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.

An Even Better Way to Make Induced Pluripotent Stem Cells


Researchers from the Centre for Genomic Regulation in Barcelona, Spain, have discovered an even faster and more efficient way to reprogram adult cells to make induced pluripotent stem cells (iPSCs).

This new discovery decreases the time it takes to derived iPSCs from adult cells from a few weeks to a few days. It also elucidated new things about the reprogramming process for iPSCs and their potential for regenerative medical applications.

iPSCs behave similarly to embryonic stem cells, but they can be created from terminally differentiated adult cells. The problem with the earlier protocols for the derivation of iPSCs is that only a very small percentage of cells were successfully reprogrammed (0.1%-2%). Also this reprogramming process takes weeks and is a rather hit-and-miss process.

The Centre for Genomic Regulation (CRG) research team have been able to reprogram adult cells very efficiently and in a very short period of time.

“Our group was using a particular transcription factor (C/EBPalpha) to reprogram one type of blood cells into another (transdifferentiation). We have now discovered that this factor also acts as a catalyst when reprogramming adult cells into iPS,” said Thomas Graf, senior group leader at the CRG and ICREA research professor.

“The work that we’ve just published presents a detailed description of the mechanism for transforming a blood cell into an iPS. We now understand the mechanics used by the cell so we can reprogram it and make it become pluripotent again in a controlled way, successfully and in a short period of time,” said Graf.

Genetic information is compacted into the nucleus like a wadded up ball of yarn. In order to access genes for gene expression, that ball of yarn has to be unwound so that the cell can find the information it needs.

The C/EBPalpha (CCAAT/Enhancer Binding Protein alpha) protein temporarily unwinds that region of DNA that contains the genes necessary for the induction of pluripotency. Thus, when the reprogramming process begin, the right genes are activated and they enable the successful reprogramming all the cells.

“We already knew that C/EBPalpha was related to cell transdifferentiation processes. We now know its role and why it serves as a catalyst in the reprogramming,” said Bruno Di Stefano, a PhD student. “Following the process described by Yamanaka the reprogramming took weeks, had a very small success rate and, in addition, accumulated mutations and errors. If we incorporate C/EBPalpha, the same process takes only a few days, has a much higher success rate and less possibility of errors, said Di Stefano.

This discovery provides a remarkable insight into stem cell-forming molecular mechanisms, and is of great interest for those studies on the early stages of life, during embryonic development. At the same time, the work provides new clues for successfully reprogramming cells in humans and advances in regenerative medicine and its medical applications.

Safe and Efficient Cell Reprogramming Inside a Living Animal


Research groups at the University of Manchester, and University College, London, UK, have developed a new technique for reprogramming adult cells into induced pluripotent stem cells that greatly reduces the risk of tumor formation.

Kostas Kostarelos, who is the principal investigator of the Nanomedicine Lab at the University of Manchester said that he and his colleagues have discovered a safe protocol for reprogramming adult cells into induced pluripotent stem cells (iPSCs). Because of their similarities to embryonic stem cells, many scientist hope that iPSCs are a viable to embryonic stem cells.

How did they do it? According to Kostarelos, “We have induced somatic cells within the liver of adult mice to transient behave as pluripotent stem cells,” said Kostarelos. “This was done by transfer for four specific gene, previously described by the Nobel-prize winning Shinya Yamanaka, without the use of viruses but simply plasmid DNA, a small circular, double-stranded piece of DNA used for manipulating gene expression in a cell.”

This technique does not use viruses, which was the technique of choice in Yamanaka’s research to get genes into cells. Viruses like the kind used by Yamanaka, can cause mutations in the cells. Kostarelos’ technique uses no viruses, and therefore, the mutagenic properties of viruses are not an issue.

Kostarelos continued, “One of the central dogmas of this emerging field is that in vivo implantation of (these stem) cells will lead to their uncontrolled differentiation and the formation of a tumor-like mass.”

However, Kostarelos and his team have determined that the technique they designed does not show this risk, unlike the virus-based methods.

“[This is the ] only experimental technique to report the in vivo reprogramming of adult somatic cells to plurpotentcy using nonviral, transient, rapid and safe methods,” said Kostarelos.

Since this approach uses circular plasmid DNA, the tumor risk is quite low, since plasmid DNA is rather short-lived under these conditions. Therefore, the risk of uncontrolled growth is rather low. While large volumes of plasmid DNA are required to reprogram these cells, the technique appears to be rather safe in laboratory animals.

Also, after a burst of expression of the reprogramming factors, the expression of these genes decreased after several days. Furthermore, the cells that were reprogrammed differentiated into the surrounding tissues (in this case, liver cells). There were no signs in any of the laboratory animals of tumors or liver dysfunction.

This is a remarkable proof-of-principle experiment that shows that reprogramming cells in a living body is fast and efficient and safe.

A great deal more work is necessary in order to show that such a technique can use useful for regenerative medicine, but it is certainly a glorious start.

 

Also involved in this paper were r, , and .

Physical Cues Push Mature Cells into Induced Pluripotent Stem Cells


Bioengineers from the laboratory of Song Li at UC Berkeley have used physical cues to help push mature cells to de-differentiate into embryonic-like cells known as induced pluripotent stem cells.

Essentially, Li and his coworkers grew skin fibroblasts from human skin and mouse ears on surfaces with parallel grooves 10 micrometers apart and 3 micrometers high, in a special culture medium. This procedure increased the efficiency of reprogramming of these mature cells four-fold when compared to cells grown on a flat surface. Growing cells in scaffolds of nanofilbers aligned in parallel had similar effects.

Li’s study could significantly advance the protocols for making induced pluripotent stem cells (iPSCs). Normally iPSCs are made by genetically engineering adult cells so that they overexpress four different genes: Oct-4, Sox-2, Klf-4, and c-Myc. To put these genes into the cells, genetically modified viruses are used, or plasmids (small circles of DNA). Initially, Shinya Yamanaka, the scientist who invented iPSCs, and his co-workers used retroviruses that contained these four genes. When fibroblasts were infected with these souped-up retroviruses, the viruses inserted their viral DNA into the genomes of the host cells and expressed these genes.

retrovirus_life_cycle

Shinya Yamanaka won the Nobel Prize for this work in Physiology or Medicine in 2012 for this work. Unfortunately, retroviruses and can cause insertional mutations when they integrate into the genome (Zheng W., et al., Gene. 2013 Apr 25;519(1):142-9), and for this reason they are not the preferred way of making iPSCs. There are other viral vectors that do not integrate into the genome of the host cell (e.g., Sendai virus; see Chen IP, et al., Cell Reprogram. 2013 Dec;15(6):503-13). There are also techniques that use plasmids, which encode the four genes but do not integrate into the genome of the host cell. Finally, synthetic messenger RNAs that encode these four genes have also been used to make iPSCs (Tavernier G,, et al., Biomaterials. 2012 Jan;33(2):412-7).

The use of physical cues to make iPSCs may replace the need for gene overexpression, just as the use of particular chemicals can replace the need for particular genes (Zhu, S. et al. Cell Stem Cell 7, 651–655 (2010); Li, Y. et al. Cell Res. 21, 196–204 (2011)). If physical cues can replace the need for the overexpression of particular genes, then this discovery could revolutionize iPSC derivation; especially since the overexpression of particular genes in mature cells tends to cause genome instability in cells (Doris Steinemann, Gudrun Göhring, and Brigitte Schlegelberger. Am J Stem Cells. 2013; 2(1): 39–51).

“Our study demonstrates for the first time that the physical features of biomaterials can replace some of these biochemical factors and regulate the memory of a cell’s identity,” said study principal investigator Song Li, UC Berkeley, Professor of bioengineering. “We show that biophysical signals can be converted into intracellular chemical signals that coax cells to change.”

a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p<0.05 (two-tailed, unpaired t-test) compared with the control flat surface. Error bars represent one standard deviation. g, Immunostaining of a stable iPSC line expanded from colonies generated on 10 μm grooves. These cells express mESC-specific markers Oct4, Sox2, Nanog and SSEA-1 (scale bar, 100 μm). h, The expanded iPSCs in g were transplanted into SCID mice to demonstrate the formation of teratomas in vivo (scale bar, 50 μm).
a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p

To boost the efficiency of mature cell reprogramming, scientists also use a chemical called valproic acid, which dramatically affects global DNA structure and expression.

“The concern with current methods is the low efficiency at which cells actually reprogram and the unpredictable long-term effects of certain imposed genetic or chemical manipulations,” said the lead author of this study Timothy Downing. “For instance, valproic acid is a potent chemical that drastically alters the cell’s epigenetic state and can cause unintended changes inside the cell. Given this, many people have been looking at different ways to improve various aspects of the reprogramming process.”

This new study confirms and extends previous studies that showed that mechanical and physical cues can influence cell fate. Li’s group showed that physical and mechanical cues can not only affect cell fate, but also the epigenetic state and cell reprogramming.

a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p<0.05 (two-tailed, unpaired t-test) compared with the control surface with random nanofibres. e, Fibroblasts were micropatterned into single-cell islands of 2,000 μm2 area with a CSI value of 1 (round) or 0.1 (elongated). After 24 h, cells were immunostained for AcH3, H3K4me2 or H3K4me3 (in green). Phalloidin staining (red) identifies the cell cytoskeleton for cell shape accuracy. The white arrowhead indicates the location of the nucleus (scale bars, 20 μm). f, Quantification of fluorescence intensity in e (n = 34, 20 and 34 for AcH3, H3K4me2 and H3K4me3, respectively). *p<0.05 (two-tailed, unpaired t-test) compared with the circular micropatterned cells (CSI = 1). Error bars represent one standard deviation.
a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p

“Cells elongate, or example, as they migrate throughout the body,” said Downing, who is a research associate in Li’s lab. “In the case of topography, where we control the elongation of a cell by controlling the physical microenvironment, we are able to more closely mimic what a cell would experience in its native physiological environment. In this regard, these physical cues are less invasive and artificial to the cell and therefore less likely to cause unintended side effects.”

Li and his colleagues are studying whether growing cells on grooved surfaces eventually replace valproic acid and even replace other chemical compounds in the reprogramming process.

“We are also studying whether biophysical factors could help reprogram cells into specific cell types, such as neurons,” said Jennifer Soto, a UC Berkeley graduate student in bioengineering who was also a co-author on this paper.

Timothy Downing, et al., Nature Materials 12, 1154–1162 (2013).  

Stomach Cells Naturally Revert to Stem Cells


George Washington University scientists from St. Louis, Missouri have found that the stomach naturally produces more stem cells than previously realized. These stem cells probably repair stomach damage from infections, the foods we eat, and the constant tissue insults from stomach acid.

The reversion of adult cells to a stem cell fate is one of the goals of stem cell research. Shinya Yamanaka’s research group at the Center for iPS Cell Research and Application and the Institute for Frontier Medical Sciences at Kyoto University won the Nobel Prize in 2012 for his work on reprogramming adult cells into embryonic-like stem cells, otherwise known as induced pluripotent stem cells (iPSCs) that was initially published in 2006.

A collaborative research effort between scientists from Washington University School of Medicine in St. Louis and Utrecht Medical Center in the Netherlands have shown that this reversion from adult cells to stem cells occurs naturally in the stomach on a regular basis.

Jason Mills, associate professor of medicine at Washington University, said, “We already knew that these cells, which are called chief cells, can change back into stem cells to make temporary repairs in significant stomach injuries in significant stomach injuries, such as a cut to damage from infection. The fact that they’re making this transition more often, even in the absence of noticeable injuries, suggests that it may be easier than we realized to make some types of mature, specialized adult cells revert to stem cells.”

Chief cells normally produce a protein called pepsinogen. In the presence of stomach acid, pepsinogen activates itself and once active, the new protein product, pepsin, degrades proteins. Pepsin in an enzyme that is most active in the acidic environment of the stomach. Another enzyme released by chief cells is chymosin, which is also known as rennet. Chymosin curdles the proteins in milk and makes them easier to degrade.

PARIETAL AND CHIEF Cells

Mills and his groups are in the process of studying the transformation of chief cells into stem cells, for injury repair. Mills would also like to investigate the possibility that the potential for growth unleashed by this change may contribute to stomach cancers.

Mills and his collaborator Hans Clevers from the Netherlands have identified stomach stem cell marker proteins that show that chief cells become stem cells even in the absence of serious injury. In the case of serious injury, either in cell culture of in animal models, more chief cells become stem cells, making it possible to repair the damage in the stomach.

Forming Induced Pluripotent Stem Cells Inside a Living Organism


A team from the Spanish National Cancer Research Centre (CNIO) has become the first research team to convert adult cells that are still within a living organism into cells that show characteristics of embryonic stem cells.

The CNIO researchers also say that these embryonic stem cells, which were obtained directly from inside an organism, have a broader capacity for differentiation than those obtained by means of an in vitro culture system. Specifically, they have the characteristics of totipotent cells, a primitive state never before obtained in a laboratory, according to the CNIO team.

Manuel Serrano, Ph.D., director of CNIO’s Molecular Oncology Program and head of the Tumor Suppression Laboratory, led this study. It was supported by Manuel Manzanares, Ph.D., and his team from the Spanish National Cardiovascular Research Centre.

The CNIO researchers say their work extends that of Nobel Prize winner Shinya Yamanaka, M.D., Ph.D., one step forward. Yamanaka opened a new horizon in regenerative medicine when, in 2006, he demonstrated that stem cells could be created from adult cells by using a cocktail of genes. But while Yamanaka induced his cells in culture in the lab (in vitro), the CNIO team created theirs directly in mice (in vivo). Generating these cells within an organism brings this technology even closer to regenerative medicine, they say.

In a study published online Sept. 11 in the journal Nature, the CNIO research team details how it used genetic manipulation techniques to create mice in which Dr. Yamanaka’s four genes could be activated at will. When these genes were activated, they observed that the adult cells were able to de-differentiate into embryonic stem cells in multiple tissues and organs.

María Abad, Ph.D., lead author of the article and a researcher in Dr. Serrano’s group, said, “This change of direction in development has never been observed in nature. We have demonstrated that we can also obtain embryonic stem cells in adult organisms and not only in the laboratory.”

Dr. Serrano added, “We can now start to think about methods for inducing regeneration locally and in a transitory manner for a particular damaged tissue.” Stem cells obtained in mice also show totipotent characteristics never generated in a laboratory. Totipotent cells can form all the cell types in a body, including the placental cells. Embryonic cells within the first couple of cell divisions after fertilization are the only cells that are totipotent.

The researchers reported that they were also able to induce the formation of pseudo-embryonic structures in the thoracic and abdominal cavities of the mice. These pseudo-embryos displayed the three layers typical of embryos (ectoderm, mesoderm, and endoderm), and extra-embryonic structures such as the vitelline membrane, which surrounds the egg, and even signs of blood cell formation, which first appears in the primary embryonic vesicle (otherwise known as the “yolk sac”).

“This data tell us that our stem cells are much more versatile than Dr. Yamanaka’s in vitro inducted pluripotent stem cells, whose potency generates the different layers of the embryo but never tissues that sustain the development of a new embryo, like the placenta,” the CNIO researcher said.  Below is a figure from their paper.  The pictures look pretty convincing.

a, Cysts in the abdominal cavity of a reprogrammable mouse. b, Frequency of embryo-like structures after intraperitoneal injection of in vivo iPS cells (3 clones), in vitro iPS cells (2 clones) and ES cells (JM8.F6). Fisher’s exact test: *P < 0.05. c, Cyst generated by intraperitoneal injection. Left panels, germ layer markers: SOX2 (ectoderm), T/BRACHYURY (mesoderm) and GATA4 (endoderm). Right panels, extraembryonic markers: CDX2 (trophectoderm), and AFP and CK8, both specific for visceral endoderm of the yolk sac. d, Cyst generated by intraperitoneal injection presenting TER-119+ nucleated erythrocytes and LYVE-1+ endothelial cells in structures resembling yolk sac blood islands.
a, Cysts in the abdominal cavity of a reprogrammable mouse. b, Frequency of embryo-like structures after intraperitoneal injection of in vivo iPS cells (3 clones), in vitro iPS cells (2 clones) and ES cells (JM8.F6). Fisher’s exact test: *P < 0.05. c, Cyst generated by intraperitoneal injection. Left panels, germ layer markers: SOX2 (ectoderm), T/BRACHYURY (mesoderm) and GATA4 (endoderm). Right panels, extraembryonic markers: CDX2 (trophectoderm), and AFP and CK8, both specific for visceral endoderm of the yolk sac. d, Cyst generated by intraperitoneal injection presenting TER-119+ nucleated erythrocytes and LYVE-1+ endothelial cells in structures resembling yolk sac blood islands.

The researchers emphasize that any possible therapeutic applications of their work are still distant, but they believe that it could mean a change of direction for stem cell research, regenerative medicine and tissue engineering.

“Our stem cells also survive outside of mice in a culture, so we can also manipulate them in a laboratory,” said Dr. Abad. “The next step is studying if these new stem cells are capable of efficiently generating different tissues such as that of the pancreas, liver or kidney.”

This paper is very interesting, but I find it rather unlikely that their approach will take regenerative medicine by storm.  Engineering mice to express these four genes in an inducible manner caused the formation of unusual tumors throughout the mice.  Maybe they can be coaxed to differentiate into kidney or heart muscle or whatever, but learning how to get them to do that will take a fair amount of in vitro work.  This is interesting, but I doubt that it will change the field overnight.

Japanese first Ever Induced Pluripotent Stem Cell Clinical Trial Given the Green Light


The first clinical trial that utilizes induced pluripotent stem cells has been given a green light. For this clinical trial six patients who suffer from age-related macular degeneration will donate skin biopsies and the cells from these skin biopsies will be used to generate induced pluripotent stem (iPS) cells in the laboratory. After those iPS cell lines are screened for safety (normal numbers of chromosomes, no mutations in critical genes, etc.), they will be differentiated into retinal cells. The retinal cells will be transplanted into the retinas of these six patients.

This clinical trial was approved by Japan Health Minister Norihisa Tamura and it will be next summer by Masayo Takahashi. Dr. Takahashi is a retinal regeneration expert and a colleague of the man who first developed iPS cells, Shinya Yamanaka. Yamanaka won the Nobel Prize for his discovery of iPSCs last year. In fact, this clinical trial epitomizes, in the eyes of many, the determination of Japanese scientists and politicians to dominate the iPS cell field. This national ambition kicked into high gear after Yamanaka shared the Nobel Prize for Physiology or Medicine last October for his iPS cell work.

Norhisa Tamura, Japanese Minister of Health
Norihisa Tamura, Japanese Minister of Health
Masayo Takahashi, MD, PhD, Riken Center for Developmental Biology.
Masayo Takahashi, MD, PhD, Riken Center for Developmental Biology.

“If things continue this way, this will be the first in-clinic study in iPS cell technology,” says Doug Sipp of the Riken Center for Developmental Biology (CDB). The CDB, Takahashi’s institute, will co-run the trial with Kobe’s Institute for Biomedical Research and Innovation. “It’s exciting.”

Sipp, however, also noted that this move has not surprised anyone in Japan, since the Japanese stem cell community has heavily invested in iPS cells. Nevertheless, since Takahashi yet to formally publish the details of her trial, some have questioned whether she is actually ready to move forward. IPS cells are viewed as the perfect compromise for regenerative medicine. They are adult, and therefore do not require the destruction of human embryos for their establishment, and they are also pluripotent like an embryonic cell, which makes them relatively powerful sources for regenerative medicine.

Critics, however, warn that iPS cells were only discovered in 2007. To date, they remain difficult to create and culture and they can become tumorous in many hands. However, many labs have a great deal of expertise and skill when it comes to handling and deriving iPS cells. These labs derive and culture iPS cells routinely. In fact, Sipp notes that Riken’s CDB alone has produced world-class work with all kinds of stem cells, including embryonic stem (ES) cells, which are the models for iPS cells.

Additionally, Sipp and others point out that a scientist who has collaborated with Takahashi in the past, Riken’s Yoshiki Sasai, is doing groundbreaking work with ES cells and the eye. The British journal Nature has called Sasai “The Brainmaker,” and has said that his research is “wowing” the world.

The Japanese government has also soundly funded Takahashi’s trail. The health ministry’s recent stimulus plan set aside more money for stem cells (in particular iPS cells) than anything else. According to the journal Nature, the Japanese government sequestered 21.4 billion yen ($215 million) for stem cell research. Of this pot of money, the health ministry provided 700 million yen ($7 million) for a cell-processing center to support Takahashi before her trial was even approved. Two centers devoted to iPS cells are slated to be built with 2.2 billion yen ($22 million). The AFP reports the prime minister has set aside a breathtaking $1.18 billion, for iPS-cell work. Yamanaka has told Nature that the Japanese government seems to be “telling us to rush iPS cell-related technologies to patients as quickly as possible.”

Robert Lanza, CSO of Advanced Cell Technology, might once have been the logical bet to be first to the clinic with iPS cells. Unlike Takahashi, he has three ES cell trials under his belt, and has started talks with the FDA about transplanting iPS cell-derived platelets, but his iPS proposal is taking longer. Lanza bitterly noted, not without justification, “We don’t have the prime minister and emperor to speed things along for us.”

Since 2007, the year that Yamanaka reported the derivation of iPS cells from adult cells, Japan has focused on iPS cells. Yamanaka showed that increasing the expression of four genes could change limited adult human cells into potent, embryonic-like cells. “At Yamanaka’s institute alone, there are at least 20 teams focusing on iPS cells now,” Sipp says. There are teams at Riken, the Universities of Tokyo and Keio, and others. “A lot is happening here.” In fact, the Center for IPS Cell Research and Application was created expressly for Yamanaka.

Takahashi has reported part of the design of her clinical trial at scientific meetings. She told the International Society for Stem Cell Research in June 2012 she had created iPS-cell derived retinal pigment epithelial (RPE) cells for transplantation. RPE cells lie behind the photoreceptors in the retina, and the photoreceptors have their ends embedded into the RPE. The RPE cells replenish and nourish the photoreceptors, and without the RPE cells, the photoreceptors die from the damage incurred by exposure to light.

Retinal Pigmented Epithelium

Death of the RPE cells cause eventual death of photoreceptors and that results in blindness. At the International Society for Stem Cell Research conference, Takahashi reported her that her iPS cell-derived RPEs possess proper structure and gene expression. They also do not produce tumors when transplanted into mice, and survive at least six months when transplanted into the retinas of monkeys. The vision of these animals, however, was not tested. She did note that some AMD patients’ sight improves when RPE cells are moved from the eye’s periphery to its center.

Retinal pigment epithelial cells derived from iPS cells.
Retinal pigment epithelial cells derived from iPS cells.

Takahashi has published many iPS and ES cell papers. These papers include two papers with Yamanaka: one on creating retinal cells from iPS cells, and one on creating safe iPS cells. However she has not published trial details, which is not required, but such a landmark trial should be transparent, as argued by many stem cell experts.

Still, according to Sipp, Takahashi has submitted a relevant paper to a top journal for review, which shows that this clinical trial is purely a determination of the safety of the procedure. Lanza has reported his trials in the journal The Lancet, and similar, but small, trials are doing well. His three ES cell trials treated Stargardt’s macular dystrophy and Age-related Macular Degeneration. Lanza’s trial, however, treated “dry” macular degeneration, while Takahashi’s trial will treat “wet” Age-related Macular Degeneration, which is good news for Takahashi.

Paul Knoepfler, a UC Davis stem cell scientist who runs a widely read blog site, has written that the ministry overseeing Takahashi’s trial will reportedly monitor some key factors: gene sequencing and tumorigenicity. But Knoepfler, like others, would like to see more details.

The Japanese Health Ministry and the US FDA recently agreed to devise a joint regulatory framework for retinal iPS cell clinical trials, which will come on line 2015. Takahashi’s trial is set for 2014.

Increased Flexibility in Induced Pluripotent Stem Cell Derivation Might Solve Tumor Concerns


Regenerative medicine depends on stem cells for the promises that it can potentially deliver to ailing patients. Training stem cells to repair injured tissues with custom-grown tissue substitutes and to replace dead cells are some of the goals of regenerative medicine. A major player in regenerative medicine is induced pluripotent stem cells (iPSCs), which are made from a patient’s own tissues. Because iPSCs are derived from a patient’s own cells, their chance of being rejected by the patient’s own immune system is rather low. Unfortunately, Shinya Yamanaka’s formula for making iPSCs, for which he was awarded last year’s Nobel Prize, utilizes a strict recipe that uses a precise combination of genes, some of which increase the risk of cancer risk, and, therefore, restricts their full potential for clinical application.

From left: Emmanuel Nivet and Juan Carlos Belmonte. Seated: Ignacio Sancho Martinez. (Source: Salk Institute for Biological Studies)
From left: Emmanuel Nivet and Juan Carlos Belmonte. Seated: Ignacio Sancho Martinez. (Source: Salk Institute for Biological Studies)

However, the laboratory Juan Carlos Izpisua Belmonte and his colleagues at the Salk Institute have published a paper in the journal Cell Stem Cell that shows that the recipe for iPSCs is much more versatile than originally thought. For the first time, Izpisua Belmonte and his colleague have replaced a gene that was once thought to be impossible to substitute in the production of iPSCs. This creates the potential for more flexible recipes that should speed the adoption of iPSCs for stem cell-based therapies.

Pluripotent stem cells come from two main sources. Embryonic stem cells (ESCs) are derived from early human blastocyst-stage embryos. The cells of the inner cell mass are extracted and these immature cells that have never differentiated into specific cell types, and are cultured, grown, and propagated to form an embryonic stem cell line. Secondly, induced pluripotent stem cells or iPSCs, are derived from mature cells that have been reprogrammed back into an undifferentiated state. In 2006 by Yamanaka introduced four different genes into a mature cell to reprogram the cell to pluripotency. This pluripotent cell can be cultured and grown into an iPSCs line. Because of Yamanaka’s initial success in iPSC production, most stem cell researchers adopted his recipe, even though variations on his protocol have been examined and used.

Izpisua Belmonte and his colleagues used a fresh approach for the derivation of iPSCs. They played around with the Yamanka protocol and in doing do discovered that pluripotency (the stem cell’s ability to differentiate into nearly any kind of adult cell) can also be programmed into adult cells by “balancing” the genes required for differentiation. What genes? Those genes that code for “lineage transcription factors,” which are proteins that direct stem cells to differentiate first into a particular cell lineage, or type, such as a blood cell versus a skin cell, and then finally into a specific cell, such as a white blood cell.

“Prior to this series of experiments, most researchers in the field started from the premise that they were trying to impose an ’embryonic-like’ state on mature cells,” says Izpisua Belmonte, who holds the Institute’s Roger Guillemin Chair. “Accordingly, major efforts had focused on the identification of factors that are typical of naturally occurring embryonic stem cells, which would allow or further enhance reprogramming.”

Despite these efforts, there seemed to be no way to determine through genetic identity alone that cells were pluripotent. Instead, pluripotency was routinely evaluated by functional assays. In other words, if it acts like a stem cell, it must be a stem cell.

That condition led the team to their key insight. “Pluripotency does not seem to represent a discrete cellular entity but rather a functional state elicited by a balance between opposite differentiation forces,” says Izpisua Belmonte.

Once they understood this, they realized the four extra genes weren’t necessary for pluripotency. Instead, adult cells could be reprogrammed by altering the balance of “lineage specifiers,” genes that were already in the cell that specified what type of adult tissue a cell might become.

“One of the implications of our findings is that stem cell identity is actually not fixed but rather an equilibrium that can be achieved by multiple different combinations of factors that are not necessarily typical of ESCs,” says Ignacio Sancho-Martinez, one of the first authors of the paper and a postdoctoral researcher in Izpisua Belmonte’s laboratory.

Izpisua Belmonte’s laboratory showed that more than seven additional genes can facilitate reprogramming adult cells to iPSCs. Most importantly, for the first time in human cells, they were able to replace a gene from the original recipe called Oct4, which had been replaced in mouse cells, but was still thought indispensable for the reprogramming of human cells. Their ability to replace it, as well as SOX2, another gene once thought essential that had never been replaced in combination with Oct4, demonstrated that stem cell development must be viewed in an entirely new way. In point of fact, Belmonte’s group showed that genes that specify mesendodermal lineage can replace OCT4 in human iPSC generation, and ectodermal lineage specifiers are able to replace SOX2 in hiPSC generation. Simultaneous replacement of OCT4 and SOX2 allows human cell reprogramming to iPSCs

“It was generally assumed that development led to cell/tissue specification by ‘opening’ certain differentiation doors,” says Emmanuel Nivet, a post-doctoral researcher in Izpisua Belmonte’s laboratory and co-first author of the paper, along with Sancho-Martinez and Nuria Montserrat of the Center for Regenerative Medicine in Barcelona, Spain.

Instead, the successful substitution of both Oct4 and SOX2 shows the opposite. “Pluripotency is like a room with all doors open, in which differentiation is accomplished by ‘closing’ doors,” Nivet says. “Inversely, reprogramming to pluripotency is accomplished by opening doors.”

This work should help to overcome one of the major hurdles in the widespread adoption of iPSC-based therapies; namely, that the original four genes used to reprogram stem cells had been implicated in cancer. “Recent studies in cancer, many of them done by my Salk colleagues, have shown molecular similarities between the proliferation of stem cells and cancer cells, so it is not surprising that oncogenes [genes linked to cancer] would be part of the iPSC recipe,” says Izpisua Belmonte.

With this new method, which allows for a customized recipe, the team hopes to push therapeutic research forward. “Since we have shown that it is possible to replace genes thought essential for reprogramming with several different genes that have not been previously involved in tumorigenesis, it is our hope that this study will enable iPSC research to more quickly translate into the clinic,” says Izpisua Belmonte.

Other researchers on the study were Tomoaki Hishida, Sachin Kumar, Yuriko Hishida, Yun Xia and Concepcion Rodriguez Esteban of the Salk Institute; Laia Miquel and Carme Cortina of the Center of Regenerative Medicine in Barcelona, Spain.

Stem Cells Made from the Brains of Cadavers


Shinya Yamanaka won the Nobel Prize this year for his discovery that the application of genetic engineering to adult cells can revert them into embryonic-like stem cells. Such cells, known as induced pluripotent stem cells (iPSCs) are made from adult cells when four different genes are transiently expressed in an adult cell. These four particular genes are all transcription factors that bind to DNA and activate the transcription of particular genes that are necessary for the acquisition of the embryonic state. Once they are derived, iPSCs can grow in culture and differentiate into any adult cell type, although the efficiency with which they do this is very cell line-dependent.

The discovery of iPSCs gave new hope to the notion that patient-specific stem cells could be used to treat genetic diseases. However, a research group has actually managed to extract live cells from dead bodies and use those cells to derive iPSC lines.

Fibroblasts are cells found in connective tissue, and they are very common in the skin and brain. Fibroblasts can be collected from cadavers and subjected to a protocol that will convert them into iPSCs. These reprogrammed stem cells can differentiate into a multitude of cell types, including the neurons found in the brain and spinal cord. Because microorganisms can colonize the body and degrade it after death, such a culturing process is tricky to carry out successfully.

Scientists from the laboratory of Thomas Hyde at the Lieber Institute for Brain Development, Johns Hopkins Medical Campus in Baltimore, Maryland have used fibroblasts from scalps and the linings that surround the brain (dura later) from 146 human brain donors and used them lake iPSCs.

Those cells extracted from the dura mater were 16 times more likely to grow successfully than those from the scalp. Hyde explained that he expected this disparity in growth potential since the scalp is prone contamination with bacterial and fungi after death. Such contaminants can inhibit the growth of fibroblasts in the laboratory.

However, scalp cells grew more rapidly than dura mater cells. “Since the skin is constantly renewing, while the turnover in dura mater is much slower,” such a result makes sense, explained Hyde.

The derivation of iPSCs from cadavers might play an important role in developing future stem cell therapies. Cadavers can provide brain, heart and other tissues for study that researchers cannot safely obtain from living people. The derivation of iPSCs from cadavers provides scientists with an excellent source of material for research that cannot be obtained elsewhere. As noted in the paper, “These tissues may be accessible through existing brain tissue collections, which is critical for studying disorders such as neuropsychiatric diseases.”

“For instance, we can compare neurons derived from fibroblasts with actual neurons from the same individual,” Hyde said. “It tells us about how reliable a given method for deriving neurons from fibroblasts is. That can be crucial if, for example, you want to create dopamine-making neurons to treat someone with Parkinson’s disease.”

Also using iPSCs made from patients who died from developmental abnormalities can greatly enlighten scientists on those maladies that are due to malfunctions in development.

“We’re very interested in major neuropsychiatric disorders such as schizophrenia, bipolar disease, autism and mental retardation,” Hyde said. “By understanding what goes wrong with the brain cells in these individuals, we could perhaps help fix that.”

Citation: Bliss LA, Sams MR, Deep-Soboslay A, Ren-Patterson R, Jaffe AE, et al. (2012) Use of Postmortem Human Dura Mater and Scalp for Deriving Human Fibroblast Cultures. PLoS ONE 7(9): e45282. doi:10.1371/journal.pone.0045282

Induced Pluripotent Stem Cells


Embryonic stem cells might provide the means to heal a variety of physical ailments. However the problem with embryonic stem cells is not necessarily in their use, but in their derivation. In order to make embryonic stem cell lines, human embryos are destroyed.

The following video shows Alice Chen from Doug Melton’s laboratory at Harvard University destroying embryos to make embryonic stem cells:  http://www.jove.com/index/details.stp?ID=574.

Now that federal funding is available to not only work with existing embryonic stem cell lines but to MAKE new lines, there is nothing to stop researchers from thawing and (I’m sorry to be so blunt) killing human embryos. Can we have our “cake and eat it too?” Can we have the benefits of embryonic stem cells and not destroy embryos? Perhaps we can.

In 2001, Masako Tada reported the fusion of embryonic stem cells with a connective tissue cell called a fibroblast. This fusion reprograms the fibroblasts so that they behave like embryonic stem cells (Current Biology 11, no. 9 (2001): 1553–8). This suggests that something within embryonic stem cells can redirect the machinery of somatic cells to become more like that of embryonic stem cells. In 2006 Kazutoshi Takahashi and Shinya Yamanaka were able to generate embryonic stem cell lines by introducing four specific genes into mouse skin fibroblasts. These “induced pluripotent stem cells” (iPSCs) shared many of the properties of embryonic stem cells derived from embryos, but when transplanted into mouse embryos, they were not able to participate in the formation of an adult mouse (Cell 126, no. 4 (2006): 663–76). This experiment showed that it is possible to convert adult cells into something that resembles an embryonic stem cell. Could we push adult cells further? In 2007, three different research groups used retroviruses to transfer four different genes (Oct3/4, Sox2, c-Myc and Klf4) into mouse skin fibroblasts and completely transformed them into cells that had all the features and behaviors of embryonic stem cells (Cell Stem Cell 1, no. 1 (2007): 55–70; Nature 448 (2007): 313–7; Nature 448 (2007): 318–24.).

These experiments drew a great deal of excitement, but there were several safety concerns that had to be addressed before iPSCs could be used in human clinical trials.  Scientists used engineered retroviruses to introduce genes into adult cells in order to reprogram them into iPSCs (Current Topics in Microbiology and Immunology 261 (2002): 31-52).  Retroviruses insert a DNA copy of their genome into the chromosomes of the host cell they have infected.  If that viral DNA inserts into a gene, it can disrupt it and cause a mutation.  This can have dire consequences (see Folia Biologia 46 (2000): 226-32; Science 302 (2003): 415-9).  Fortunately this is not an intractable problem.  The conversion of adult cells into iPSCs only requires the transient expression of the inserted genes.  Secondly, scientists have created retroviruses that self-inactivate after their initial insertion (Journal of Virology 72 (1998): 8150-7; Virology 261, (1999).  One laboratory has also discovered a way to make iPSCs with a virus that does not insert into host cell chromosomes (Science 322 (2008): 945-9).  Other researchers have designed ingenious ways to move the necessary genes into adult cells without using viruses (Science 322 (2008): 949-53).  Both procedures avoid the dangers associated with the use of retroviruses.

A second concern involves the genes used to convert re-program adult cells into iPSCs.  One of these genes, c-Myc, is found in multiple copies in human and animal tumors.  Thus increasing the number of copies of the c-Myc gene might predispose such cells to form tumors (Recent Patents on Anticancer Drug Discovery 1 (2006): 305-26; Seminars in Cancer Biology 16 (2006): 318-30). Indeed, the increased ability of iPSCs made by Yamanaka to cause tumors in laboratory animals underscore this concern (Hepatology 46, no 3 (2009): 1049-9).  Several groups, however, have succeeded in making iPSCs from adult cells without the use of the c-Myc gene (Science 321 (2008): 699­-702; Nature Biotechnology 26 (2008): 101-6; Science 318 (2007): 1917–20), although the conversion is much less efficient.  Additionally, several groups have established that particular chemicals, in combination with the addition of a subset of the four genes originally used, can effectively transform particular cells into iPSCs (Cell Stem Cell 2 (2008): 525-8).   Thus the larger safety concerns facing iPSCs have been largely solved.

Finally, patient-specific iPSCs have been made in several labs, even though they have not been used in clinical trials to date.  Here is a short list of some of the diseases for which patient-specific iPSCs have been made:

Amylotrophic Lateral SclerosisScience 321 (2008): 1218­21.

Spinal Muscular AtrophyNature 457 (2009): 277­81.

Parkinson’ DiseaseCell 136, no. 5 (2009): 964­77.

Adenosine deaminase deficiency-related severe combined immunodeficiency – Cell 134, no. 5 (2008): 877­86.

Shwachman-Bodian-Diamond syndrome – Cell 134, no. 5 (2008): 877­86.

Gaucher disease – Cell 134, no. 5 (2008): 877­86.

Duchenne and Becker muscular dystrophy – Cell 134, no. 5 (2008): 877­86.

Huntington disease – Cell 134, no. 5 (2008): 877­86.

Juvenile-onset type 1 diabetes mellitus – Cell 134, no. 5 (2008): 877­86.

Down syndrome – Cell 134, no. 5 (2008): 877­86.

Lesch-Nyhan syndromeCell 134, no. 5 (2008): 877­86.

Thus iPSCs represent an exciting, embryo-free alternative to embryonic stem cells that provide essentially all of the opportunities for regenerative medicine without destroying embryos.