Brain Stem Cells Reprogrammed to Treat Mice With Multiple Sclerosis

Scientists at the University of Rochester Medical Center and the University at Buffalo have published a technique for harvesting and using a patient’s own stem cells to treat Multiple Sclerosis. This same technique could potentially find additional applications for treating other rare, fatal children’s diseases.

Cells in the central nervous system capable of generating nerve impulses are known as neurons. Neurons have long extensions called axons and nerve impulses are propagated down the axon, away from the central body of the neuron. In order for nerve impulses to move much faster through the axons of the central nervous system than they normally would, many neurons have a an insulating sheath called the “myelin sheath” that surrounds the axon. Without a myelin sheath, nerve impulses move at as speeds of about 1 meter per second. With a myelin sheath, nerve impulses move much faster; faster than 120 meters per second.

In the peripheral nervous system, the myelin sheath is made by cells called Schwann cells. In the central nervous system, the myelin sheath is made by cells called oligodendrocytes. Oligodendrocytes are derived from “oligodendrocyte progenitor cells” or OPCs. This New York research group isolated and directed stem cells from the human brain to become oligodendrocytes. They injected these cultured OPCs into the brains of mice that were born without the ability to make myelin. 12 weeks later, the implanted cells had become oligodendrocytes and had coated more than 40 percent of the brain’s neurons with myelin. This same group had used a similar strategy in two papers published in Cell Stem Cell and Nature Medicine. However, this recent reported a four-fold increase in myelination.

Steven Goldman, research team leader and chair of the Department of Neurology at the University of Rochester Medical Center, noted that these implanted OPCs are presently the best candidates for treating patients with multiple sclerosis and other myelin disorders. Goldman noted, “These cells migrate more effectively throughout the brain, and they myelinate other cells more quickly and more efficiently than any other cells assessed thus far. Now we finally have a cell type that we think is safe and effective enough to propose for clinical trials.”

Fraser Sim, the first author of the paper, who is an assistant professor of Pharmacology and Toxicology at the University at Buffalo, and Rochester graduate student Crystal McClain, ran extensive analyses of gene activity in different types of stem cells. These data led to the conclusion that stem cells that express a protein called CD140a on their surfaces seemed to be most likely to become OPCs. Sim said, “Characterizing and isolating the exact cells to use in stem cell therapy is one key to ultimately having success. You need to have the right cells in hand before you can even think about getting to a clinical trial to treat people. This is a significant step.”

In order to make this experiment work, the research team needed to know how to direct brain stem cells into becoming OPCs. They turned to a decade of research by the Goldman lab that has tested the effects of many growth factors, small molecules and other factors on brain stem cell differentiation. The group used two specific growth factors, BMP4, which directs brain stem cells to become support cells called astrocytes, and Noggin, which drives the astrocyte-like cells to become OPCs. These cultured cells are very responsive to chemical cues from their local environment. It is important to select the right type of stem cell, but it is also just as important to generate a milieu that has the proper molecular signals to produce the type of cell needed for a particular treatment.

This current work focuses on the creation of myelin, and while myelin loss plays a large role in multiple sclerosis, but myelin loss also plays a central role in other childhood disorders. For example, cerebral palsy, diabetes, high blood pressure, and some cases of stroke also include myelin loss. While Goldman’s team has had previous success remyelinating the brains of mice born without myelin, these new results identify a specific subset of cells that appear to be the most efficient yet at producing myelin and improve the hope of developing cell therapy as a way to treat these diseases.

Multiple Sclerosis treatment might involve the injection of stem cells to create myelin in the brains of patients. In fact, one of the authors of this paper, Martha Windrem, has developed methods to inject cells into the brain so that they will migrate throughout a large swath of the brain, laying down myelin on neurons as they go. Sim suggests that another approach might “involve using certain medications to turn on these cells already present in the brains of patients and thereby create new myelin. The use of the new techniques described in this work will permit us to better understand how human cells behave in the brain and help us predict which medications may be successful in the treatment of myelin loss.”

Geron Corporation Announces the Preliminary Results of Its Phase I Study with GRNOPC1

Geron Corporation has announced data from its GRNOPC1 phase I study. In this study, four different patients who had experienced spinal cord injury were treated with injections of the embryonic stem cell-derived cell line GRNOPC1. This cell line is a stable, oligodendrocyte progenitor cell (OPC) line. Oligodendrocytes surround the axons for many central nervous system neurons. This insulation allows the nerve impulses that travel through these axons to travel much faster than they normally would. This insulation is essential for proper physiological function. However during spinal cord injury, oligodendrocytes are destroyed and the insulation attenuates, thus leading to the continuing decrease in neurological function after spinal cord injury. Implantation OPCs have been shown in mice to increase neurological function after a spinal cord. Therefore, the implantation of OPCs in humans who have suffered from spinal cord might improve their neurological function.

Geron introduced OPCs from the embryonic stem cell-derived cell line GRNOPC1 into the spinal cords of four human patients who had suffered from a recent spinal cord injury. These patients were all classified as American Spinal Injury Association (ASIA) Impairment Scale grade A thoracic spinal cord injuries. ASIA grade A is the worse category and indicates a complete spinal cord injury where no motor or sensory function has been preserved in the sacral segments S4-S5. These patients all received a dose of two million GRNOPC1 cells. These cells were injected into the lesion site, and the injection utilized a syringe positioning device that Geron actually designed. The OPC cells were administered between 7 and 14 days after injury, and a drug that suppresses the immune system called tacrolimus was given for to temporarily suppress the immune response from the time of injection until 46 days after the injection. After this the tacrolimus dose tapered and withdrawn completely by 60 days after the stem cell injection.

The patients were evaluated by determining the sensory and motor ability of the patient’s legs. There were also MRI scans of the spinal cord, but patients are at different stages in the evaluation period. One patient has experienced the 365-day follow-up whereas another patient has just had the 30-day follow up. The patients will be examined for five years after the procedure and after that, they will be interviewed by telephone until nine years after the procedure.

To date, the examinations have shown no surgical complications during or after the procedures, no adverse events related to the injection procedures or to GRNOPC1, a few mild adverse events related to tacrolimus, n evidence of cavitation in the spinal cord at the injury sites on MRI, no unexpected neurological changes, no evidence of immune responses to GRNOPC1.

There is no evidence to date of immune rejection of GRNOPC1, an allogeneic cell therapy, including after withdrawal of the immunosuppressive drug.

Stem Cell-Mediated Transfer of a Human Artificial Chromosome Can Treat Muscular Dystrophy in Mice

Duchenne muscular dystrophy (DMD) is a horrific disease characterized by relentless, progressive muscle loss. The effects of this disease are insidious and it leads to a terrible wasting away that ends in a slow, painful death. This muscle loss is very difficult to halt or reverse, and even though there have been tremendous advances in the cloning, sequencing and manipulation of the DMD gene, a variety of attempts to treat this disease have failed.

In 1986, Dr. A.P. Monaco et al. of Dr. L. Kunkel’s group isolated the DMD gene (Monaco et al., Nature 1986 22;323(6089):646-50). The DMD gene is extremely long (it is 2,500 kb long and consists of 79 exons that cover 1% of the x-chromosome, and it transcribed to form a transcript that is 14 kilobases long). In 1987, Dr. E.P. Hoffman and co-workers identified a 427 kilodalton protein encoded by the DMD gene. He named this protein “dystrophin”, which is absent from the skeletal muscle of most DMD patients (Hoffman et al., Cell 1987 ;51(6):919-28).

Success in cloning the DMD gene led to gene therapy trials to correct the genetic defect in the DMD gene. “Anti-sense Oligonucleotides” or AOs are short, easy to produce stretches of nucleic acid that specifically bind to the messenger RNA (mRNA) made from the DMD gene and influence the processing of that RNA. By changing the manner in which the messenger RNA is processed, a new mRNA is made that encodes a normal dystrophin protein rather than an abnormal dystrophin that does not work (Wu B, et al., Proc Natl Acad Sci U S A. 2008 105(39):14814-9; also see Wells KE, et al., FEBS Lett. 2003 552(2-3):145-9). The second strategy is to use modified viruses of deliver a normal copy of the DMD gene into the muscle cells (Goyenvalle A, et al., Science. 2004 306(5702):1796-9). While these techniques showed hopeful results in laboratory animals (mdx mice and DMD dogs), trials in human patients have been less successful and lead researchers to be less sanguine about gene therapy to treat DMD. As it turns out, the immune system of DMD patients views introduced dystrophin as foreign and mounts an immune response to it. Therefore, a new strategy is required.

Now, work by Francesco Saverio Tedesco and colleagues at the Division of Regenerative Medicine, Stem Cells and Gene Therapy at the San Raffaele Scientific Institute in Milan, Italy combines stem cell and gene therapy to deliver an artificial human chromosome into implanted stem cells to overcome these challenges in the mdx mouse model of DMD.

Previously, this research team had identified a blood vessel stem cell called a ”mesoangioblast.” Mesangioblasts have the ability to form blood vessels, but they can also cross blood vessel walls and differentiate into a many different types of mesodermal cell types, which includes muscle cells. Since the discovery of this stem cell, researchers in this lab constantly wondered if mesangioblasts could deliver a replacement dystrophin gene to abnormal muscles in mdx mice? Tedesco and his colleagues used a human artificial chromosome vector that was engineered to carry the entire normal human DMD gene (including the large regulatory regions). Then they transferred this vector into cultured mesangioblast from mdx mice, and injected the corrected mesoangioblasts directly into the skeletal muscles of recipient mdx mice that had a poorly functioning immune system. The use of mdx mice with compromised immune systems is an important step in preventing the immune system from rejecting the implanted mesangioblasts. The authors showed that the transplanted mesoangioblasts effectively engrafted into the muscles of mdx mice and expressed normal dystrophin protein. The muscle fibers from mdx mice transplanted with the modified mesangioblasts did not show any signs of muscular dystrophy, but, instead, were functional muscle fibers that lacked all DMD pathology. They also found that the transplanted mesoangioblasts differentiated into muscle satellite cells. Muscle satellite cells are the working muscle stem cell pool that produces new muscle cells under normal conditions.

However, in order to treat DM patients, all the muscles must be treated and not just injected muscles. To address this concern, the authors injected corrected mesoangioblasts into the arterial circulation of mdx mice. They found that the cells were able to not only able to cross blood vessel walls and home to dystrophic muscles, but they could contribute to the formation of new dystrophin-expressing muscle fibers. In further tests, mice that had received the mesoangioblast transplants showed reduced fiber fragility, increased force, and greater motor capacity on treadmill and freewheel tests.

There are still technical and regulatory hurdles that must be addressed before this strategy is used in DMD patients. For one, the immune response against dystrophin must be addressed. Nevertheless, stem cell-mediated transfer of a normal DMD gene by means of a human artificial can chromosome does show promise as a potential treatment for this tragic and ultimately fatal disease.

Stimulating Stem Cells from Bone Marrow Prevents Rejection of Transplanted Livers

Patients who need new organs and receive a transplanted organ must take anti-rejection drugs for the rest of their lives. These drugs suppress the immune system and cause the patient to be susceptible to various infections and cancers. However, researchers from The Johns Hopkins University have developed a technique to stimulate stem cells in the body of a rat after a liver transplant that prevents rejection of the new organ without the need for lifelong immunosuppressive drugs.

In this procedure, researchers transplanted portions of the livers from dark agouti (DA) rats into another rat strain (Lewis-type). After the transplant, they gave the rats a seven-day treatment of low-dose tacrolimus (an immunosuppressant, trade name is Prograf), or plerixafor (a stem-cell stimulator, the trade name is Mozobil) or a combination of the two drugs. They only gave a very low, short-term dose of the immunosuppressive drug (that prevented immediate immunological rejection of the liver), and four doses of the medication that mobilizes bone marrow stem cells in the recipient’s body. These mobilized stem cells seek out and populate the donor organ. The stem cells also hold the immune response at bay and prevent rejection of the transplanted liver. Twelve of the 13 rats that received a combination of the two drugs had long-term liver function and survived more than 180 days, while nearly all of the remaining rats rejected their new livers after 12 days. The surviving rats had not received any immunosuppressive drugs other than the initial low-dose treatment for one week. Essentially, the Hopkins research group transformed the donor liver from a foreign object under attack by the rat’s immune system into an organ tolerated by the recipient’s immune system — all in a matter of three months from the date of transplant. Presently, this same research group is testing the method on other transplanted organs, including kidneys, in rats and other larger animals.

The technique, if replicated in humans, could mark a major shift in the process of organ transplantation. The leader of this study, Zhaoli Sun, associate professor of surgery at the Johns Hopkins University School of Medicine, said: “It is the dream for all scientists in the transplant field to erase the need for lifelong immunosuppressant drugs. Currently, if a patient survives for 10 or 20 years with a new liver, that organ is still seen as foreign inside its new body because immunosuppression puts blinders on the immune system that must stay on to prevent rejection. Our idea was to find a way to turn that organ into something that ‘belongs’ and is never at risk of rejection.”

Thousands of people with end-stage liver disease have received lifesaving liver transplants, but transplant rejection remains a chronic risk for these patients. Anti-rejection drugs also are quite expensive and increase the chance of developing severe infections and many kinds of cancers. Also, because anti-rejection drugs tend to cause patients to feel poorly, many patients are not compliant with their drugs, which is to say that they do not take them regularly.  Because anti-rejection drugs must be taken every day, failure to comply increases the risk of rejection of the transplant.  Typically, organ transplant recipients receive full doses of immunosuppressant drugs, such as tacrolimus, immediately after they receive new livers, since without these drugs, rejection would quickly result and patient would die.

The Lewis rats in this experiment received the equivalent of one-tenth the standard dose of tacrolimus.  The main goal was to induce some tissue rejection by the new liver, but not enough to kill it.  This “controlled rejection,” appears to create injury signals in the body that signal for stem cells to move from the bone marrow to the organ and repair the damage.  The stem cells also prevent the new liver from regenerating itself with cells from the donor because those cells are under attack by the recipient’s immune system.  This leaves an opening for the recipient’s stem cells to populate the liver and regenerate the liver.

Sun and his colleagues used plerixafor, a relatively new drug, which is known to free stem cells from the bone marrow and release them to circulate in the bloodstream. The drug is currently approved for patients about to undergo chemotherapy whose stem cells are harvested frozen and then returned to the body after cancer treatment. Many of these stem cells travel to the damaged liver and repopulate it with cells from the recipient, slowly taking over for the donor cells. The mechanism that brings the stem cells into the liver is becoming better understood, while the mechanisms by which stem cells become liver cells remain elusive. The stem cells also appear to modulate the immune response by increasing the number of regulatory T-cells, which helps reduce the chances of rejection.

Sun said, “In our study, the risk of organ rejection is eventually eliminated because the liver is no longer a foreign object, but comprised of many of the recipient’s own cells. Once the recipient’s stem cells take over, the body sees the regenerated liver as its own and works to protect it; not attack it.” Within three months, Sun and his colleagues found that the majority of the liver cells in the transplanted organ belonged to the recipient, not the donor. When they used whole livers instead of partial livers, the process took a year. This suggests that the transformation process is jump started by using partial livers for transplant, because the organ already “needs” to regenerate itself to most effectively function, he says.

Sun cautions that clinical trials with human organ transplant patients might be years away, but only if further research in animals confirms the method’s safety and value. The technique might prove useful not only at the time of a new transplant, but even after years of immunosuppressant drug use.

Seeking Superior Stem Cells

Wellcome Trust Sanger Institute researchers have discovered that if they add two additional genes to the four that are already normally used to convert adult cells into induced pluripotent stem cells, the reprogramming process is more efficient and the quality of the stem cells generated in the process are better.

Read about it here.

Induced Pluripotent Stem Cells Safer Than Originally Thought

Readers of this blog will recognize that I have been very excited about induced pluripotent stem cells (iPSCs). iPSCs are made from adult cells. When particular genes are introduced into adult cells, these cells de-differentiate into embryonic-like cells and a fraction of them become so similar to embryonic stem cells that they are called induced pluripotent stem cells. This re-programming is the induced by the introduced genes drives the cells into this embryonic state. Because this technique can make stem cells without destroying human embryos, they are an attractive alternative to embryonic stem cells (ESCs).

The traditional manner in which iPSCs are made makes use of viruses that insert their viral DNA into the genome of the host cell. Reprogramming uses these viruses to overexpress four different genes (c-Myc, Oct4, Klf4, and Sox2) into the genomes of host cells, and these cause the cells to dedifferentiate into an embryonic state. One of the problems with this technique is that is generates cells that have foreign DNA inserted into the genome in random places. These random insertions can produce mutations if they insert into a protein-coding region. Therefore, researchers have used safer techniques that are not as efficient, but do not leave foreign DNA in the host cells.

Unfortunately, several studies have shown that the process by which iPSCs are made and cultured actually introduces mutations into the cells. Five different studies have established that the reprogramming process and subsequent culture of iPSCs in vitro often induces genetic (DNA sequence) and epigenetic (chromosome structure) abnormalities in these cells (Hussein, S. M. et al. Nature 471, 58–62 (2011); Gore, A. et al. Nature 471, 63–67 (2011); Lister, R. et al. Nature 471, 68–73 (2011); Mayshar, Y. et al. Cell Stem Cell 7, 521–531 (2010); Laurent, L. C. et al. Cell Stem Cell 8, 106–118 (2011). The Hussein paper examined copy number variation (CNV) across the genome during iPSC generation. CNV refers to duplications of various portions of chromosomes. Duplications represent a form of gross chromosomal mutation. Duplications tend to occur in cancer cells or other types of cells that have experience significant stresses like ionizing radiation, ultraviolet radiation, drugs that break DNA or other types of major stresses. The paper by Gore and colleagues searched for point mutations (single base changes) in iPSCs by using genome-wide sequencing of protein-coding regions. Lister and co-workers examined DNA methylation, which is a chemical modification of DNA bases (an epigenetic mark) across the genomes of ESCs and iPSCs at the single-base level. In addition to these studies, Mayshar et al have examined changes in chromosome numbers in iPSCs. All these data, with comparisons of CNV in ESCs and iPSCs by Laurent and colleagues have lead to the conclusion that reprogramming and subsequent expansion of iPSCs in culture lead to the accumulation of diverse abnormalities at the chromosomal, subchromosomal and single-base levels.

A new paper in Cell Stem Cell, however, that utilized sophisticated new techniques to analyze the genomic DNA of iPSCs shows that these deep fears might be unwarranted. Kristin Baldwin, associate professor at The Scripps Research Institute’s Dorris Neuroscience Center, said, “We’ve shown that the standard reprogramming method can generate induced pluripotent stem cells that have very few DNA structural mutations, which are often linked to dangerous cell changes such as tumorigenesis,”

Researchers at Scripps Research and the University of Virginia used the latest chromosomal error-mapping methods to determine how many mutations are actually introduced by iPSC induction. The new methods include a high-resolution version of a DNA-error-finding technique known as paired-end mapping, and an advanced algorithm called “Hydra,” for handling the mapping data.

To generate iPSCs, the team followed the standard, four-gene reprogramming procedure, but in order to minimize other potential sources of mutations they selected host cells that were not decades old as in the other papers. Instead they selected relatively error-free fibroblast cells from fetal mice. These fibroblasts were only kept in lab dishes for a very brief time before reprogramming. When the team analyzed these iPSCs they used two different strategies to distinguish which mutations were present in rare donor fibroblast cells and which were newly acquired during reprogramming. Their advanced techniques also allowed them to find more kinds of mutations, across a wider range of the genome. However, instead of finding more mutations, they found almost none. “We sequenced three iPSC lines at very high resolution, and were surprised to find that very few changes to the chromosomal sequence had appeared during reprogramming,” says Michael J. Boland, a research associate in Baldwin’s lab.

Each of the iPSC lines contained only a single mutation that may have originated from the reprogramming process. Mutations inherited from the donor fibroblast cell were present in one pair of lines, and a second line “inherited” none. The researchers noted the complete absence of new mutations caused by mobilization of retrovirus-like sequences that burrowed into the mammalian genome long ago, can become active again in certain cell types. Because these retrovirus-like sequences can actually jump from one location in the genome to another new location, they can cause severe mutations that even include breakage of chromosomes and loss or duplication of chromosomal material. Fortunately, all cells have ways to suppress such “retroelements,” but the suppression mechanisms in normal cells are different from those in stem cells. Therefore, the researchers worried that retroelements would be allowed to escape suppression during the transition to a stem cell state. While no previous surveys of iPSCs could detect these mutations, the study showed that despite very sensitive detection of controls, no retroelements had become active during reprogramming.

In order to establish that they had in fact made iPSCs, they implanted their reprogrammed cell lines into mouse embryos, and they made live, fertile mice. Baldwin added, “The mice generated from these cells have survived to a normal lab-mouse lifespan without obvious diseases that might arise from new DNA mutations.”

The Baldwin lab now is trying to determine if a similar reprogramming method could also yield relatively error-free human iPSCs. Baldwin concluded, “If our results with these mouse cells are applicable to human cells, then selecting better donor cells and using more sensitive genome-survey techniques should allow us to identify reprogramming methods that can produce human iPSCs that will be safer or more useful for therapies than current lines.”

UN Group Releases Pro-Life Papers

A set of documents called the San Jose Articles were released at the United Nations on October 6, 2011. These documents are intended to remind nations that they are under no obligation when it comes to international law to liberalize their laws regarding abortion.

The San Jose Articles are named for the city in Costa Rica where the authors of these documents drafted them. They wrote these articles in response to a long campaign of misinformation waged by abortion lobbyists who argue that international law recognizes a right to abortion. As a result of this campaign, Columbia recently legalized abortion and Mexico is strongly considering doing so.

This group of scholars, however, after surveying international law, discovered that the corpus of international law only recognizes a right to life from the moment of fertilization.

International human rights lawyer Yuri Mantilla noted, “There seems to be a global narrative promoting the killing of a preborn baby, the smallest member of the human family, that you have a right to do that. That’s happening at the U.N., mainly because European nations and the U.S. promote it.”

Joseph Rees, former U.S. Ambassador to East Timor and one-time U.S. representative to the U.N. Economic and Social Council, said at a press briefing Thursday he’d seen those efforts firsthand. “When I was in Timor I witnessed a sustained effort by some international civil servants and representatives of foreign NGOs to bully a small developing country into repealing its pro-life laws,” he said. “The problem is that people on the ground, even government officials, have little with which to refute the extravagant claim that abortion is an internationally recognized human right. The San Jose Articles are intended to help them fight back.”