Inhibition of signaling pathway stimulates adult muscle satellite cell function


Stem cell researcher Michael Rudnicki and his team from the University of Ottawa in Ontario, Canada has done it again. Rudnicki works on muscle stem cells and his work has greatly expanded our understanding of muscle satellite cells.

Muscle satellite cells are found in skeletal muscle, and they are a prime example of a “unipotent” stem cell, or a stem cell that can differentiate into only one cell type. Muscle satellite cells can only form skeletal muscle, but they can be isolated from skeletal muscle and grown in culture. When muscle is injured by exercise or shear forces, satellite cells move into action and divide to form muscle cells that fuse with existing muscle cells and firm them up. Lifting weights will also increase the activity of satellite cells and they will divide and contribute to the formation of new muscle fibers.

As we age, our capacity to regenerate damaged muscle slows way down. As someone who lifted weights in high school and then on and of after high school, I can attest to this as I have entered my later years. My joints get sore faster and I cannot handle heavier weights any more. Also, I do not get big from lifting anymore. This is due to the reduction in muscle repair and I have become older.

Rudnicki and others have identified a reduced capacity in adult mice to repair their muscles, and this reduction in muscle regenerative ability has been directly linked to reduced muscle satellite cell activity. Aged mice have muscle satellite cells that show a diminished ability to contribute to muscle regeneration and repopulate themselves.

In a recent paper published in the journal Nature Medicine, Rudnicki and his colleagues compared used gene expression profiles in the satellite cells of older and younger mice. Curiously, they identified the genes that encode the components of a cell signaling pathway called the “JAK-STAT” pathway that are more highly expressed in the satellite cells of older mice than in those of younger mice.

These data suggested that inhibition of the JAK-STAT pathway in the satellite cells of older mice might lead to higher satellite cell activity in older mice. Fortunately, there are drugs that will inhibit the JAK-STAT signaling pathway.

Knockdown of the activity of the Jak2 or Stat3 proteins significantly stimulated satellite stem cell divisions in culture (the satellite cells were grown in cultured muscles). When Jak2 of Stat3 were inhibited genetically (by introducing loss-of-function mutations in these genes), the isolated satellite cells showed a markedly ability to repopulate local satellite cell populations after they were transplanted into a wounded muscle.

Inhibition of Jak2 and Stat3 activity with drugs also stimulated the engraftment of satellite cells in a living animal. If these same rugs were injected into the muscle of older laboratory mice, these mice showed marked enhancement of muscle repair and force generation after injury.

Thus, these results from the Rudnicki lab show that they is an intrinsic property of satellite cells that separate the satellite cells of younger animals with those of older animals. These results also suggest a promising therapeutic avenue for the treatment of muscle-wasting diseases.

Directly Reprogramming Skin Cells into White Blood Cells


Scientists from the Salk Institute have, for the first time, directly converted human skin cells into transplantable white blood cells, which are the soldiers of the immune system that fight infections and invaders. This work could prompt the creation of new therapies that introduce new white blood cells into the body that can attack diseased or cancerous cells or augment immune responses for other conditions.

This work, which shows that only a small amount of genetic manipulation could prompt this direct conversion, was published in the journal Stem Cells.

“The process is quick and safe in mice,” says senior author Juan Carlos Izpisua Belmonte, who holds the Salk’s Roger Guillemin Chair. “It circumvents long-standing obstacles that have plagued the reprogramming of human cells for therapeutic and regenerative purposes.”

The problems that Izpisua Belmonte mentions, includes the long time (at least two months) numbingly tedious cell culture work it takes to produce, characterize and differentiate induced pluripotent stem (iPS) cells. Blood cells derived from iPSCs also have other obstacles: they engraft into organs or bone marrow poorly and can cause tumors.

The new method designed by Izpisua Belmonte and his team, however, only takes two weeks, does not produce tumors, and engrafts well.

“We tell skin cells to forget what they are and become what we tell them to be—in this case, white blood cells,” says one of the first authors and Salk researcher Ignacio Sancho-Martinez. “Only two biological molecules are needed to induce such cellular memory loss and to direct a new cell fate.”

This faster reprogramming technique developed by Belmonte’s team utilized a form of reprogramming that does not go through a pluripotency stage. Such techniques are called indirect lineage conversion or direct reprogramming. Belmonte’s group has demonstrated that such approaches can reprogram cells to form the cells that line blood vessels. Thus instead of de-differentiating cells into an embryonic stem cell-type stage, these cells are rewound just enough to instruct them to form the more than 200 cell types that constitute the human body.

Direct reprogramming used in this study uses a molecule called SOX2 to move the cells into a more plastic state. Then, the cells are transfected with a genetic factor called miRNA125b that drives the cells to become white blood cells. Belmonte and his group are presently conducting toxicology studies and cell transplantation proof-of-concept studies in advance of potential preclinical and clinical studies.

“It is fair to say that the promise of stem cell transplantation is now closer to realization,” Sancho-Martinez says.

Study co-authors include investigators from the Center of Regenerative Medicine in Barcelona, Spain, and the Centro de Investigacion Biomedica en Red de Enfermedades Raras in Madrid, Spain.

The First Patient Treated with iPSC-Derived Cells


Nature News has reported that a Japanese patient was received the first treatment derived from induced pluripotent stem cells.

Ophthalmologist Masayo Takahashi from the Riken Center for Developmental Biology and her team used genetic engineering techniques to reprogram skin fibroblasts from this patient into induced pluripotent stem cells. These cultured iPSCs were then differentiated into retinal pigment epithelium cells. Takahashi’s colleagues, led by Yasuo Kurimoto at Kobe City Medical Center General Hospital, then implanted those retinal pigment epithelium cells into the retina of this female patient, who suffers from age-related macular degeneration.

It is unlikely that this procedure will restore the woman’s vision. However, because age-related macular degeneration is a progressive process, Takahashi and her research team will be examining if this procedure prevents further deterioration of her sight. Takahashi’s Riken team has extensively tested this procedure in laboratory animals and recently received human trial clearance. Takahashi’s team will also be looking particularly hard at the side effects of this procedure; such as immune reaction or cancerous growth.

“We’ve taken a momentous first step toward regenerative medicine using iPS cells,” Takahashi says in a statement, according to Nature News. “With this as a starting point, I definitely want to bring [iPS cell-based regenerative medicine] to as many people as possible.”

Transplanted Mesenchymal Stem Cells Prevent Bladder Scarring After Spinal Cord Injury


A collaborative research effort between laboratories from Canada and South Korea have shown that a cultured mesenchymal stem cell line called B10 can differentiate into smooth muscle cells and improve bladder function after a spinal cord injury.

Spinal cord injury can affect the lower portion of the urinary tract. Overactive bladder, urinary retention, and increased bladder thickness and fibrosis (bladder scarring) can result from spinal cord injuries. Human mesenchymal stem cells (MSCs) can differentiate under certain conditions into smooth muscle. For this reason, MSCs have therapeutic potential for patients who have suffered from spinal cord injuries.

Seung U. Kim and his colleagues from Gachon University Gil Hospital in Inchon, South Korea have made an immortalized human mesenchymal stem cell line by transfecting primary cell cultures of fetal human bone marrow mesenchymal stem cells with a retroviral vector that contains the v-myc oncogene. This particular cells line, which they called HM3.B10 (or B10 for short), grows well in culture and can also differentiates into several different cell types.

In this present study, which was published in the journal Cell Transplantation, Kim and his colleagues and collaborators injected B10 hMSCs directly into the bladder wall of mice that had suffered a spinal cord injury but were not treated showed no such improvement.

“Human MSCs can secrete growth factors,” said study co-author Seung U. Kim of the Division of Neurology at the University of British Columbia Hospital, Vancouver, Canada. “In a previous study, we showed that B 10 cells secrete various growth factors including hepatocyte growth factor (HGF) and that HGF inhibits collagen deposits in bladder outlet obstructions in rats more than hMSCs alone. In this study, the SCI control group that did not receive B10 cells showed degenerated spinal neurons and did not recover. The B10-injected group appeared to have regenerated bladder smooth muscle cells.”

Four weeks after the initial spinal cord injury, the mice in the B10-treated group received injections of B10 cells transplanted directly into the bladder wall. Kim and his team used magnetic resonance imaging (MRI) to track the transplanted B10 cells. The injected B10 cells had been previously labeled with fluorescent magnetic particles, which made them visible in an MRI.

“HGF plays an essential role in tissue regeneration and angiogenesis and acts as a potent antifibrotic agent,” explained Kim.

These experiments also indicated that local stem cell injections rather than systemic, intravenous infusion was the preferred method of administration, since systemic injection caused the hMSCs get stuck largely in the blood vessels of the lungs instead of the bladder.

The ability of the mice to void their bladders was assessed four weeks after the B10 transplantations. MRI analyses clearly showed strong signals in the bladder as a result of the labeled cells that had been previously transplanted. Post-mortem analyses of the bladders of the transplanted group showed even more pronounced differences, since the B10-injected animals had improved smooth muscle cells and reduced scarring.

These results suggest that MSC-based cell transplantation may be a novel therapeutic strategy for bladder dysfunction in patients with SCI.

“This study provides potential evidence that an human [sic] stable immortalized MSC line could be useful in the treatment of spinal cord injury-related problems such as bladder dysfunction.” said Dr. David Eve, associate editor of Cell Transplantation and Instructor at the Center of Excellence for Aging & Brain Repair at the University of South Florida. “Further studies to elucidate the mechanisms of action and the long-term effects of the cells, as well as confirm the optimal route of administration, will help to illuminate what the true benefit of these cells could be.”

Bioinformatic Analysis Leads to Gene Combination that Makes Clinical Quality Mouse iPSCs


Adult cells can be de-differentiated so that they resemble embryonic stem cells by genetically engineering them to overexpress particular genes. Such reprogrammed cells are known as induced pluripotent stem cells or iPSCs, and these cells might have the potential to cure damaged nerves, regrow limbs and organs, and precisely model a patient’s particular disease. Unfortunately, the very process of reprogramming triggers replication stress, which causes iPSCs to acquire serious genetic and epigenetic abnormalities that lower the cells’ quality and limit their therapeutic usefulness.

When iPSCs were first derived in 2006, the efficiency of their derivation was quite low, since only a fraction of a percentage of reprogrammed cells successfully grew to become cell lines. Thus some of the earliest work with iPSCs tried to increase the efficiency of reprogramming. These experiments provided a greater understanding of the reprogramming process and demonstrated that many different variables, including the ratio of reprogramming factors and the reprogramming environment, could also greatly affect the quality of the iPSCs that were derived.

A research group from the Whitehead Institute, which includes founding member Rudolf Jaenisch, in collaboration with scientists from Hebrew University, has shown that the reprogramming factors themselves greatly influence the reprogramming efficiency and the quality of the resulting cells. This work was published in the current issue of the journal Cell Stem Cell.

“Postdoctoral researcher Yosef Buganim and Research Scientist Styliani Markoulaki show that a different combination of reprogramming factors may be less efficient than the original, but can produce higher quality iPSCs,” says Jaenisch, who is also a professor of biology at MIT. “And quality is a really important issue. At this point, it doesn’t matter if we get one colony out of 10,000 or one out of 100,000 cells, as long as it is of high quality.”

In order to derive iPSCs from mature adult cells, scientists transfect adult cells to a cocktail of genes. The genes used are all active in embryonic stem cells. By pushing cells to overexpress these embryonic stem cell-specific genes, adult cells can become iPSCs, which can then be differentiated into almost any other cell type, such as nerve, liver, or muscle cells. The original gene combination included Oct4, Sox2, Klf4, and Myc or (OSKM). This combination efficiently reprograms cells, but a relatively high percentage of the resulting cells have serious genomic aberrations, including aneuploidy, and trisomy 8, which make them unsuitable for use in clinical research.

Buganim and Markoulaki used bioinformatic analysis of a network of 48 genes that are integral to the reprogramming process. With this analysis, Buganim and Markoulaki designed a new reprogramming gene cocktail: Sall4, Nanog, Esrrb, and Lin28 (SNEL). With this gene combination, approximately 80% of SNEL colonies made from mouse cells were of high quality and fulfilled the tetraploid complementation assay, which is the most stringent pluripotency test available. As a comparison, only 20-30% of high quality OSKM passed the same test. Buganim hypothesizes that SNEL reprograms cells better because, unlike OSKM, the cocktail does not rely on a potent oncogene like Myc, which might be the source of some of the genetic problems produced by the reprogramming process. Even importantly, the cocktail does not rely on the potent key master regulators Oct4 and Sox2 that seem to abnormally activate some regions in the adult cell genome.

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Buganim and Markoulaki also analyzed SNEL colonies down to the genetic and epigenetic level. On their DNA, SNEL cells have deposits of the histone protein H2AX in locations very similar to those in ESCs, and the position of H2AX seems to predict the quality of the cell. This characteristic might be a fast way to quickly screen for high quality colonies.

It must be stressed that this SNEL gene combination was designed for mouse cells; it is unable to reprogram human cells, which are generally more difficult to manipulate than mouse cells. However, the same bioinformatic analysis might provide the proper insights to find the right combination for human cells that produce clinical quality iPSCs.

“We know that SNEL is not the ideal combination of factors,” says Buganim, who is currently a Principal Investigator at Hebrew University in Jerusalem. “This work is only a proof of principle that says we must find this ideal combination. SNEL is an example that shows if you use bioinformatics tools you can get better quality. Now we should be able to find the optimal combination and try it in human cells to see if it works.”

Yale Scientists Find Marker for High-Quality Induced Pluripotent Stem Cells


Pluripotent stem cells can be made by genetically engineering adult cells into less mature cells that have pluripotency. These induced pluripotent stem cells or iPSCs can potentially differentiate into any cell type in the adult body and because they are made from the patient’s own cells, they have a lower risk of being rejected by the patient’s immune system.

However, iPSCs suffer from an increased mutation rate when they are made and these increased mutation rate increases their risk of causing tumors and being rejected by the patient’s immune system. Having said that, not all iPSCs are created equal, and the safety of iPSCs seems to be very line-specific. Thus, how do you know a good stem cell from a bad one?

Yale Stem Cell Center researchers led by Andrew Xiao Yale have published a report in the Sept. 4 issue of Cell Stem Cell in which they describe an indicator that seems to predict which batch of personalized stem cells will differentiate into patient-specific tissue types and which will develop into unusable placental or tumor-like tissues.

Xiao’s group identified a variant histone protein called H2A.X that seems to predict the developmental path of iPSC cells in mice. Histone proteins assemble into tiny spools around which DNA winds. This DNA spooling allows cells to tightly package their DNA into a tight, compact structure that is easily stored called “chromatin.” Histones that are commonly used include histones H2A, H2B, H3 and H4.

Core histones

Two copies of each of these proteins assemble into a globular structure called a core histone and the DNA of the cell winds around this core histone to form a “nucleosome.” Then linker histones (H1 or H5) take these nucleosomes package them into spiraled coils.

DNA solenoids

H2A.X is a variant version of histone H2A is modified when DNA damage occurs. Modified H2A.X signals to the DNA repair machinery to fix the broken DNA (see TT Paull, and others, Curr. Biol. 10(15):886–95).

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According to the data from Xiao’s research team, in pluripotent stem cells, H2A.X is specifically targeted to those genes typically expressed in cells used to make the placenta, and it helps suppress differentiation of pluripotent stem cells into cells of the placental lineage. Given this distribution in mouse embryonic stem cells, H2A.X deposition pattern is a functional marker of the quality of iPSCs. Conversely, defective H2A.X deposition predisposes iPSCs toward differentiating into placental-type cells and tumors.

“The trend is to raise the standards and quality very high, so we can think about using these cells in clinic,” Xiao said. “With our assay, we have a reliable molecular marker that can tell what is a good cell and what is a bad one.”

Neuralstem Treats Final Patient in Phase 2 ALS Stem Cell Trial


NeuralStem, Inc. has announced that the final patient in its Phase 2 clinical trial that assessed the efficacy of its NSI-566 spinal cord-derived neural stem cell line in the treatment of amyotrophic lateral sclerosis (ALS), which is otherwise known as Lou Gehring’s disease.

ALS is a rapidly progressive, invariably fatal neurological disease that attacks the nerve cells responsible for controlling voluntary muscles; that is, muscle action we are able to control, such as those in the arms, legs, and face, etc.  ALS is a member of those disorders known as motor neuron diseases, all of which are characterized by the gradual degeneration and death of motor neurons.

Motor neurons are nerve cells located in the brain, brain stem, and spinal cord that serve as controlling units and vital communication links between the nervous system and the voluntary muscles of the body. Messages from motor neurons in the brain (so-called upper motor neurons) are transmitted to motor neurons in the spinal cord (so-called lower motor neurons) to particular muscles. In ALS, both the upper motor neurons and the lower motor neurons degenerate or die, and stop sending messages to muscles. Unable to function, the muscles gradually weaken, waste away (atrophy), and have very fine twitches (called fasciculations). Eventually, the ability of the brain to start and control voluntary movement is lost.

ALS causes weakness with a wide range of disabilities. Eventually, all muscles under voluntary control are affected, and individuals lose their strength and the ability to move their arms, legs, and body. When muscles in the diaphragm and chest wall fail, people lose the ability to breathe without ventilatory support. Most people with ALS die from respiratory failure, usually within 3 to 5 years from the onset of symptoms. However, about 10 percent of those with ALS survive for 10 or more years.

Although the disease usually does not impair a person’s mind or intelligence, several recent studies suggest that some persons with ALS may have depression or alterations in cognitive functions involving decision-making and memory.

ALS does not affect a person’s ability to see, smell, taste, hear, or recognize touch. Patients usually maintain control of eye muscles and bladder and bowel functions, although in the late stages of the disease most individuals will need help getting to and from the bathroom.

In this multicenter Phase 2 trial, 15 patients who still had the ability to walk were treated in five different dosing cohorts. The first 12 of these patients received injections only in the cervical regions of the spinal cord in increasing doses (5 injections of 200,000 cells per injection to injections of 4000,000 cells each . In the cervical region, these injected stem cells could potentially preserve the nerves that mediate breathing and this is precisely that this part of the trail aims to test.

spinal cord regions

In the final three patients injected in this trial, patients received a total of 40 injections of 400,000 cells each into both cervical and lumbar regions (a total of 16 million cells were injected. This is in contrast to the patients who participated in the Phase 1 study who received 15 injections of 100,000 cells each (total of 1.5 million cells). This trial will continue until six months past the final surgery, after which the data will be analyzed.

“By early next year, we will have six-month follow-up data on the last patients who received what we believe will be the maximum safe tolerated-dose for this therapy,” said Dr. Eva Feldman, principal investigator in this clinical trial, and a member of the ALS Clinic at the University of Michigan. Dr. Feldman also serves as an unpaid consultant to Neuralstem.