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.


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Producing blood cells from stem cells could yield a purer, safer cell therapy

The journal Stem Cells Translational Medicine has published a new protocol for reprogramming induced pluripotent stem cells (iPSCs) into mature blood cells. This protocol uses only a small amount of the patient’s own blood and a readily available cell type. This novel method skips the generally accepted process of mixing iPSCs with either mouse or human stromal cells. Therefore, is ensures that no outside viruses or exogenous DNA contaminates the reprogrammed cells. Such a protocol could lead to a purer, safer therapeutic grade of stem cells for use in regenerative medicine.

The potential for the field of regenerative medicine has been greatly advanced by the discovery of iPSCs. These cells allow for the production of patient-specific iPSCs from the individual for potential autologous treatment, or treatment that uses the patient’s own cells. Such a strategy avoids the possibility of rejection and numerous other harmful side effects.

CD34+ cells are found in bone marrow and are involved with the production of new red and white blood cells. However, collecting enough CD34+ cells from a patient to produce enough blood for therapeutic purposes usually requires a large volume of blood from the patient. However, a new study outlined But scientists found a way around this, as outlined by Yuet Wai Kan, M.D., FRS, and Lin Ye, Ph.D. from the Department of Medicine and Institute for Human Genetic, University of California-San Francisco has devised a way around this impasse.

“We used Sendai viral vectors to generate iPSCs efficiently from adult mobilized CD34+ and peripheral blood mononuclear cells (MNCs),” Dr. Kan explained. “Sendai virus is an RNA virus that carries no risk of altering the host genome, so is considered an efficient solution for generating safe iPSC.”

“Just 2 milliliters of blood yielded iPS cells from which hematopoietic stem and progenitor cells could be generated. These cells could contain up to 40 percent CD34+ cells, of which approximately 25 percent were the type of precursors that could be differentiated into mature blood cells. These interesting findings reveal a protocol for the generation iPSCs using a readily available cell type,” Dr. Ye added. “We also found that MNCs can be efficiently reprogrammed into iPSCs as readily as CD34+ cells. Furthermore, these MNCs derived iPSCs can be terminally differentiated into mature blood cells.”

“This method, which uses only a small blood sample, may represent an option for generating iPSCs that maintains their genomic integrity,” said Anthony Atala, MD, Editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “The fact that these cells were differentiated into mature blood cells suggests their use in blood diseases.”

A More Efficient Way to Make Human Induced Pluripotent Stem Cells

Stem cell researchers at the University of California, San Diego have designed a simple, reproducible, RNA-based method of generating human induced pluripotent stem cells (iPSCs). This new technique broad applications for the successful production of iPSCs for use in therapies and human stem cell studies.

Human iPSCs are made from adult cells by genetically engineering adult cells to overexpress four different genes (Oct4, Klf4, Sox2, and c-Myc). This overexpression drives the cells to de-differentiate into pluripotent stem cells that have many of the same characteristics as embryonic stem cells, which are made from embryos. However, because iPSCs are made from the patient’s own cells, the chances that the immune system of the patient will reject the implanted cells is low.

The problem comes with the overexpression of these four genes. Initially, retroviruses have been used to reprogram the adult cells. Unfortunately, retroviruses plop their DNA right into the genome of the host cell, and this change is permanent. If these genes get stuck in the middle of another gene, then that cell has suffered a mutation. Secondly, if these genes are stuck near another highly-expressed gene, then they too might be highly expressed, thus driving the cells to divide uncontrollably.

Several studies have shown that in order to reprogram these cells, these four genes only need to be overexpressed transiently. Therefore, laboratories have developed ways of reprogramming adult cells that do not use retroviruses. Plasmid-based systems have been used, adenovirus and Sendai virus-based systems, which do not integrate into the genome of the host cell, have also been used, and even RNA has been used (see Federico González, Stéphanie Boué & Juan Carlos Izpisúa Belmonte, Nature Reviews Genetics 12, 231-242).

The UC San Diego team led by Steven Dowdy has used Venezuelan equine virus (VEE) that they engineered to express the reprogramming genes required to make iPSCs from adult cells. Because this virus does not integrate into the host genome, and expresses RNA in the host cell only transiently, it seems to be a safe and effective way to make buckets of messenger RNA over a short period of time.

The results were impressive. The use of this souped-up VEE produced good-quality iPSCs very efficiently. Furthermore, it worked on old and young human cells, which is important, since those patients who will need regenerative medicine are more likely to be young patients than old patients. Also, changing the reprogramming factors is rather easy to do as well.

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.

A New Automated Protocol to Prepare and Purify Induced Pluripotent Stem Cell Lines

Induced pluripotent stem cells (iPSCs) come from adult cells and not embryos. By genetically engineering adult cells to express a cadre of genes that are normally found in early embryonic cells, scientists can de-differentiate the adult cells into cells that resemble embryonic stem cells in many (although not all) ways.

Generating iPSCs from human adult cells is tedious and not terribly efficient, but there are ways to increase the efficiency of iPSC generation (see here). Additionally, iPSCs can show a substantial tendency to form tumors, but this tendency is cell line-specific (see here and here). Furthermore, there are ways to screen iPSC lines for tumorgenicity.

Because iPSCs are directly from the patient’s cells, the chances of rejection by the immune system are less likely (see here). Therefore, many stem cells scientists believe that iPSCs may represent one of the best future possibilities for regenerative medicine. However, a hurdle in iPSC development is the ability to generate and evaluation iPSC lines in a rapid, but reliable manner. Once adult cells are induced to become iPSCs, the iPSC cultures are a mixed bag of iPSCs, undifferentiated adult cells that failed to make the transition to iPSCs, and partially reprogrammed cells. Selecting the iPSCs by merely eye-balling the cells through the microscope is tricky and fraught with errors. If the scientist wants to select iPSCs for toxicity studies and not partially differentiated cells, selecting the wrong cells for the experiment can be fatal to the experiment itself.

Scientists from the New York Stem Cell Foundation (NYSCF) Research Institute have developed a protocol for iPSC generation and evaluation is automated and efficient, and may bring us closer to the goal of using iPSCs in the clinic some day. This protocol is the culmination of three and a half years of work. This protocol uses a technology called “fluorescence activated cell sorting” or FACS to identify fully reprogrammed cells. FACS sorts the cells according to their expression of two specific cell surface molecules and the absence of another cell surface molecule. This negative selection for a cell surface molecule found in partially reprogrammed cells but not iPSCs is a very powerful technique for purifying iPSCs.

David Kahler, the NYSCF director of laboratory automation, said, “To date, this protocol has enabled our group to derive (and characterize over) 228 individual iPS cell lines, representing one of the largest collections derived in a single lab.” Kahler continued: “This standardized method means that these iPS cells can be compared to one another, an essential step for the use in drug screens and the development of cell therapies.”

This particular cell selection technique provides the basis for a new technology developed by NYSCF, the Global Stem Cell Array, which is a fully automated, robotic platform to generate cell lines in parallel.

Underway at the NYSCF Laboratory, the Array reprograms thousands of adult cells from kin and blood samples taken from healthy donors and diseased patients into iPSC lines. Sorting and characterizing cells at an early stage of reprogramming allows efficient development of iPSC clones and derivation of adult cell types.

“We are excited about the promise this protocol holds to the field. As stem cells move towards the clinic, Kahler’s work is a critical step to ensure safe, effective treatments for everyone.” said Susan L. Solomon, who is the Chief Executive Officer of NYSCF.

Increasing Reprogramming Efficiency by Turning Off One Gene

The removal of one genetic roadblock could improve the efficiency of adult cell reprogramming by some 10 to 30 fold, according to research by stem cell scientists at the Methodist Hospital Research Institute and two other institutions.

Rongfu Wang, the principal investigator and director of the Center for Inflammation and Epigenetics, said this about his group’s findings: “The discovery six years ago that scientist can convert adult cells into inducible pluripotent stem cells, or iPSCs, bolstered the dream that a patient’s own cells might be reprogrammed to make patient-specific iPSCs for regenerative medicine, modeling human diseases in Petri dishes, and drug screening. But reprogramming efficiency has remained very low, impeding its applications in the clinic.”

Wang and his group identified a protein encoded by a gene called Jmjd3, which is known as KDM6B, acts as an impediment to the reprogramming of adult cells into iPSCs. Jmjd3 is involved in several different biological processes, including the maturation of nerve cells and immune cell differentiation (Popov N, Gil J. Epigenetics. 2010 5(8):685-90).

These findings by Wang’s team are the first time anyone has identified a role for Jmjd3 in the reprogramming process. According to Wang, fibroblasts that lack functional Jmjd3 showed greatly enhanced reprogramming efficiency.

Helen Wang, one of the co-principal authors of this study, said, “Our findings demonstrate a previously unrecognized role of Jmjd3 in cellular reprogramming and provide molecular insight into the mechanisms by which the Jmjd3-PHF20 axis controls this process.’

While teasing apart the roles of Jmjd3 in reprogramming, Wang and his colleagues discovered that this protein regulates cell growth and cellular aging. These are two previously unidentified functions of Jmjd3, and Jmjd3 appears to work primarily by inactivating the protein PHF20. PHF20 is a protein that is required for adult cell reprogramming, and cells that lack PHF20 do not undergo reprogramming to iPSCs.

Rongfu Want explained it like this: “So when it comes to increasing iPSC yields, knocking down Jmjd3 is like hitting two birds with one stone.”

Jmjd3 is almost certainly not the only genetic roadblock to stem cell conversion. Wang noted, “Removal of multiple roadblacks could further enhance the reprogramming efficiency with which researchers can efficiently generate patient-specific iPSCs for clinical applications.”

While this is certainly an exciting finding, there is almost certainly a caveat that comes with it. increased reprogramming efficiency almost certainly brings the potential for increased numbers of mutations. Other studies have shown that iPSC generation is much more efficient if the protein P53 is inhibited, but P53 is the guardian of the genome. It prevents the cell from dividing if there is substantial amounts of DNA damage. Inhibiting P53 activity allows iPSC generation even if the cells have excessive amounts of DNA damage. Therefore, inhibiting those cellular processes that are meant to guard against excessive cell proliferation and growth can lead to greater numbers of mutations. Thus, before Jmjd3 inactivation is used to generate iPSCs for clinical uses, extensive animal testing must be required to ensure that this procedure does make iPSCs even less safe than they already are.