Repopulation of Damaged Livers With Skin-Derived Stem Cells


Patients with severe liver disease must receive a liver transplant. This major procedure requires that the patient survives major surgery and then takes anti-rejection drugs for the rest of their lives. In general, liver transplant patients tend to fair pretty well. The one-year survival rate of liver transplant patients approaches 90% (see O’Mahony and Goss, Texas Heart Institute Journal 2012 39(6): 874-875).

A potentially better way to treat liver failure patients would be to take their own liver cells, convert them into induced pluripotent stem cells (iPSCs), differentiate them into liver cells, and use these liver cells to regenerate the patient’s liver. Such a treatment would contain a patient’s own liver cells and would not require anti-rejection drugs.

Induced pluripotent stem cells or iPSCs are made from genetically-engineered adult cells that have had four specific genes (Oct4, Klf4, Sox2, and c-Myc) introduced into them. As a result of the heightened expression of these genes, some of the adult cells dedifferentiate and are reprogrammed into cells that resemble embryonic stem cells. Normally, this procedure is relatively inefficient, slow, and induces new mutations into the engineered cells. Also, when iPSCs are differentiated into liver cells (hepatocytes), they do not adequately proliferate after differentiation, and they also fail to properly function the way adult hepatocytes do.

New work from laboratories at the University of California, San Francisco (UCSF), has differentiated human hepatocytes by means of a modified technique that bypasses the pluripotency stage. These cells were then used to repopulate mouse livers.

“I really like this paper. It’s a step forward in the field,” said Alejandro Soto-Gutiérrez, assistant professor of pathology at the University of Pittsburgh, who was not involved in the work. “The concept is reprogramming, but with a shortcut, which is really cool.”

Research teams led by Holger Willenbring and Sheng Ding isolated human skin cells called fibroblasts and infected them with engineered viruses that forced the expression of three genes: OCT4, SOX2, and KLF4. These transduced cells were grown in culture in the presence of proteins called growth factors and small molecules in order to induce reprogramming of the cells into the primary embryonic germ layer known as endoderm. In the embryo, the endoderm is the inner-most layer of cells that forms the gastrointestinal tract and its associated structures (liver, pancreas, and so on). Therefore, the differentiation of adult cells into endodermal progenitor cells provides a handy way to form a cell type that readily divides and can differentiate into liver cells.

“We divert the cells on their path to pluripotency,” explained coauthor Holger Willenbring, associate professor of surgery at UCSF. “We still take advantage of what is intrinsic to reprogramming, that the cells are becoming very plastic; they’ve become flexible in what kind of cell type they can be directed towards.”

The authors called these cells induced multipotent progenitor cells (iMPCs). The iMPCs were easily differentiated into endodermal progenitor cells (iMPC-EPCs). These iMPC-EPCs were grown in culture with a cocktail of small molecules and growth factors to increase iMPC-EPC colony size while concomitantly maintain them in an endodermal state. Afterwards, Willenbring and others cultured these cells with factors and small molecules known to promote liver cell differentiation. When these iMPC-Hepatocytes (Heps) were transplanted into mice with damaged livers, the iMPC-Hep cells continued to divide at least nine months after transplantation. Furthermore, the transplanted cells matured and displayed gene expression profiles very similar to that of typical adult hepatocytes. Transplantation of iMPC-Heps also improved the survival of a mouse model of chronic liver failure about as well as did transplantation of adult hepatocytes.

“It is a breakthrough for us because it’s the first time that we’ve seen a cell that can actually repopulate a mouse’s liver,” said Willenbring. Willenbring strongly suspects that iMPCs are better able to repopulate the liver because the derivation of iMPC—rather than an iPSC—eliminates some steps along the path to generating hepatocytes. These iMPCs also possess the ability to proliferate in culture to generate sufficient quantities of cells for therapeutic purposes and, additionally, can functionally mature while retaining that proliferative ability to proliferate. Both of these features are important prerequisites for therapeutic applications, according to Willenbring.

Before this technique can enter clinical trials, more work must be done. For example: “The key to all of this is trying to generate cells that are identical to adult liver cells,” said Stephen Duncan, a professor of cell biology at Medical College of Wisconsin, who was not involved in the study. “You really need these cells to take on all of the functions of a normal liver cell.” Duncan explained that liver cells taken directly from a human adult might be able to repopulate the liver in this same mouse model at levels close to 90 percent.

Willenbring and his colleagues observed repopulation levels of 2 percent by iMPC-Heps, which is substantially better than the 0.05 percent repopulation typically accomplished by hepatocytes derived from iPSCs or embryonic stem cells. However: “As good as this is, the field will need greater levels of expansion,” said Ken Zaret of the Institute for Regenerative Medicine at the University of Pennsylvania, who did not participate in the work. “But the question is: What is limiting the proliferative capacity of the cells?”

Zaret explained that it is not yet clear whether some aspect of how the cells were programmed that differed from how they normally develop could have an impact on how well the population expands after transplantation. “There still is a ways to go [sic],” he said, “but [the authors] were able to show much better long-term repopulation with human cells in the mouse model than other groups have.”

See S. Zhu et al., “Mouse liver repopulation with hepatocytes generated from human fibroblasts,” Nature, doi:10.1038/nature13020, 2014.

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).  

100% Reprogramming Rates


For the first time, stem cell scientists have reprogrammed cultured skin cells into induced pluripotent cells (iPSCs) with near-perfect efficiency.

Even several laboratories have examined protocols to increase the efficiency of cellular reprogramming, a research team at the Weizmann Institute of Science in Rehovot, Israel has managed to increase the conversion rate to almost 100%, ten times the rate normally achieved, by removing a single proteins called Mbd3. This discovery can potentially allow scientists to generate large volumes of stem cells on demand, which would accelerate the development of new treatments.

In 2006, scientists from the laboratory of Shinya Yamanaka showed that mature cells could be reprogrammed to act like embryonic stem cells (ESCs). These reprogrammed adult cells could grow in culture indefinitely and differentiate into any type of cell in the body. However the creation of iPSc lines was notoriously inefficient and labor-intensive. Low cell-conversion rates have slowed the study of the reprogramming process itself. It has also discouraged the development of protocols for producing iPSCs under GMP or “Good Manufacturing Practice” conditions for use in human patients.

However, in a series of experiments that were published in the journal Nature, Weizmann Institute stem-cell researcher Jacob Hanna and his team have reprogrammed cells with nearly 100% efficiency. Moreover, Hanna and his group showed that reprogrammed cells transition to pluripotency on a synchronized schedule.

“This is the first report showing that you can make reprogramming as efficient as anyone was hoping for,” says Konrad Hochedlinger, a stem-cell scientist at Harvard Medical School in Boston, Massachusetts. “It is really surprising that manipulating a single molecule is sufficient to make this switch, and make essentially every single cell pluripotent within a week.”

To make iPSCs from adult cells, scientists typically transfect them with a set of four genes. These genes turn on the cells’ own pluripotency program, which converts them into iPSCs. But even established techniques convert less than 1% of cultured cells. Many cells get stuck in a partially reprogrammed state, and some become pluripotent faster than others, which makes the whole reprogramming process difficult to monitor.

Hanna and his team investigated the potential roadblocks to reprogramming by working with a line of genetically-engineered mouse cells. In these cells, the reprogramming genes were already inserted into the genomes of the cells and could be activated with a small molecule. Such cells normally reprogram at rates below 10%. But when a gene responsible for producing the protein Mbd3 was repressed, reprogramming rates soared to nearly 100%.

Hanna says that the precise timing of embryonic development led him to wonder whether it is possible to “reprogram the reprogramming process.” Cells in an embryo do not remain pluripotent indefinitely, explained Hanna. Usually, Mbd3 represses the pluripotency program as an embryo develops, and mature cells maintain their expression of Mbd3. However, during cellular reprogramming, those proteins expressed from the inserted pluripotency genes induce Mbd3 to repress the cells’ own pluripotency genes.

This hamstrings reprogramming, says Hanna. “It creates a clash, and that’s why the process is random and stochastic. It’s trying to have the gas and brakes on at the same time.” Depleting the cells of Mbd3 allows reprogramming to proceed unhindered.

The team also reprogrammed cells from a human, using a method that does not require inserting extra genes. This technique usually requires daily doses of RNA over more than two weeks. With Mbd3 repressed, only two doses were required.

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.

Making Induced Pluripotent Stem Cells With Small Molecules


A Journal article in the August 9th edition of Science Magazine features work from the laboratories of Yang Zhao and Hongkui Deng, both of whom are from the College of Life Sciences and Peking-Tsinghua Center for Life Sciences at Peking University in Beijing, China. Zhao and Deng and colleagues used small molecules to transform adult cells into induced pluripotent stem cells.

To review, induced pluripotent stem cells are derived from adult cells by genetically engineering the adult cells to express a cocktail of four genes (OCT4, Klf4, Sox2, and c-Myc). To introduce these genes into cells, viruses are normally used, but other techniques are also available. The resultant cells look and act like embryonic stem cells, but they do not require the death of embryos.

In this paper, Deng and colleagues took mouse embryonic fibroblasts (skin cells cultured from mouse embryos) and used them to screen over 10,000 small molecules for their ability to substitute for the OCT4 gene in the production of iPSCs. If this sounds labor intensive, that’s because it is. To conduct the screen, they used mouse embryonic fibroblasts that were infected with viruses that expressed Sox2, Klf4, and c-Myc. These genes are not enough to convert adult cells into iPSCs. However, with these chemicals, these three genes could produce iPSCs from mouse embryonic fibroblasts (MEFs). They identified at least three molecules; Forskolin, 2-methyl-5-hydroxytryptamine and a synthetic molecule called D4476, that could substitute for OCT4.

Thus, by using chemicals, they could get away from using one of the genes required to de-differentiate adult cells into iPSCs. Could they whittle down the number of genes even further? Previously, Deng and Zhao published a paper in which a chemical cocktail was used to substitute for the other three genes so that conversion into iPSCs was achieved by introducing only the OCT4 gene into cells (Li, YQ et al., CELL RESEARCH 21(1): 196-204. They called this cocktail “VC6T.” Therefore, they used VC6T and Forskolin, on their MEFs and the beginnings of de-differentiation occurred, but not much else.

Could chemicals be identified that would take the cells the rest of the way to iPSCs? Another chemical screen examined this possibility. In this test, the MEFs were rigged so that they expressed OCT4 when the cells were treated with the antibiotic doxycycline. By giving the cells doxycycline for 4-8 days, and then testing chemicals to take the cells the rest of the way, they identified a slew of compounds that, when given to the OCT4-expressing MEFs, they became iPSCs.

Then came the real test – make iPSCs with just chemicals and no introduced genes. Could it be done? When they gave the MEFs some of the chemicals identified in the last screen (they called it DZNep), plus VC6T, the expression of OCT4 went up, but the cells simply did not look like iPSCs. So, they changed the culture medium to a “2i” culture system that inhibits some key regulatory proteins in the cells. When they used this same chemical cocktail in a 2i culture system, it worked and iPSCs were produced. Deng and Zhao called these stem cells “chemically induced pluripotent stem cells” or CiPSCs.

(A and B) Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B) plus chemicals or Oct4. Error bars, mean ± SD (n = 3 biological repeat wells). (C) Morphology of MEFs for chemical reprogramming on day 0 (D0) and a GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical treatment. (D) Numbers of GFP-positive colonies induced after DZNep treatment on day 36. Error bars, mean ± SD (n = 2 biological repeat wells). (E to G) Morphology of a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment (E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC generation. Scale bars, 100 μm. For (D), cells for reprogramming were replated on day 12.
(A and B) Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B) plus chemicals or Oct4. Error bars, mean ± SD (n = 3 biological repeat wells). (C) Morphology of MEFs for chemical reprogramming on day 0 (D0) and a GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical treatment. (D) Numbers of GFP-positive colonies induced after DZNep treatment on day 36. Error bars, mean ± SD (n = 2 biological repeat wells). (E to G) Morphology of a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment (E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC generation. Scale bars, 100 μm. For (D), cells for reprogramming were replated on day 12.

Next, they optimized the dosages of these chemicals in order to increase the efficiency of iPSC production. They were able to increase the efficiency of iPSC production to 5% (1 of every 20 colonies of cells), which is respectable. They also identified yet another small molecule that beefed up iPSC production by another 40-fold. Also, this chemical cocktail was able to make iPSCs from mouse adult fibroblasts, fat-derived stem cells, and fibroblasts from newly born mice.

When the CiPSC lines were characterized, they made all the right genes to be designated as pluripotent stem cells, and they had normal numbers of normal-looking chromosomes all the way through 13 passages.

When injected into mice with dysfunctional immune systems, the CiPSCs made tumors that were mixtures of tissues of all over the body. When they were transferred into early mouse embryos, they could contribute to the bodies of developing mice, and they could even contribute to the production of eggs and sperm, When baby mice were completely made from CiPSCs, those mice were fertile and had babies of their own. This is the ultimate test of pluripotency and the CiPSCs passed it with flying colors.

A) Hematoxylin and eosin staining of CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2 offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n, total numbers of chimeras studied.
A) Hematoxylin and eosin staining of CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2 offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n, total numbers of chimeras studied.

Other experiments in this paper examined why these chemicals induced pluripotency in adult cells, but these experiments, though interesting, are lost in the fact that this research group has generated iPSCs without using any viruses, or genetic engineering technology. These CiPSCs are true pluripotent stem cells and they were generated without killing any embryos or introducing genes that might drive cells to become abnormal.

If this can be replicated with human cells, it would be earth-shattering for regenerative medicine.

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.