Added Netrin-1 Increases Induced Pluripotent Stem Cell Production Without Affecting Stem Cell Quality

Since 2006, stem cell researchers have succeeded in generating induced pluripotent cells (iPS cells) from mature, adult cells. These cells have enormous potential applications, particularly for regenerative medicine. However, the process by which these cells are made still requires further tweaking in order to increase its efficiency and safety. Recently, two teams of researchers from Inserm, CNRS, Centre Léon Bérard and Claude Bernard Lyon 1 University have discovered a molecule that seems to favor the production of iPS cells. Their work was published in the journal Nature Communications.

Reprogramming an already specialized cell into a pluripotent stem cell was discovered in 2006 by the Japanese scientist Shinya Yamanaka. His iPS cells were capable of differentiating into any type of cell from the human body. Yamanaka and his colleagues made iPS cells by introducing into adult cells a cocktail of four genes (Oct4, Klf4, Sox2, and c-Myc). iPS cells, like embryonic stem cells, which are made from human embryos, are pluripotent, which means that they can differentiate into any mature adult cell type. iPS cells represent a promising medical advance, since they might be able to ultimately replace diseased organs with new organs that were derived from the patient’s own cells. Such technology will create tissues and organs that match the tissue types of the patient from whom the adult cells were isolated, which would eliminate all risks of transplantation rejection. The use of iPS cells would also circumvent the inherent ethical problems raised by the use of embryonic stem cells, which are derived from the destruction of human embryos.

Despite this success, cell reprogramming is besets by some problems. First of all, it is not terribly efficient; many cells undergo programmed cell death and this restricts the number of iPS cells produced. To increase the efficiencies of iPS cell production, Fabrice Lavial’s team, in collaboration with Patrick Mehlen’s team, identified new regulators of the derivation of iPS cells. They examined those genes that are regulated by the four inducing genes involved in the initiation of reprogramming. From this list of genes, they selected those genes known to have a role in programmed cell death, and whose expression varies over the course of reprogramming. This screening process yielded a gene that encodes a protein called netrin-1.

Netrin-1 is a protein naturally secreted by the body. Interestingly, netrin-1 can prevent programmed cell death, among other things. In the early days of reprogramming mouse cells, the researchers observed that their production of netrin-1 was strongly reduced, which limited the efficacy of the reprogramming process. Next, these research teams tested the effects of adding extra netrin-1 to cells during the early phases of reprogramming. This increased the quantity of iPS cells produced from mouse cells. When they repeated this experiment with human cells, the reprogramming process generated fifteen times more iPS cells than those produced by protocols without added netrin-1.

From a therapeutic point of view, it was important to determine whether this treatment affected the quality of cell reprogramming. Genomic tests, however, failed to show any deleterious effects of the use of netrin-1 on reprogrammed cells. “According to several verifications, netrin-1 treatment does not seem to have any impact on the genomic stability the iPS cells or on their ability to differentiate into other tissues,” says Fabrice Lavial, Inserm Research Fellow.

These research teams continue to test the effects of netrin-1 on the reprogramming of other types of cells. They would like to gain a better understanding of the mode of action of this molecule in stem cell physiology.

Identifying Barriers to Cell Reprogramming

A new study from the laboratory of Miguel Ramalho-Santos, associate professor of obstetrics, gynecology and reproductive sciences at the University of California, San Francisco (UCSF), might lead to a faster way to derive stem cells that can be used for regenerative therapies.

Induced pluripotent stem cells or iPSCs, which are made from adult cells by means of genetic engineering and cell culture techniques, behave much like embryonic stem cells. These adult cell-derived stem cells are pluripotent and can be differentiated into heart, liver, nerve and muscle cells. This present work by Ramalho-Santos and his colleagues builds upon the reprogramming protocols that have been developed to de-differentiate mature adults cells into iPSCs.

Ramalho-Santos and his co-workers have been interested in understanding the reprogramming process more completely in order to increase the efficiency and safety of this process. In particular, the Ramalho-Santos laboratory has been examining the cellular barriers that prevent adult cells from being reprogrammed in order to circumvent them and increase the efficiency of stem-cell production. In this present work, Ramalho-Santos’ group identified many of these cellular barriers to reprogramming.

“Our new work has important implications for both regenerative medicine and cancer research,” said Ramalho-Santos, who is also a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.

In 2012, Shinya Yamanaka from Kyoto University won the Nobel Prize in Physiology or Medicine for his discovery of iPSCs. Yamanaka discovered ways to turn back the clock on adult cells, but the protocol that he developed and others have used for years is inefficient, slow, and tedious. The percentage of adult cells successfully converted to iPS cells is usually rather low, and the resultant cells often retain traces of their earlier lives as mature, fully-differentiated cells.

To make iPSCs, researchers force the expression of pluripotency-inducing genes in adult cells. These four genes (Oct4, Klf4, Sox2, cMyc) have become known as the so-called “Yamanaka factors” and they work to turn back the clock on cellular maturation. However, as Ramalho-Santos explained: “From the time of the discovery of iPS cells, it was appreciated that the specialized cells from which they are derived are not a blank slate. They express their own genes that may resist or counter reprogramming.”

So what are those barriers? Ramalho-Santos continued: “Now, by genetically removing multiple barriers to reprogramming, we have found that the efficiency of generation of iPS cells can be greatly increased.” This discovery will contribute to accelerating the production of safe and efficient iPSCs and other types of other reprogrammed cells, according to Ramalho-Santos.

Instead of identifying individual genes that act as barriers to reprogramming, Ramalho-Santos and others discovered that sets of genes acted in combination to establish barriers to reprogramming. “At practically every level of a cell’s functions there are genes that act in an intricately coordinated fashion to antagonize reprogramming,” Ramalho-Santos explained. These existing mechanisms probably help mature, adult cells maintain their identities and functional roles. Ramalho-Santos explained it this way: “Much like the Red Queen running constantly to remain in the same place in Lewis Carroll’s ‘Through the Looking-Glass,’ adult cells appear to put a lot of effort into remaining in the same state.” Ramalho-Santos also added that apart from maintaining the integrity of our adult tissues, the barrier genes probably serve important roles in other diseases, including in the prevention of certain cancers

To identify these barriers, Ramalho-Santos and his team had to employ cutting-edge genetic, cellular and bioinformatics technologies. They collaborated with other UCSF labs headed by Jun Song, assistant professor of epidemiology and biostatistics, and Michael McManus, associate professor of microbiology and immunology.

They conducted genome-wide RNAi screens that revealed known and novel barriers to human cell reprogramming. Of these, a protein called ADAM29 antagonizes reprogramming as does clathrin-mediated endocytosis, which antagonizes reprogramming by enhancing TGF-β signaling. Also it became apparent that different barrier pathways have a combined effect on reprogramming efficiency. Additionally, genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport, and cell adhesion also act as barriers to reprogramming.

Barriers to reprogramming

The hopes are that this knowledge will produce iPSCs faster that are safer to use and differentiate more completely.

Patient-Specific Stem Cells Made More Easily?

A Michigan State University research team uncovered the function of an already characterized gene that could be linchpin in the derivation of patient-specific stem cells that might be able to save millions of lives by differentiating into practically any cell in the body.

The gene is known as ASF1A, and even though it was not discovered by the team, ASF1A is one of the genes responsible for the mechanism of cellular reprogramming. Cellular reprogramming de-differentiates adult cells into less mature stem cells that have the capacity to differentiate into any cell type in the adult body.

This work was published in the journal Science. In this paper, the MSU team analyzed more than 5,000 genes from a human egg (oocyte) and determines that ASF1A in combination with another gene known as OCT4 and another molecule were primarily responsible for reprogramming.

Human oocytes
Human oocytes

“This has the potential to be a major breakthrough in the way we look at how stem cells are developed,” said Elena Gonzalez-Munoz, a former MSU post-doctoral researcher and first author of the paper. “Researchers are just now figuring out how adult somatic cells such as skin cells can be turned into embryonic stem cells. Hopefully this will be the way to understand more about how that mechanism works.”

An MSU team identified the thousands of genes expressed in oocytes in 2006. From this list of genes, the genes responsible for cellular reprogramming were then identified.

In 2007, a Japanese research team led by Shinya Yamanaka found that by introducing four other genes into adult cells, they could derive embryonic-like stem cells without the use of a human egg. These cells are called induced pluripotent stem cells, or iPSCs. “This is important because the iPSCs are derived directly from adult tissue and can be a perfect genetic match for a patient,” said Jose Cibelli, an MSU professor of animal science and a member of the team.

Apparently, ASF1A and OCT4 work in together in combination with a hormone-like substance that also is produced in the oocyte called GDF9 to facilitate the reprogramming process. “We believe that ASF1A and GDF9 are two players among many others that remain to be discovered which are part of the cellular-reprogramming process,” Cibelli said.

“We hope that in the near future, with what we have learned here, we will be able to test new hypotheses that will reveal more secrets the oocyte is hiding from us,” he said. “In turn, we will be able to develop new and safer cell-therapy strategies.”

Induced Pluripotent Stem Cells Used to Make New Bone In Monkeys

Cynthia Dunbar, MD and her colleagues at the National Heart, Lung, and Blood Institute, which is a division of the National Institutes of Health (NIH) in Bethesda, Maryland have shown for the first time that it is possible to make new bone from induced pluripotent stem cells that are derived from a patient’s own skin cells.

This study, which was done in monkeys, shows that there is some risk that induced pluripotent stem cells (iPSCs) can form tumors, but that the risk of tumor formation is less than what was shown in immuno-compromised mice.

iPSCs are made from adult cells by means of a process called “reprogramming.” To reprogram adult cells, genetic engineering techniques are used to introduce specific genes into adult cells. These introduced genes drive the adult cells to de-differentiate into a less mature state, until they eventually become pluripotent, much like embryonic stem cells.

Originally, discovered by Nobel-prize winner Shinya Yamanaka, reprogramming was initially done with genetically engineered viruses that insert genes into the genome of cells. Even though these viruses do a passable job of reprogramming cells, they also introduce insertion mutations. Yamanaka and others originally used four transcription factors (Oct4, Sox2, Klf4, c-Myc) to reprogram adult cells. Several of these genes are overexpressed in a variety of tumors, and therefore, the use of these genes does create a risk of forming cells that overgrown and become tumorous. Secondly, The reprogramming process does put cells under the types of stresses that increase the mutation rate, and these mutations can also increase the risk of forming tumor cells. However, it is clear that not all reprogramming protocols cause the same rate of mutations, and that the mutation rate of iPSCs was originally overestimated. What is required is a good way to screen iPSC lines for mutations and for safety, especially since not all iPSC lines are equal when it comes to their safety.

The advantage of using iPSCs over embryonic stem cells is that the immune system of the patient should not reject tissues and cells made from iPSCs. This would eliminate the need for immune suppression drugs, which can be rather toxic.

Cynthis Dunbar from the National Heart, Lung, and Blood Institute said of her experiments, “We have been able to design an animal model for testing of pluripotent stem cell therapies using the rhesus macaque, a small monkey that is readily available and has been validated as being closely related physiologically to humans.

Dr. Dunbar continued: “We have used this model to demonstrate that tumor formation of a type called a ‘teratoma’ from undifferentiated autologous iPSCs does occur; however, tumor formation is very slow and requires large numbers of iPSCs given under very hospitable conditions. We have also shown that new bone can be produced from autologous iPSCs as a model for their possible clinical application.”

Dunbar and her team used a excisable polycistronic lentiviral vector called STEMCCA (Sommer et al., 2010) that expressed four genes: human OCT4, SOX2, MYC, and KLF4 to make iPSCs from skin cells. After they had derived culturable iPSCs from rhesus monkeys (made under feeder-free conditions), Dunbar and her group seeded them on ceramic scaffolds that are used by reconstructive surgeons to fill in or rebuild bone. Interestingly, these cells regrew bone in the monkeys.

The differentiated iPSCs formed no teratomas, but monkeys that had received implantations of undifferentiated iPSCs formed teratomas in a dose-specific manner.

Dunbar and her colleagues note that this approach might be beneficial for people with large congenital bone defects or other types of traumatic injuries. Having said that, it is doubtful that bone replacement therapies will be the first human iPSC-based treatment, since bone defects are not life-threatening, even though they can seriously compromise the quality of a patient’s life.

“A large animal preclinical model for the development of pluripotent or other high-risk/high-reward generative cell therapies is absolutely issues of tissue integration of homing, risk of tumor formation, and immunogenicity,” said Dunbar. “The testing of human-derived cells in vitro or in profoundly immunodeficient mice simply cannot model these crucial preclinical safety and efficiency issues.”

This NIH team is now collaborating with other labs to differentiate macaque iPSCs into liver, heart, and white blood cells for to test them for eventual pre-clinical trials in hepatitis C, heart failure, and chronic granulomatous disease, respectively.

Stimulus-Triggered Acquisition of Pluripotency Cells: Embryonic-Like Stem Cells Without Killing Embryos or Genetic Engineering

Embryonic stem cells have been the gold standard for pluripotent stem cells. Pluripotent means capable of differentiating into one of many cell types in the adult body. Ever since James Thomson isolated the first human embryonic stem cell lines in 1998, scientists have dreamed of using embryonic stem cells to treat diseases in human patients.

However, deriving human embryonic stem cell lines requires the destruction or molestation of a human embryo, the smallest, youngest, and most vulnerable member of our community. In 2006, Shinya Yamanaka and his colleges used genetic engineering techniques to make induced pluripotent stem (iPS) cells, which are very similar to embryonic stem cells in many ways. Unfortunately, the derivation of iPSCs introduces mutations into the cells.

Now, researchers from Brigham and Women’s Hospital (BWH), in Boston, in collaboration with the RIKEN Center for Developmental Biology in Japan, have demonstrated that any mature adult cell has the potential to be converted into the equivalent of an embryonic stem cell. Published in the January 30, 2014 issue of the journal Nature, this research team demonstrated in a preclinical model, a novel and unique way to reprogram cells. They called this phenomenon stimulus-triggered acquisition of pluripotency (STAP). Importantly, this process does not require the introduction of new outside DNA, which is required for the reprogramming process that produces iPSCs.

“It may not be necessary to create an embryo to acquire embryonic stem cells. Our research findings demonstrate that creation of an autologous pluripotent stem cell – a stem cell from an individual that has the potential to be used for a therapeutic purpose – without an embryo, is possible. The fate of adult cells can be drastically converted by exposing mature cells to an external stress or injury. This finding has the potential to reduce the need to utilize both embryonic stem cells and DNA-manipulated iPS cells,” said senior author Charles Vacanti, MD, chairman of the Department of Anesthesiology, Perioperative and Pain Medicine and Director of the Laboratory for Tissue Engineering and Regenerative Medicine at BWH and senior author of the study. “This study would not have been possible without the significant international collaboration between BWH and the RIKEN Center,” he added.

The inspiration for this research was an observation in plant cells – the ability of a plant callus, which is made by an injured plant, to grow into a new plant. These relatively dated observations led Vacanti and his collaborators to suggest that any mature adult cell, once differentiated into a specific cell type, could be reprogrammed and de-differentiated through a natural process that does not require inserting genetic material into the cells.

“Could simple injury cause mature, adult cells to turn into stem cells that could in turn develop into any cell type?” hypothesized the Vacanti brothers.

Vacanti and others used cultured, mature adult cells. After stressing the cells almost to the point of death by exposing them to various stressful environments including trauma, a low oxygen and acidic environments, researchers discovered that within a period of only a few days, the cells survived and recovered from the stressful stimulus by naturally reverting into a state that is equivalent to an embryonic stem cell. With the proper culture conditions, those embryonic-like stem cells were propagated and when exposed to external stimuli, they were then able to redifferentiate and mature into any type of cell and grow into any type of tissue.

To examine the growth potential of these STAP cells, Vacanti and his team used mature blood cells from mice that had been genetically engineered to glow green under a specific wavelength of light. They stressed these cells from the blood by exposing them to acid, and found that in the days following the stress, these cells reverted back to an embryonic stem cell-like state. These stem cells then began growing in spherical clusters (like plant callus tissue). The cell clusters were introduced into developing mouse embryos that came from mice that did not glow green. These embryos now contained a mixture of cells (a “chimera”). The implanted clusters were able to differentiate into green-glowing tissues that were distributed in all organs tested, confirming that the implanted cells are pluripotent.

Thus, external stress might activate unknown cellular functions that set mature adult cells free from their current commitment to a particular cell fate and permit them to revert to their naïve cell state.

“Our findings suggest that somehow, through part of a natural repair process, mature cells turn off some of the epigenetic controls that inhibit expression of certain nuclear genes that result in differentiation,” said Vacanti.

Of course, the next step is to explore this process in more sophisticated mammals, and, ultimately in humans.

“If we can work out the mechanisms by which differentiation states are maintained and lost, it could open up a wide range of possibilities for new research and applications using living cells. But for me the most interesting questions will be the ones that let us gain a deeper understanding of the basic principles at work in these phenomena,” said first author Haruko Obokata, PhD.

If human cells can be made into embryonic stem cells by a similar process, then someday, a simple skin biopsy or blood sample might provide the material to generate embryonic stem cells that are specific to each individual, without the need for genetic engineering or killing the smallest among us. This truly creates endless possibilities for therapeutic options.

Vascular Progenitors Made from Induced Pluripotent Stem Cells Repair Blood Vessels in the Eye Regardless of the Site of Injection

Johns Hopkins University medical researchers have reported the derivation of human induced-pluripotent stem cells (iPSCs) that can repair damaged retinal vascular tissue in mice. These stem cells, which were derived from human umbilical cord-blood cells and reprogrammed into an embryonic-like state, were derived without the conventional use of viruses, which can damage genes and initiate cancers. This safer method of growing the cells has drawn increased support among scientists, they say, and paves the way for a stem cell bank of cord-blood derived iPSCs to advance regenerative medical research.

In a report published Jan. 20 in the journal Circulation, Johns Hopkins University stem cell biologist Elias Zambidis and his colleagues described laboratory experiments with these non-viral, human retinal iPSCs, that were created generated using the virus-free method Zambidis first reported in 2011.

“We began with stem cells taken from cord-blood, which have fewer acquired mutations and little, if any, epigenetic memory, which cells accumulate as time goes on,” says Zambidis, associate professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center. The scientists converted these cells to a status last experienced when they were part of six-day-old embryos.

Instead of using viruses to deliver a gene package to the cells to turn on processes that convert the cells back to stem cell states, Zambidis and his team used plasmids, which are rings of DNA that replicate briefly inside cells and then are degraded and disappear.

Next, the scientists identified and isolated high-quality, multipotent, vascular stem cells that resulted from the differentiation of these iPSC that can differentiate into the types of blood vessel-rich tissues that can repair retinas and other human tissues as well. They identified these cells by looking for cell surface proteins called CD31 and CD146. Zambidis says that they were able to create twice as many well-functioning vascular stem cells as compared with iPSCs made with other methods, and, “more importantly these cells engrafted and integrated into functioning blood vessels in damaged mouse retina.”

Working with Gerard Lutty, Ph.D., and his team at Johns Hopkins’ Wilmer Eye Institute, Zambidis’ team injected these newly iPSC-derived vascular progenitors into mice with damaged retinas (the light-sensitive part of the eyeball). The cells were injected into the eye, the sinus cavity near the eye or into a tail vein. When Zamdibis and his colleagues took images of the mouse retinas, they found that the iPSC-derived vascular progenitors, regardless of injection location, engrafted and repaired blood vessel structures in the retina.

“The blood vessels enlarged like a balloon in each of the locations where the iPSCs engrafted,” says Zambidis. Their vascular progenitors made from cord blood-derived iPSCs compared very well with the ability of vascular progenitors derived from fibroblast-derived iPSCs to repair retinal damage.

Zambidis says that he has plans to conduct additional experiments in diabetic rats, whose conditions more closely resemble human vascular damage to the retina than the mouse model used for the current study, he says.

With mounting requests from other laboratories, Zambidis says he frequently shares his cord blood-derived iPSC with other scientists. “The popular belief that iPSCs therapies need to be specific to individual patients may not be the case,” says Zambidis. He points to recent success of partially matched bone marrow transplants in humans, shown to be as effective as fully matched transplants.

“Support is growing for building a large bank of iPSCs that scientists around the world can access,” says Zambidis, although large resources and intense quality-control would be needed for such a feat. However, Japanese scientists led by stem-cell pioneer Shinya Yamanaka are doing exactly that, he says, creating a bank of stem cells derived from cord-blood samples from Japanese blood banks.

Induced Pluripotent Stem Cells Self-Repair a Chromosome Abnormality

Japanese and American scientists have made a fantastic discovery. An incurable type of chromosomal abnormality, in which one end of the chromosome has fused with the other, is known as a “ring chromosome.” The presence of ring chromosomes often correlates with neurological abnormalities. The collaborative work by these two researcher groups has shown that if induced pluripotent stem cells are derived from abnormal adult cells that contain ring chromosomes will spontaneously repair themselves.

This research team, which included researchers from Kyoto University professor and iPS cell pioneer Shinya Yamanaka, also included Yohei Hayashi and other researchers from the U.S.-based Gladstone Institutes.

“I was very surprised at the results,” said Hayashi. “There still remains a risk, but the findings may lead to the development of breakthrough treatment for chromosomal abnormalities.”

Normal chromosomes pairs consist of two rod-shaped chromosomes, but in the case of a ring chromosome, the arms of one of the two chromosomes are fused to form a ring.

Ring chromosomes tend to be associated with mental disabilities and growth retardation, and there are no therapeutic strategies for ring chromosomes.

Hayashi and his colleagues developed iPS cells from skin cells of patients with ring chromosome disorder to study the effects of this chromosomal defect in stem cells. However, the ring chromosomes could not be observed in the engineered iPS cells made from this patient’s cells.

For reasons still unknown, only normal chromosomes survived, according to the researchers. Typically, a cell that contains ring chromosomes divides into two abnormal cells in the normal mitotic process.

These findings were published in the online edition of the British science journal Nature on Jan. 13, 2014.