New Method Derived Skeletal Muscle Cells from Pluripotent Stem Cells

A University of Wisconsin research team led by Masatoshi Suzuki has devised a new protocol for the production of large quantities of skeletal muscle cells from pluripotent stem cells.

Suzuki and his team used embryonic stem cells lines and induced pluripotent stem cells to generate large quantities of muscles and muscle progenitor.

Suzuki adapted a technique used to make brain cells to derive his muscle cells in culture. He grew the stem cells as floating spheres in high concentrations of two growth factors: fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF). This combination of growth factors directed the stem cells to differentiate into skeletal muscle cells and muscle progenitors.

To replace damaged or diseased muscles in the clinic, physicians will require large quantities of muscle cells. Therefore, there was an ardent search to design a technique that was efficient, but also fast and relatively simple. Even though several protocols have been devised to differentiate pluripotent stem cells into muscle cells, not all of these protocols are practical for clinical use. For example, some protocols are simply too cumbersome for clinical use. Still others make use of genetically engineered cells that have not been approved for clinical use.

Earlier, Suzuki transplanted lab-engineered skeletal muscle into mice that had a form of amyotrophic lateral sclerosis. These animals had better muscle function and survived better than the control animals.

The muscle progenitors generated in Suzuki’s laboratory could potentially play a similar role in human patients with Lou Gehring’s disease. Suzuki’s method can grow muscle progenitor cells, which can grow in culture, from induced pluripotent stem cells, which are derived from the patient’s own cells. Such cells could be used as a model system to study the efficacy of particular treatments on the patient’s muscles, or they could be used to treat patients who have muscle defects.

“Our protocol can work in multiple ways and so we hope to provide a resource for people who are exploring specific neuromuscular diseases in the laboratory,” said Suzuki.

The advantages of Suzuki’s protocol are manifold. First, the cells are grown in a defined medium devoid of animal products. Secondly, the stem cells are grown as spheres, and these grow faster when grown as spheres than they do with other techniques. Third, 40-60 percent of the cells grown in this culture system differentiate into skeletal muscle cells or muscle progenitor cells. This is a very high proportion of muscle cells when compared to other protocols.

Suzuki hopes that by toying with the culture system, he and his colleagues can increase this proportion of muscle cells that form from the initial stem cell culture. This would enhance the potential of using these cells for clinical purposes.

Muscle Fusion Protein Identified – Better Muscular Disease Treatment

A research laboratory lead by Jean-François Côté at the Institut de Recherches Cliniques de Montréal, Montreal, Canada has identified an elusive protein that mediates the fusion of muscle precursor cells into mature muscle.

The development of skeletal muscles depends on the migration of muscle precursor cells called “myoblasts” to migrate to the right location and then fuse with each other to form the multi-nucleate skeletal muscle cells. This finding has the potential to improve the treatment of muscular diseases such as myopathies and muscular dystrophies.

“For several years, we have been studying myogenesis, a process by which muscles are formed during development,” said Côté.

In the fruit fly Drosophila melanogaster, muscle fusion is rather well understood. A protein called “Myoblast City” and a scaffolding protein called “ELMO” activate the Rac protein in response the muscle precursor cells adhering to surfaces. Rac initiates the intracellular mechanisms that culminate in muscle fusion.  In vertebrates, the ELMO protein exists in muscle precursor cells and a vertebrate version of the myoblast city protein called DOCK1. However, identifying the receptor that kicks this process off had proven difficult.

Drosophila myoblast fusion

Myoblast fusion plays a central role in muscle development because it determines muscle size. Also, the fusion of existing muscle fibers with muscle stem cells helps regenerate and maintain adult muscles. This fusion process has always been a poorly understood process.

Myoblast fusion

However, Côté and his co-workers have identified a receptor called BAI3 as one of the crucial links in myoblast fusion. BAI3 activates a signaling process that initiates the fusion of nearby myoblasts.

In 2008, Côté and his colleagues elucidated the role of two proteins – DOCK1 and DOCK5 – in the development of muscles. DOCK1 and DOCK5 regulate myoblast fusion. When the interaction between BAI3 and the DOCK signaling proteins is inhibited, myoblast fusion is also inhibited.

Côté pointed out that this work could have far-reaching implications, since the delivery of functional proteins to diseased muscle is typically carried out by introducing genetically engineered stem cells into the muscle that fuse with the disease muscle. By increasing the efficiency of the muscle fusion process, the delivery of genes to diseased muscles could become routine rather than painstakingly inefficient.

For Treating Heart Attacks, Satellite Cells Lacking MyoD are Superior to Those With MyoD

Atsushi Asakura and his colleagues at the University of Minnesota Stem Cell Institute have extended some of their earlier findings in a paper that appeared in PLoS One last year. This paper is almost a year old by now, but its results are fascinating and are definitely worth examining.

In 2007, Asakura published a paper with the Canadian researcher Michael A. Rudnicki in the Proceedings of the National Academy of Sciences. In this paper, Asakura and his colleagues examined the ability of muscle satellite cells from MyoD- mice to integrate into injured muscle. I realize that last sentence just sounded like gobbledygook, to some of you, but I will try to put the cookies on a lower shelf.

Satellite cells constitute a stem cell population within skeletal muscle. They are a small population of muscle-making stem cells found in skeletal muscle and they express a whole host of muscle-specific genes (e.g., desmin, Pax7, MyoD, Myf5, and M-cadherin). Satellite cells are responsible for muscle repair, but previous work has shown that there are at least two populations of satellite cells in skeletal muscle. One population rapidly contributes to muscle repair, whereas the other population is more stem cell-like and remains longer in an undifferentiated state in the recipient muscle (see Beauchamp JR , et al (1999) J Cell Biol 144:1113–1122; Kuang S , et al (2007) Cell 129:999–1010). Presently, it is not clear which population is more efficient in repairing continuously degenerating muscle.

MyoD is a gene that encodes a protein that binds to DNA and activates the expression of particular genes. It plays a vital role in regulating muscle differentiation, and belongs to a family of proteins known as myogenic regulatory factors or MRFs. All MRFs are bHLH or basic helix loop helix transcription factors, and they act sequentially in muscle differentiation. MRF family members include MyoD, Myf5, myogenin, and MRF4 (Myf6). MyoD is one of the earliest genes that indicates a cell has committed to become a muscle cell. MyoD is expressed in activated satellite cells, but not in quiescent (sleeping) satellite cells. Strangely, even though MyoD marks myoblast commitment, muscle development is not dramatically prevented in mouse mutants that lack the MyoD gene. However, this is likely to result from functional redundancy from Myf5. Nevertheless, the combination of MyoD and Myf5 is vital to the success of muscle production.


Therefore, Asakura and his crew decided to isolated muscle satellite cells from mice that lacked functional copies of the MyoD gene. Making such mice is labor intensive, but doable with mouse embryonic stem cell technology. When such MyoD- mice were made, Asakura and others isolated the satellite cells from these mice and characterized them. They discovered in their 2007 paper, that the satellite cells from the MyoD- mice were much more stem cell-like than satellite cells from MyoD+ mice. The MyoD- satellite cells grew better in culture, integrated into injured muscles better and survived better than their MyoD+ counterparts.

Why is this important? Because when it comes to treating degenerative muscle diseases like muscular dystrophy, finding the best cell is crucial. MyoD+ satellite cells have been used, but they are limited in the amount of muscle repair they provide. MyoD- cells might be a better option for treating a disease like muscular dystrophy.

Or for that matter, what about the heart? Finding the right cell to treat the heart after a heart attack has proven difficult. There are some things bone marrow cells do well, and other things they do not do well when it comes to regenerating the heart. Likewise, there are some things mesenchymal do well and other things they do not do well when placed in a damaged heart. Can MyoD- satellite cells do a better job than either of these types of stem cells?

That was the question addressed in the 2012 Nakamura paper that was published in PLoS One. Clinical trials that have treated heart attack patients with injections of MyoD+ satellite cells into the heart have shown that such treatments can improve heart function, but usually only transiently. They also prevent remodeling of the heart after a heart attack. However, two larger studies failed to produce significant improvements in heart function compared to the placebo, and patients who received the satellite cell transplants were also susceptible to very fast heart beats (tachycardia). Because of these downsides, the excitement for transplanting muscle satellite cells into the heart has waned.

So how did MyoD- satellite cells do? All the laboratory animals used in this experiment (BALB/c mice) were given heart attacks, and injected with either MyoD+ or MyoD- satellite cells. The hearts of animals injected with MyoD- satellite cells were compared with animals whose hearts were injected with MyoD+ satellite cells.

In culture, the MyoD- satellite cells grew better than the MyoD+ cells. When injected into the heart, the MyoD- cells integrated into the heart muscle and spread throughout the heart muscle much more robustly than the MyoD+ cells. The MyoD- cells were also much less susceptible to cell death and survived better than their MyoD+ counterparts.

(A) These panels show MI induced by left coronary artery ligation. Wild-type and MyoD−/− myoblasts were directly injected into the peri-infarct regions of LV. After 1 week, X-gal staining of whole heart indicated that more MyoD−/− myoblasts engrafted than wild-type myoblasts (arrows). Arrowheads indicate left coronary artery ligation points. X-gal staining of cross sections indicated that more MyoD−/− myoblasts than wild-type myoblasts engrafted in both injured and uninjured areas of the heart. Arrows indicate engrafted lacZ+ wild-type and MyoD−/− myoblasts. Scale bars = 1 mm.
(A) These panels show MI induced by left coronary artery ligation. Wild-type and MyoD−/− myoblasts were directly injected into the peri-infarct regions of LV. After 1 week, X-gal staining of whole heart indicated that more MyoD−/− myoblasts engrafted than wild-type myoblasts (arrows). Arrowheads indicate left coronary artery ligation points. X-gal staining of cross sections indicated that more MyoD−/− myoblasts than wild-type myoblasts engrafted in both injured and uninjured areas of the heart. Arrows indicate engrafted lacZ+ wild-type and MyoD−/− myoblasts. Scale bars = 1 mm.

Functionally speaking for the heart, animals that had received transplantations of MyoD- satellite cells had higher ejection fractions, small areas of dead heart tissue, lower end systolic and end diastolic volumes, and more normal echocardiograms. Even though MyoD- cells differentiated into skeletal muscle and not heart muscle (no surprise there), the MyoD- cells induced a very substantial quantity of new blood vessels to sprout in the scar area.

(A) Two weeks post-transplantation, immunofluorescence staining of heart cross sections showed that the progeny of lacZ+ wild-type and MyoD−/− myoblasts formed nestin+ multinucleated skeletal myotubes. Laminin (red) indicates cardiomyocytes and skeletal myotubes. Arrows indicate lacZ+ donor cell-derived nuclei in nestin+ myotubes. (B) Comparison of the relative numbers of lacZ+/nestin+ myotubes for wild-type and MyoD−/− myoblasts 2 weeks after injection (n = 3).
(A) Two weeks post-transplantation, immunofluorescence staining of heart cross sections showed that the progeny of lacZ+ wild-type and MyoD−/− myoblasts formed nestin+ multinucleated skeletal myotubes. Laminin (red) indicates cardiomyocytes and skeletal myotubes. Arrows indicate lacZ+ donor cell-derived nuclei in nestin+ myotubes. (B) Comparison of the relative numbers of lacZ+/nestin+ myotubes for wild-type and MyoD−/− myoblasts 2 weeks after injection (n = 3).

From these experiments, it seems that the MyoD- satellite cells are superior to the MyoD+ satellite cells for treating heart after a heart attack. These cells secrete a whole host of factors that aid the heart in healing and also structurally support the heart and prevent remodeling.

Might it be possible to use such cells in human trials? Asakura notes that engineering MyoD- satellite cells would be impractical for human clinical purposes, but it might be possible to downregulate MyoD expression with drugs (bromodeoxyuridine) or other reagents (RNAi or Id protein transformation).

This work shows that there is a better way to use muscle satellite cells for heart treatments. It simply requires you to remove MyoD function, and the cells will grow and spread throughout the heart better, and more robustly augment heart function and healing.

See NakamuraY, et al PLoS One 7(7) 2012:e41736.

Injecting Mesenchymal Stem Cells After Exercise Increase Blood Vessels in Muscles

The laboratory of Marni D. Boppart at the Beckman Inst. for Advanced Science and Technology in Urbana, Illinois has published a very interesting study that shows that injections of mesenchymal stem cells (MSCs) into mice after they have exercised increases the number of new blood vessels formed in skeletal muscle.

Early last year, Boppart’s laboratory showed that exercise in mice induces a population of mesenchymal stem cells located near blood vessels (pericytes) to migrate into muscles and form new muscle fibers. A particular adhesion molecule known as integrin α7 is responsible for the movement of MSCs into the muscle. Boppart and his colleagues showed that engineering mice that made extra α7 integrin had muscles that were filled with more stem cells making new muscle than their normal litter mates after exercise.

More recently, Boppart and his colleagues have injected cultured MSCs into the muscles of mice after the mice had exercised by running downhill. The mice injected with MSCs had more blood vessels in their muscles than mice than received no such injections.  The blood vessels in the MSC-injected mice were also larger.  Further work showed that the MSCs produced a veritable smorgasbord of angiogenic factors, which are molecules that induce the formation of blood vessels.

Thus MSCs, in response to exercise, can increase muscle mass as a result of exercise and increase the density and diameter of blood vessels.

Dr. Centeno at the Regenexx blog site wonders if stem-cell doping will be the next trick athletes will use to increase their muscle size and strength.  Such a trick would be completely undetectable with contemporary technology, and it seems as though this might be a genuine possibility.

Exercise Triggers Muscle Stem Cells

New findings from researchers from the University of Illinois showed that adult stem cells in muscle are responsive to exercise. This discovery might provide a link between exercise and muscle health, and could provide the impetus for therapeutic techniques that use muscle-specific stem cells to heal injured muscles and prevent or restore muscle loss with age.

Mesenchymal stem cells (MSCs) in skeletal muscles have been known to be important for muscle repair in response to injury. Experiments that demonstrate the roles of mesenchymal stem cells in muscle repair have use chemical-induced injuries that initiate damage muscle tissue and inflammation. However, exercise also stresses muscle, and a research group led by kinesiology and community health professor Marni Boppart investigated whether MSCs also responded to exercise-induced stress.

According to Boppart, “Since exercise can induce some injury as part of the remodeling process following mechanical strain, we wondered if MSC accumulation was a natural response to exercise and whether these cells contributed to the beneficial regeneration and growth process that occurs post-exercise.”

Boppart’s group found that muscle-based MSCs respond to mechanical strain. In fact, mice subjected to vigorous exercise showed robust accumulation after exercise. They also found that MSCs do not directly contribute to new muscle fibers, but, instead, they release growth factors that spur other cells in muscle to fuse and generate new muscle.

Boppart’s research group isolated muscle-based MSCs after the mice exercised, and then they stained the MSCs with a fluorescent marker and injected them into other mice to see how they coordinated with other muscle-building cells. In addition to examining MSCs in vivo, Boppart’s laboratory examined the response of MSCs to strain on different substrates. They discovered that MSC response is very sensitive to the mechanical environment, indicating that conditions under which muscles are strained affects the activity of the cells.

Boppart added, “We’ve identified an adult stem cell in muscle that may provide the basis for muscle health with exercise and enhanced muscle healing with rehabilitation/movement therapy. The fact that MSCs in muscle have the potential to release high concentrations of growth factor into the circulatory system during exercise also makes us wonder if they provide a critical link between enhanced whole-body health and participation in routine physical activity.”

Since, preliminary data suggest MSCs become deficient in muscle with age; the group hopes to determine if these cells contribute to the decline in muscle mass over a person’s lifetime. The team hopes to develop a combinatorial therapy that utilizes molecular and stem-cell-based strategies to prevent age-related muscle loss.

Cell Transplantation Treatments for Amyotrophic Lateral Sclerosis (ALS)

Because so many of you commented on the ALS entry, I decided to write more about stem cell treatments for this disease.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from death of so-called “upper” and “lower” motor neurons. Motor neurons enervate skeletal muscles, and the activity of motor neurons allows those muscles to contract. Without contraction of skeletal muscles, the skeleton cannot move and ambulatory movement as we know it, becomes impossible.

There is only one treatment for ALS and that is a drug called riluzole (Rilutek). When neurons start to die, they dump enormous quantities of neurotransmitters into the spaces surrounding the cells, and this neurotransmitter dump causes nearby neurons to die from neurotransmitter overdose. Blocks the glutamate receptor and prevents large quantities of glutamate from binding to the surfaces of neurons en mass and killing them. Riluzole, however, only buys ALS patients time and increases survival by a matter of months (3-5 months). ALS patients die approximately within three-to-five years after receiving their diagnosis. Death typically results from the weakness of those skeletal muscles that are responsible for airway and respiratory control (See Borasio, G. & Miller, R. Clinical characteristics and management of ALS. Semin. Neurosci. 2002;21:155–166).

As you can see, better treatment options are required, and cell transplantation has recently been proposed as a treatment for various neurological disorders (see Miller, R. H. The promise of stem cells for neural repair. Brain Res. 2006;1091:258–264). In 2009, the FDA approved the first phase I trial of intraspinal stem cells as a treatment of ALS. This trial is ongoing, with completion anticipated sometime in 2012.

What causes motor neurons to all of a sudden start dying off? In a small subset of cases, genetic mutations in genes such as super¬oxide dismutase 1 are the reason for motor neuron die-off (see Rothstein, J. D. Current hypotheses for the underlying biology of amyotrophic lateral sclerosis. Ann. Neurol. 2009;65(Suppl. 1):S3–S9 & Ilieva, H., Polymenidou, M. & Cleveland, D. W. Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J. Cell Biol. 2009;187:761–772). In the vast majority of cases, the exact mechanisms of motor neuron degeneration in ALS are poorly understood. ALS pathogenesis involves multiple cell types and many mechanisms. The events that cause neuronal death in ALS patients include inflammation, oxidative stress, overdose of the neurotransmitter glutamate, and loss of neurotrophic support. Therefore, the spinal is converted into a toxic waste dump that is completely inhospitable for the survival of neurons. The best way to treat this disease is to maintain or restore motor neuron function and roll back the toxic environment in the spinal cord. Also replacing dead neurons is the goal of cell transplantation therapies.

Cell transplantation strategies come in two forms:  A) transplantation of neurons (the conductive cells in the nervous system; and B) transplantation of glial cells (the support cells in the nervous system).  Neuron transplantation is possible, since neurons can be derived from embryonic stem cells or from existing neural stem cell lines.  Neuron transplantation has been studied in an ALS model in rodents.  See the following papers:  a)  Bonner, J. F., Blesch, A., Neuhuber, B. & Fischer, I. Promoting directional axon growth from neural progenitors grafted into the injured spinal cord. J. Neurosci. Res. 88, 1182–1192 (2010).  b)  Silani, V., Calzarossa, C., Cova, L. & Ticozzi, N. Stem cells in amyotrophic lateral sclerosis: motor neuron protection or replacement? CNS Neurol. Disord. Drug Targets 9, 314–324 (2010).  c)  Xu, L., Ryugo, D. K., Pongstaporn, T., Johe, K. & Koliatsos, V. E. Human neural stem cell grafts in the spinal cord of SOD1 transgenic rats: differentiation and structural integration into the segmental motor circuitry. J. Comp. Neurol. 514, 297–309 (2009).  d)  Yan, J. et al. Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord. PLoS Med. 4, e39 (2007).  Unfortunately, when it comes to transplanting motor neurons, there are some daunting practical issues:  grafted neurons must receive functional synapses, send axons through inhibitory white matter, and direct axons over long distances to the target muscles in order to retain neuromuscular function.  Given these limitations, direct replacement of motor neuron populations is unlikely to provide a viable treatment option for ALS.

Transplantation of glial cells, such as astrocytes and microglia is a much more practical possibility for ALS treatment.  Astrocytes and microglia contribute to ALS pathology by impaired metabolic support, compromised neuron–glia crosstalk, or release of toxic metabolites.  By replacing diseased glia cells, the pathology of ALS can be effectively short-circuited and the environment of the spinal cord is ameliorated.  Experiments, once again in rodents, have shown that transplantation of astrocytes that express the wild-type SOD1 allele can reduce the degeneration and death of motor neurons expressing mutant SOD1 (see Boucherie, C., Schafer, S., Lavand’homme, P., Maloteaux, J. M. & Hermans, E. Chimerization of astroglial population in the lumbar spinal cord after mesenchymal stem cell transplantation prolongs survival in a rat model of amyotrophic lateral sclerosis. J. Neurosci. Res. 2009;87:2034–2046; & Clement, A. M. et al. Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice. Science 2003;302:113–117).  Other experiments that transplanted glial-restricted progenitor (GRP) cells into the spinal cords of mutant SOD1 rats showed that such GRPs differentiates into astrocytes that restored the levels of astrocyte physiology, decreased glutamate levels in the spinal cord and extended the survival of the transplanted rats (Lepore, A. C. et al. Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease. Nat. Neurosci. 2008;11:1294–1301).  These experiments indicate that cellular replacement therapies might support motor neurons in ALS by maintaining a more hospitable microenvironment in the spinal cord.

Another therapeutic strategy for ALS patients is to use growth factors to protect the axons that extend from the motor neurons in the spine to the skeletal muscles in the limbs and body wall.  Axonal defects that include degeneration of the neuromuscular junction and distal axon are some of the earliest hallmarks of ALS.  Degeneration of axons occurs before the onset of symptoms and the death of the motor neurons (Fischer LR & Glass JD. Axonal degeneration in motor neuron disease. Neurodegener. Dis. 4, 431–442 (2007).  This suggests that axonal dysfunction is a consequence of a loss of trophic support.  To understand trophic support, consider that you have to take a very long trip to bring something to a client.  Without gas stations along the way, you would never make it to your destination, since your care would run out of gas.  Axons that extend from neurons are the same way.  In order to make it all the way to their target muscle, they need gas stations along the way.  Once they attach to their target muscle, the muscle secreted molecules that allows the axon to survive.  However, glial cells along the way provided molecules that helped the axon survive as well.  Without this constant input of pro-survival molecules (so-called “trophic factors”), the axons retract and lose their neuromuscular junction with the muscle.  This is what is meant by trophic support.

Transplanted stem cells that secrete neurotrophic factors might provide a strategy to protect the diseased neurons.  Trophic support of axons by growth factors and insulin-like growth factor I (IGF‑I) have been shown to provide neuroprotection in both in vitro and in vivo models of ALS and reduce motor neuron degeneration (see Sakowski, S. A. et al. Neuroprotection using gene therapy to induce vascular endothelial growth factor‑A expression. Gene Ther. 16, 1292–1299 (2009) & Sakowski, S. A., Schuyler, A. D. & Feldman, E. L. Insulin-like growth factor‑I for the treatment of amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. 10, 63–73 (2009).  Unfortunately, recent clinical trials showed that subcutaneous delivery of IGF‑I had no therapeutic benefit in ALS, since the growth factor could not reach the motor neurons in the spinal cord (Sorenson, E. J. et al. Subcutaneous IGF‑1 is not beneficial in 2‑year ALS trial. Neurology 71, 1770–1775 (2008).  Intraspinal transplantation of stem cells that are capable of secreting IGF‑I could potentially overcome this limitation and provide therapeutic levels of IGF‑I directly to motor neurons.

More and more data point to the efficacy of treatments that improvement the spinal cord environment.  Transplantation of stem cells that secrete growth factors might do exactly this task.  Cortical human neural progenitor cells engineered to secrete glial cell-derived neurotrophic factor (GDNF) provide protection for motor neurons after transplantation into the spinal cords of SOD1G93A transgenic rats (which develop a rodent form of ALS; see Suzuki, M. et al. GDNF secreting human neural progenitor cells protect dying motor neurons, but not their projection to muscle, in a rat model of familial ALS. PLoS ONE 2, e689 (2007).  These cells also rapidly differentiated into astrocytes and replaces diseased glial cells.  Therefore, transplanted stem cells provided multiple therapeutic benefits.  Similarly, transplantation of neural progenitor cells producing either GDNF or IGF‑I into SOD1G93A mice decreases loss of motor neuron loss (Park, S. et al. Growth factor-expressing human neural progenitor cell grafts protect motor neurons but do not ameliorate motor performance and survival in ALS mice. Exp. Mol. Med. 41, 487–500 (2009).  Intramuscular delivery of GDNF-producing mesenchymal stem cells in a rat model of ALS also increases neuro­muscular contacts, motor neuron survival and lifespan (Suzuki, M. et al. Direct muscle delivery of GDNF with human mesenchymal stem cells improves motor neuron survival and function in a rat model of familial ALS. Mol. Ther. 16, 2002–2010 (2008).  Cellular therapies might, therefore, represent a source of neurotrophic support for diseased motor neurons in ALS.

Human spinal stem cells are derived from spinal cord progenitors and differentiate into both neurons and glia.  Several rodent studies have confirmed the therapeutic potential of intraspinal HSSC transplantation.  Following transplantation, these cells express excitatory amino acid transporters that can restore functional glutamate reuptake around vulnerable motor neurons.  Additionally, HSSCs also release neurotrophic factors (Yan, J. et al. Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord. PLoS Med. 4, e39 (2007).  Grafted HSSCs express several growth factors, including GDNF and brain-derived neurotrophic factor, and also form synaptic contacts with host motor neurons (Xu, L., Ryugo, D. K., Pongstaporn, T., Johe, K. & Koliatsos, V. E. Human neural stem cell grafts in the spinal cord of SOD1 transgenic rats: differentiation and structural integration into the segmental motor circuitry. J. Comp. Neurol. 514, 297–309 (2009).  Several publications show that transplantation of Intraspinal HSSCs delays symptom onset and extends the lifespan in rodent ALS models (see following papers:  a)  Xu, L. et al. Human neural stem cell grafts ameliorate motor neuron disease in SOD‑1 transgenic rats. Transplantation 82, 865–875 (2006); b)  Yan, J. et al. Combined immunosuppressive agents or CD4 antibodies prolong survival of human neural stem cell grafts and improve disease outcomes in amyotrophic lateral sclerosis transgenic mice. Stem Cells 24, 1976–1985 (2006); c) Xu, L., Shen, P., Hazel, T., Johe, K. & Koliatsos, V. E. Dual transplantation of human neural stem cells into cervical and lumbar cord ameliorates motor neuron disease in SOD1 transgenic rats. Neurosci. Lett. 494, 222–226 (2011).  Thus, transplanted HSSCs differentiate into multiple cell types, improve the spinal cord milieu, provide neurotrophic support, and form functional synaptic contacts with motor neurons in the spinal cord, forming a multifaceted attack on ALS progression.  HSSCs are the ideal stem cell for ALS treatment.

Since cellular therapies have the ability to improve survival and motor function of ALS rodents, the next stop is human trials.  In 2009, the FDA approved a human trial that involved intraspinal injection of HSSCs.  Choosing the right stem cell for ALS patients will require several clinical trials, but with this trial, HSSCs were chosen.  They were injected at two levels in the spinal cord; lumbar and cervical levels.  Some patients were injected at both levels and others were injected at one but not the other level.  Injections were performed with a special device designed specifically for stabilized injection into the spinal cord at specific locations.

a | Platform anchored to patient’s spine consists of two bridge rails (blue), one of which is scored at 2-mm intervals to aid regular positioning of injections. Gondola (green) compensates for slight movements in the platform application. Mechanical Z drive (orange) allows precise raising and lowering of a floating cannula. b | Cannula tip is positioned 1 mm medial to dorsal root entry zone. c | Needle penetrates into spinal cord ~4 mm from pial surface. d | Once needle tip is positioned at the target, metal outer sleeve is pulled up, leaving flexible tubing exposed.

This figure comes from Nicholas M. Boulis, Thais Federici, Jonathan D. Glass, J. Simon Lunn, Stacey A. Sakowski & Eva L. Feldman. Translational stem cell therapy for amyotrophic lateral sclerosis.  Nature Reviews Neurology, advance online publication, Published online 13 December 2011 | doi:10.1038/nrneurol.2011.191.

The clinical trial will be completed in 2012 and the data should be published soon thereafter.  A new era in ALS treatment is dawning and stem cells are leading the way.