Reprogramming Skin Cells into Neural Stem Cells By Introducing One Gene

Transforming skin cells into nerve cells that interconnect and send nerve impulses to each other requires an extensive amount of reprogramming. The production of induced pluripotent stem cells is rather labor-intensive and introduces some risks. However, a new procedure designed by Yadong Huang at the Gladstone Institutes has shown that the introduction of a single gene into skin cell can generate nerve cells from skin cells.

This single gene, Sox2, transforms skin cells within days into early-stage brain stem cells known as induced neural stem cells or iNSCs. In culture, iNSCs self-renew and mature into neurons that can connect with each other and then transmit electro-chemical signals between each other. When the iNSCs were cultured for one month, they had already formed a completely new neural network.

An excited Huang made these points: “Many drug candidates, especially those developed for neurodegenerative diseases, fail in clinical trials because current models don’t accurately predict the drug’s effects on the human brain. Human neurons derived from reengineered skin cells could help assess the efficacy and safety of these drugs, thereby reducing risks and resources associated with human trials.”

Huang’s findings build on the work of Japanese research Shinya Yamanaka, who was the first scientist to publish the production of induced pluripotent stem cells. Since that time, other researchers have used genetic engineering techniques to directly reprogram adult cells into other types of adult cells without passing through the embryonic-stem-cell stage. Last year, Sheng Ding managed to use a combination of small molecules and genes to transform skin cells directly into neural stem cells. Huang’s technique now simplifies this technique even more so that only one gene is required to reprogram skin cells into neural stem cells. By avoiding the induced pluripotent stem cell stage, Huang and Ding hope to avoid the risk of tumor formation and the mutations induced by the production of induced pluripotent stem cells.

Karen Ring, a graduate student in Biomedical Sciences at the University of California, San Francisco, who was the lead author on this paper vouched for the safety of the iNSCs: “We wanted to see whether these newly generated neurons could result in tumor growth after transplanting them into mouse brains. Instead, we saw the reprogrammed cells integrate into the mouse’s brain, and not a single tumor developed.”

Huang’s paper also addresses the function Sox2 in the reprogramming of the skin cells. Huang and his research team also want to identify similar regulators that direct the development of specific types of neurons in the brain that tend to degenerate in the case of particular types of neurodegenerative diseases. Huang noted: “If we can pinpoint which genes control the development of each neuron type, we can generate them in the Petri dish from a single sample of human skin cells. We could then test drugs that affect different neuron types, such as those involved in Parkinson’s disease.” Huang added that such a discovery would help drug developers design treatments for neurodegenerative diseases that are much more specific, and the drug design would probably occur much faster.

Alzheimer’s disease still afflicts 5.4 million people in the US alone and this number is thought to triple by 2050. There are still no medications that can reverse the devastation wrought by this disease. Huang’s data might provide the means to test such new drugs.

Dental Stem Cells for Therapeutic Purposes

Brazilian and American scientists have made induced pluripotent stem cells (iPSCs) from stem cells found in teeth. These adult stem cells are immature enough so that forming iPSCs from that is relatively easy.

Human immature dental pulp stem cells (IDPSCs) are found in dental pulp. Dental pulp is the soft living tissue inside a tooth, and it houses various stem cell populations. These stem cells express a whole cluster of genes normally found in very young and immature cells. Therefore, IDPSCs are “primal” cells that are very young and undifferentiated.

According to Dr. Patricia C.B. Bealtrao-Braga of the National Institute of Science and Technology in Stem and Cell Therapy in Ribeirao Preto, Brazil, human IDPSCs are easily isolated from adult or baby teeth during routine dental visits. IDPSCs are not viewed as foreign by the immune system and can be used in the absence of any drugs that suppress the immune system. They have very valuable cell therapy applications, including the reconstruction of large cranial defects.

Another research project in the Republic of Korea, at the college of Veterinary Medicine, Gyeongsang National University, Republic of Korea have examined a stem cell population from third molars called human dental papilla stem cells (DpaSCs). DpaSCs can form dentin and dental pulp, but they also have biological features that are similar to those of bone marrow-derived mesenchymal stem cells (MSCs).

MSCs have been very heavily studied. While these stem cells have remarkable therapeutic capabilities, they have the disadvantage of only being able to grow in culture or a short period of time. After growing in culture for about a week, MSCs tend to go to sleep and not grow anymore.

DPaSCs, however, have a remarkable capacity to grow in culture. Data from work done in the laboratory of Gyu-Jin Ryo has shown they can grow for a longer period of time than MSCs in culture without going to sleep. Therefore, they not only can form a greater number of progeny, but they can also, potentially, form larger tissues and structures.

Based on their increased culture capabilities, DPaSCs can provide a source of stem cells for tooth regeneration and repair and, possibly, a source of cells for a wide variety of regenerative medical applications.

Muscle Cells Made from Induced Pluripotent Stem Cells Successfully Treat Mice With Muscular Dystrophy

Work by researchers at the Lillehei Heart Institute at the University of Minnesota have demonstrated the ability of induced pluripotent stem cells (iPSCs) to make muscle-forming cells, and that these cells can be used to treat muscular dystrophy.

Muscular dystrophy refers to a group of inherited diseases that causes muscle fibers to be structurally weak and highly susceptible to damage. The progressive muscle damage causes the muscles to become gradually weaker and weaker until the patient will eventually require a wheelchair.

There are several different types of muscular dystrophy. Most of the varieties of muscular dystrophy causes symptoms appear during childhood, but others cause symptoms to arise during adulthood. The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD). The symptoms begin early in life (once the child learns to walk), and include frequent falls, difficulty getting up from a lying or sitting position, trouble running and jumping, waddling gait, large calf muscles, and learning disabilities. A less severe and slower progressing form of muscular dystrophy is Becker muscular dystrophy (BMD). Symptoms usually being in the teenage years, but might also not occur until the mid-20s or later. Other types of muscular dystrophy include myotonic (inability to relax muscles at will, most often begins in early adulthood, muscles of the face are usually the first to be affected), Limb-girdle (hip and shoulder muscles are first affected), congenital (apparent at birth or becomes evident before age 2 and varies in severity), fascioscapulohumeral (shoulder blades stick out like wings when the person raises his or her arms, onset occurs in teens or young adults), and oculopharyngeal (drooping of the eyelids and weakness of the muscles of the eye, face and throat, symptoms first appear in a person’s 40s or 50s).

In order to treat muscular dystrophy (MD), many researchers have tried to use gene therapy to place normal versions of the muscular dystrophy gene (which encodes a protein called Dystrophin) into the muscles of MD patients (Romero NB, et al., Hum Gene Ther. 2004;15(11):1065-76 & Mendell JR, et al., Ann Neurol. 2009;66(3):290-7. These types of experiments have met with limited success, since the immune system of muscular dystrophy patients tends to attack the muscles that express dystrophin (Mendell JR, et al., New England Journal of Medicine 2010 7;363(15):1429-37).

In light of the failure of gene therapy trials, researchers have tried stem cell treatments in MD mice. Scientists in the laboratory of Rita Perlingeiro have used muscle precursor cells made from mouse embryonic stem cells to treat MD mice (Radbod Darabi, et al., Exp Neurol. 2009; 220(1): 212–216). Given this early success, Perlingeiro and her co-workers have used mouse iPSCs to make muscle-forming cells that have been used to treat muscular dystrophy in MD mice. In this experiment, suppression of the immune system was not necessary, since the muscle cells were made from cells that came from the patients.

Perlingeiro said of the experiment, “One of the biggest barriers to the development of cell-based therapies for neuromuscular disorders like muscular dystrophy has been obtaining sufficient muscle progenitor cells to produce a therapeutically effective response. Up until now, deriving engraftable skeletal muscle stem cells from human pluripotent stem cells hasn’t been possible. Our results demonstrate that it is indeed possible and sets the stage for the development of a clinically meaningful treatment approach.”

Once transplanted, the muscle-forming cells (myogenic progenitor cells to be exact) moved into the damaged muscles and integrated into them. They formed skeletal muscle and provided extensive and long-term muscle regeneration that resulted in improved muscle function. To make the iPSC cell lines, Perlingeiro and her laboratory workers genetically modified to human iPSC lines with a gene called PAX7. PAX7 encodes a transcription factor that is essential for muscle formation and muscle regeneration. PAX7, with PAX3, designates cells as myogenic progenitor cells. Therefore, inserting the PAX7 gene into iPSCs would drive them to become myogenic progenitor cells.

Once Perlingeiro’s lab perfected the protocol for making myogenic progenitor cells from iPSCs, they found that they could make buckets and buckets of them. The iPSC-derived muscle forming cells were much more efficient at integrating into the muscles and regenerating them than other cell types. Muscle-forming stem cells from human muscle biopsies, for example, failed to persist in the muscle.

Perlingeiro concluded, “Seeing long-term maintenance of these cells without major side effects is exciting. Our research proves that these differentiated stem cells have real staying power in the fight against muscular dystrophy.”

Induced Pluripotent Stem Cell Mutations Do Not Cluster in Protein-Coding Genes

Induced pluripotent stem cells (iPSCs) are made by introducing genes into adult cells that push the adult cell into an embryonic-like state. The earliest iPSCs were made with engineered retroviruses that actually inserted their genomes into the chromosomes of the host cell. The use of such tools to form iPSCs produces cells with large segments of DNA inserted into their chromosomes, which can cause mutations. This is not a desirable trait if such are to be used in a clinical setting. Additionally, detailed genetic examinations of iPSCs have shown that the very process of reprogramming adult cells to form cells with embryonic characteristics causes mutations (Gore, et al., Nature 471 (2011): 63–67).

Thus, iPSCs tend to harbor a variety of mutations that range from base sequence changes in their DNA to changes in the number of copies of various genes. These types of mutations can make iPSCs rather dangerous to use clinically. In fact one study suggest that the mutations generated by making iPSCs can potentially illicitly activate the expression of particular genes.  These inappropriately activated genes can induce the immune system of the person who donated the adult cells from which the iPSCs ere made to attack and reject the iPSCs (Zhao, et al., Nature 474, (2011): 212–215).

One issue that has not been properly addressed to date is the status of iPSCs made by alternative methods.  The Gore paper examined 22 iPSC lines and three of them were derived by methods that do not use viruses that insert themselves into the genome of the host cell.  Their data suggested that these iPSC lines also had higher numbers of mutations than the cells from which they were derived, but the tables in the Gore paper tend to show that the mRNA-derived iPSCs had lower numbers of mutations than those derived from more traditional means. Another issue is that the human genome has a tremendous amount of empty space.  Mutations that do not occur within the coding region of a gene is likely to not cause a problem.

Into the fray comes a paper from the laboratory of Linzhao Cheng at the Johns Hopkins Institute for Cell Engineering.  Cheng and his co-workers made iPSCs from bone marrow stem cells and discovered that while they possessed more mutations than the cells from which they were made, those mutations were typically not in genes that will affect the function of the cells. Cheng and his colleagues also used techniques to make iPSCs that did not utilize viruses that insert into the genomes of the host cell.

Cheng’s group took the bone marrow-derived iPSCs and differentiated them into mesenchymal stem cells, and then sequenced their genomes to determine the new mutations that were caused by the reprogramming.  They discovered that there were 1,000 to 1,800 new mutations in each cell line, but the mutations rarely occurred in protein coding regions.

On the average, each iPSC had six mutations that occurred in coding regions, but each mesenchymal stem cell made from the iPSC lines had about 12 mutations per cell in coding regions.  While this sounds awful, we must remember that some mutations are very consequential for the proteins that are encoded by genes, but many are not.  For example, the sickle-cell disease is due to one mutation in the hemoglobin gene that causes the hemoglobin protein to form chains that deform the red blood cell.  However, there are many other mutations in the hemoglobin gene that do not affect its function in the least.

When Cheng and his colleagues examined where the mutations occurred, they found that none of the mutations in protein coding regions that they had detected were in genes that would cause the iPSCs to grow uncontrollably or predispose the cells to form cancerous tumors.

Based on his findings, Cheng thinks that iPSCs form a smaller risk than was previously thought.  His results are published in Cell Stem Cell.

Pluripotency Genes Play Distinctly Different Roles in Mouse and Human Stem Cells

In 2000, scientists began identifying and characterizing proteins that help drive cells into the unique properties of embryonic stem cells. In August 2006, Shinya Yamanaka and colleagues at Kyoto University used four of these genes, all of which encoded transcription factors, Oct3/4, Sox2, c-Myc, and Klf4, to reprogram mouse skin cells to stem cells that exhibited most but not all of the properties of embryonic stem cells. The following year, Yamanaka’s team and James Thompson’s team at the University of Wisconsin–Madison concomitantly made induced pluripotent stem cells using Oct3/4, Sox2, Lin28, and Nanog.

Since this time, researchers have defined the function of these genes in detail in mouse embryonic stem cells (mESCs). Underneath this research was the assumption that the function of these genes in mESCs was the same in human cells. New research, however, has seriously challenged this notion.

Natalia Ivanova and her colleagues at Yale University in New Haven, CT, planned to study other genes that might be involved in maintaining pluripotency in human ESCs (hESCs). They used gene silencing to define the function of individual genes in hESCs. When they first silenced three of the genes that encode traditional pluripotency factors (Nanog, Oct4, and Sox2) they were very surprised to find that the stem cells did not act as they had expected. In mouse ESCs, Nanog, Oct4, and Sox2 bind the same locations on the genome and act cooperatively to maintain stem cell self-renewal and pluripotency. When any one of those factors is unable to perform its function, mESCs differentiate into extraembryonic tissues, such as placenta.

However, when they silenced each of the three factors in three different hESC lines, Ivanova and her team identified fundamentally different roles for these proteins in hESCs. First, the three factors prevent hESCs from transforming into non-embryonic tissues. Specifically, Nanog appears to prevent cells from becoming neuroectoderm, which is a tissue that eventually becomes the nervous system. Sox2 prevents cells from becoming mesoderm, which forms connective tissue and muscle.

Oct4 has varying roles depending on the presence of a protein called BMP4. In the absence of BMP4, inactivated Oct4 induces ectoderm, the outer layer of an embryo that forms the nervous system and skin, but in the presence of BMP4, it specifies extraembryonic cell fates. Also, Ivanova found that the trio does not function as a complex. Instead, Sox2 is the “odd man out,” since silencing Sox2 does not prevent hESCs from maintaining pluripotency in hESCs since Sox3, a related protein, compensates for Sox2’s absence. “It just shows you that in human [ESCs], repression by these three factors works in a completely different way” than in mouse ESCs, said Ivanova.

The findings could have a major impact on embryonic stem cell research. Mouse ESCs are traditionally easier to grow in culture than hESCs. One can grow sufficient mESCs for use in an experiment in two days, while it may take two months to grow enough hESCs. By understanding how human ESCs are regulated by these factors may help scientists fine-tune and speed up the expansion of hESCs in culture.

Also, because Nanog and Oct4 appear to be involved in the differentiation of the ectoderm, scientists may be able to use this knowledge to come up with new ways to inhibit these factors to improve differentiation of hESCs into neurons, which are quite valuable in a number of medical and scientific applications.

For their next project, the Yale team plans to get back to their original investigation of additional factors involved in hESC pluripotency. “Some other genes may be contributing to the regulation of self-renewal and differentiation,” said Ivanova. “We’re going to try to look at what these other players might be, to find out what else regulates this process.”

Neuronal Stem Cells Made from Mature Skin Cells

Stem cell researcher Hans Schöler and his colleagues at the Max Planck Institute for Molecular Biomedicine in Münster, Germany, have successfully isolated neural stem cells from completely differentiated skin cells. Workers and Schöler’s lab procured skin cells from mice and exposed them to a cocktail of special proteins called “growth factors,” and concurrently subjected them to specific culture conditions. This induced the skin cells to differentiate into neuronal somatic stem cells. Schöler noted that their research “shows that reprogramming somatic cells does not require passing through a pluripotent stage.” These new approaches to regenerative medicine can produce stem cells in a shorter time period and are also safer for human clinical use.

Pluripotent stem cells have definitely been the darling of stem cell science since their discovery. When exposed to the right environment, pluripotent stem cells differentiate into every type of cell in the body. However, the pluripotency of these cells, while being their grace is also their curse. According to Schöler, “pluripotent stem cells exhibit such a high degree of plasticity that under the wrong circumstances they may form tumors instead of regenerating a tissue or an organ.” However reprogrammed stem cells can provide a way around these dangers, since they are not pluripotent, but Multipotent (they can only give rise to select subset of cell types rather than any cell type). This can give them an edge in terms of safety and therapeutic potential.

To convert skin cells into stem cells, the Max Planck researchers invented an ingenious protocol that combined several different growth factors (proteins that direct cellular growth) in a culture system that grows the cells and encourages their differentiation into stem cells. One of these growth factors is called Brn4, and Brn4 had never been used in reprogramming experiments before. However, Schöler’s group discovered that Brn4 is one of the most powerful inducers of the stem cell fate in skin cells. The reprogramming of mature skin cells into neuronal stem cells is even more effective if the growth factor-treated skin cells are grown in specific culture conditions. Such culture conditions drive the cells to divide faster and, according to Schöler, the cells gradually “lose their molecular memory that they were once skin cells.” Only after a few cell divisions, the newly produced neuronal somatic stem cells are, for the most part, indistinguishable from neuronal stem cells extracted from neural tissue.

There are other reasons that this work from Schöler’s laboratory might be readily applicable to clinical settings. According the Schöler, “The fact that these cells are multipotent dramatically reduces the risk of neoplasm formation, which means that in the not-too-distant future they could be used to regenerate tissues damaged or destroyed by disease or old age; until we get to that point, substantial research efforts will have to be made.” However, these experiments were done with mouse skin cells. In order to show that this protocol could work for human regenerative medicine, Schöler and his colleagues must demonstrate that human skins cells can also undergo a similar transformation. Additionally, it is crucial to show that these skin cell-derived neuronal stem cells are stable over long periods of time in culture and when implanted into laboratory animals.

Schöler concluded with these remarks: “Our discoveries are a testament to the unparalleled degree of rigor of research conducted here at the Münster Institute. We should realize that this is our chance to be instrumental in helping shape the future of medicine.” At this point, the project is still in its initial, basic science stage although “through systematic, continued development in close collaboration with the pharmaceutical industry, the transition from the basic to the applied sciences could be hugely successful, for this as well as for other, related, future projects. The blueprints for this framework are all prepped and ready to go – all we need now are for the right political measures to be ratified to pave the way towards medical applicability.”

Induced Pluripotent Stem Cells Form Red Blood Cells

Concerns over the mutations that occur when adult cells are reprogrammed into induced pluripotent stem cells has caused scientists to step back and take a second look at this technology. Can such a technology be used to treat human patients safely?

Some cells in our bodies lack nuclei. For example, platelets and red blood cells do not have nuclei, and therefore, they lack a human genome. If red blood cells can be made from pluripotent stem cells, they could potentially treat patients who suffer from anemia. The red blood cells will not harbor any mutations because they do not have DNA. Thus, induced pluripotent stem cells could potentially be used to treat patients.

A paper in Stem Cells and Development by Jessica Dias and colleagues in the laboratory of Igor Slukvin at the University of Wisconsin, Madison has reported the generation of red blood cells from human induced pluripotent stem cells (J. Dias, et al., Stem Cells and Dev 20, no 9 (2011): 1639-47).

To make red blood cells from induced pluripotent stem cells (iPSCs), they made human iPSCs from skin cells called “fibroblasts” that were taken from new-born babies.  They made they iPSCs with methods that did not use viruses.  Instead they placed in the fibroblasts, small circles of DNA that contained all the genes necessary to create iPSCs.  These small circles of DNA are called “episomes.” and they can create iPSCs without maintaining themselves in the cells.  That is to say, once the episomes convert the adult cells into iPSCs, they are lost and do contaminate the genome of the iPSCs.

After making iPSCs, they grew them for seven days with two other cells; human embryonic stem cells and a mouse bone marrow cell line called OP9.  This combination converted the iPSCs into bone marrow stem cells.  The bone marrow stem cells were isolated and cultured for five days with chemicals that are known to push bone marrow stem cells to become red blood cells.  These chemicals (erythropoietin, stem cell factor, thrombopoietin, interleukin-3, dexamethasone, insulin, interleukin-6, and iron), drove the stem cells to become red blood cell-like cells.  Because these cells were also grown under conditions that prevented them from attaching they grew and differentiated.  After five days, the cells were maintained on another mouse bone marrow cells line called MS5 cells.

Dias and her colleagues also used an alternative technique that worked just as well that did not include isolating the bone marrow stem cells, but subjected the cells to a Percoll centrifugation that also isolated the differentiating cells from the other cells.  This technique seemed faster and less troublesome.

Neither of these techniques could be employed if these cells were to be used for human treatments.  The use of animal cells lines could contaminate the iPSCs with animal viruses or animal proteins.  Both of these would cause the human immune system to react adversely to the cells (Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid.Nat Med. 2005 Feb;11(2):228-32).  Therefore, some other protocol will need to be devised if this type of treatment is employed for anemic humans.

Nevertheless, this culture did generate red blood cells that expressed mainly embryonic and fetal types of hemoglobin.  While there was some adult hemoglobin made, it was the minority molecule.  All of the cells produced by this cell culture system were of the same type as those that produce red blood cells (erythroid), and not of those that make white blood cells (myeloid).  This shows that it is feasible to make red blood cells from iPSCs, and it might even be feasible to produce them in a culture system that makes large quantities of them.  Other uses for culture systems like this could include making red blood cells to grow malarial parasites for drug research.  Clearly this is a remarkable discovery that may lead to a source of red blood cells for patients and laboratories alike.