatal disease. Doctors determined that the best hope for Molly was a cell transplant from a relative whose cells matched Molly's, but without anemia. So Molly's parents produced fifteen embryos by IVF, only one of which had the right genetic material. It was implanted in Mrs. Nash who gave birth to Adam. Adam's stem cells were taken from his umbilical cord and implanted in his sister. Despite all the success of the treatment and the medical justification, the fact remains that Adam was conceived, not just to be a son, but a medical treatment. Adam was a means valuable only insofar as he carried the right genetic material. If he hadn't, he would have been rejected like the other fourteen discarded embryos. The undeniable conclusion is that we are growing humans for body parts.

8. Contemporary moral issues often follow the flow of money

Stem cell research and human cloning are about transforming the mystery and majesty of life into a mere malleable and marketable commodity. In the short term, this is big business and offers great fame and fortune to the pioneers and biotech companies who master their secrets and harness the power of life through ESCR.

9. ESCR currently has major disadvantages

The promises of ESCR are right now nothing more than hoped for possibilities. Successful clinical trials for people are years away at best. Why? The reality is that the scientific evidence so far does not support public statements.

First, one minor complication is that use of human embryonic stem cells requires lifelong use of drugs to prevent rejection of the tissue. Second, another more serious disadvantage is that using embryonic stem cells can produce tumors from rapid growth when injected into adult patients. A third disadvantage reported in the March 8, 2001, New England Journal of Medicine was of tragic side effects from an experiment involving the insertion of fetal brain cells into the brains of Parkinson's disease patients. Results included uncontrollable movements: writhing, twisting, head jerking, arm-flailing, and constant chewing. Fourth, a recent report in the Journal Science reported that mice cloned from ESC were genetically defective. If human ESC are also genetically unstable, that could materially compromise efforts to transform cells extracted from embryos into successful medical therapies. Finally, the research may be hampered because many of the existing stem cell lines were grown with the necessary help of mouse cells. If any of this research is to turn into treatments, it will need approval from the FDA, which requires special safeguards to prevent transmission of animal diseases to people. It is unclear how many of these cell lines were developed with the safeguards in place. This leads to a host of problems related to transgenic issues.

10. The Success and Promise of Adult Stem Cell Research

In all fairness, adult stem cells have restricted differentiation potential and do not proliferate as well as ESC. On the other hand, while ESCR yields, at best, meager results, and has only far distant possibilities of successful clinical applications, current clinical applications of adult stem cells are abundant! They include treatments for the following: corneal restoration, brain tumors, breast cancer, ovarian cancer, liver disease, leukemia, lupus, arthritis, and heart disease. Thousands of patients are treated and cured using adult stem cells. Alternative sources for adult stem cells include: placenta, cord blood, bone marrow organ donors, and possibly fat cells.

For these ten reasons my conclusion is that more dollars should be invested in adult stem cell research and the macabre research associated with ESCR should be abandoned entirely.


Discussion

We have further investigated a new strategy to restore dystrophin expression in mdx mouse muscles by an intraarterial injection of normal purified Sca-1, CD34 muscle-derived stem cells. Marker transgenic DNA was found in muscles of mdx/mdx mice demonstrating the migration of these cells from the blood vessels. These muscle-derived cells crossed the vessel barriers thus leading to a mosaic expression of the marker gene in muscles of the injected hindlimb of adult mice. The precise pathway used by the transplanted cells to home to host muscle tissues is not known. However, the adhesion-initiating vascular diapedesis of these cells is high in capillaries. Based on our findings, the cells might have actively migrated in response to chemoattractants, as described previously, after bone marrow transplantation (Mazo et al. 1998 Deog-Yeon et al. 2000).

The fate of injected Sca-1, CD34 positive cells was evaluated by examining tissues of five mdx/mdx mice. We found transcription and expression of LacZ and normal dystrophin genes in muscles of the injected hindlimbs. Unexpectedly, PCR and RT-PCR analysis revealed the absence of injected cells in other muscles and tissues of the treated mice. Histochemical analysis of muscles showed colocalization of dystrophin and β-gal in muscle fibers. Peripherally nucleated double-positive fibers were generally grouped in some segments of the muscles and always seemed to exist in small groups, suggesting that there might have been clonal proliferation of some donor cells, later contributing to the formation of several muscle fibers. Thus, muscle-derived cells injected into arterial circulation of a congenital dystrophinopathy partly restored normal dystrophin production in striated muscles.

In this study, we used a muscle culture system for facilitating the enrichment and purification of Sca-1 + and CD34 + cells. We found a lower content of desmin-positive cells in a population of purified muscle-derived cells that display both CD34 and Sca-1 as markers of progenitors cells. This population was obtained with a purification method described in Qu et al. 1998, but with a different medium and growth conditions. However, these cells showed a poor ability of myogenic differentiation, indicating that they are distinct from the pp6 cells described in Qu et al. 1998. These cells could represent a pool of stem cells capable of commitment to more than one lineage (hemopoietic, endothelial, and muscular) given the right environmental cues. However, the major problem encountered in this study was the poor ability of the pp6 cells to undergo muscle differentiation when injected directly in the muscle. It is conceivable that engraftment efficiency may depend on the inflammatory consequences of muscle directly injected compared with the arterial injection of the hindlimb where histologic features of muscles are invaried. These data do not preclude the possibility of the muscle-derived Sca-1, CD34 double-positive cells representing the immediate progenitors to satellite cells. The descendants of the activated satellite cells, myogenic precursor cells, undergo multiple rounds of division before fusing to existing or new fibers. Quiescent satellite cells express c-met and M-cadherin proteins, but do not express markers of committed myoblasts such as Myf-5, MyoD, and desmin (Cornelison and Wold 1997 Sabourin et al. 1999). The total number of satellite cells in muscle remains relatively constant, suggesting that a capacity for self-renewal in the satellite cell compartment maintains the population of quiescent cells (Bischoff 1994). However, the mechanism by which satellite cells undergo self-renewal in skeletal muscle is poorly understood. Muscle stem cells may undergo asymmetric cell division to generate two daughter cells: a committed myogenic precursor and a pluripotent “self.” Moreover, it remains possible that muscle-derived stem cells represent an independent stem cell population separate from the satellite cells. Additionally, circulating pluripotent cells derived from the bone marrow might be the common putative stem cell population present in many different tissues.

To analyze the potential role of the endothelial CD34 + progenitor cells of our purified muscle-derived cells, we performed in vivo studies focusing on the muscle injections of endothelial cells. Interestingly, endothelial cells injected intramuscularly remained as mononuclear cells under the basal lamina of the host muscle fiber in the same location as muscle satellite cells and fused poorly with host fibers. Few LacZ- and vimentin-positive cells were also located at the perithelial sides of muscle vessels.

It is extremely interesting to note that the efficiency of Sca-1, CD34 muscle engraftment increased significantly to 12% when TA muscle of the injected hindlimb was damaged 48 h after intraarterial injection. This observation suggests that these cells first attached to the capillaries as Sca-1, CD34 positive stem cell remaining in the muscle without differentiation and participate actively in muscle formation only after muscle damage. Thus, the myogenic potentialities of the injected cells are determined by the location of the precursors at the beginning of muscle and blood vessel damage. In another experiment, the damage to the leg muscles of mdx mice 48 h after the intraarterial injection of TnILacZ Sca-1 + CD34 + cells (pp6) was done by a swimming exercise which was shown to damage the muscle fibers of mdx mice. 1 mo later, we observed only 1% of β-gal–positive fibers in the leg injected arterially. There are two possible hypotheses to interpret the results of these two experiments with muscle damage produced with a needle and with exercise. The first is that the Sca-1 + CD34 + cells are attached to the capillaries and participate in muscle regeneration when not only the muscle fibers, but also the blood vessels, are damaged. The second hypothesis is that the Sca-1 + CD34 + cells are still present in the blood circulation and are attracted to the damaged muscle when the blood vessels are damaged. The first hypothesis means that there are two types of muscle regeneration. The first type (low regeneration), which would occur after damage only to the muscle fibers, would involve the participation of only the satellite cells attached to the muscle fibers. The second type of muscle regeneration (intense regeneration) would occur after a damage to the muscle involving not only the muscle fibers, but also the blood vessels. With this more intensive damage, the regeneration would involve not only the participation of satellite cells, but also of stem cells already attached to the blood vessels or present in the blood circulation. The low regeneration would occur after intense muscle activity. The intense regeneration would occur after a major injury to the muscle (an accident, a bite, or a crush). The presence of precursor cells attached to the blood vessels and participating in muscle regeneration only after damage to these vessels would explain why Grounds and McGeachie 1992 observed that within a few hours after a muscle crush there were more myogenic cells than satellite cells. This intense regeneration hypothesis also explains why there is muscle regeneration after muscle irradiation when notexin is injected intramuscularly (Heslop et al. 2000), since these injections also damaged the blood vessels. More importantly, this hypothesis would account for the absence of muscle regeneration in DMD patients despite the presence of stem cells originating from the bone marrow or other sources already attached to the blood vessels and capable of forming myogenic cells.

During embryonic vasculogenesis, primordial perithelial cells, which later would generate smooth muscle and connective tissue of the vessel wall, develop in the outermost portion of vasculogenic clusters (DeRuiter et al. 1997). It is possible that these multipotent mesodermal progenitors remain associated to the tissue vasculature as perivascular cells and acquire a commitment depending on the tissue (Bianco and Cossu 1999). In this model, the mesodermal progenitors would represent the source of cells to develop and regenerate tissue. Thus, when muscle stem cells are injected by intraarterial circulation, they would become associated with the vasculature and would be able to differentiate into the myogenic lineage after muscle and blood vessel damage. In contrast, the direct muscle injection of these cells did not give the correct lineage relationship between endothelial and perithelial progenitor cells.

This raised the possibility that the precursor Sca-1, CD34 muscle-derived cells with characteristics of bilineage-committed precursors (hemopoietic and muscular) may prevent irreversible pathological damage and allow engraftment in many distant areas affected by the disease. It would also be important to know the muscle-derived signals governing cell migration. Thus, further accurate studies of the Sca-1, CD34 muscle-derived cells will be essential to improve transplantation efficiency.