STAP Cells: The Plot Thickens Even More


You might remember that Charles Vacanti and researchers at the RIKEN Institute in Japan reported a protocol for reprogramming mature mouse cells into pluripotent stem cells that could not only integrate into mouse embryos, but could also contribute to the formation of the placenta. To convert mature cells into pluripotent cells, Vacanti and others exposed the cells to slightly acidic conditions or other types of stressful conditions and the cells reverted to a pluripotent state.

Even though Vacanti and others published these results in the prestigious journal Nature, as other scientists tried to replicate the results in these papers, they found themselves growing more and more frustrated. Also, some gaffes with a few of the figures contributed to a kind of pall that has hung over this research in general.

The original makers of these cells, stress-acquired acquisition of pluripotency or STAP cells, have now made a detailed protocol of how they made their STAP cells publicly available at the Nature Protocol Exchange. Already. it is clear that a few things about the original paper are generating many questions.

First of all, Charles Vacanti’s name does not appear on the protocol. He was the corresponding author of the original paper. Therefore the absence of his name raises some eyebrows. Secondly, the authors seem to have backed off a few of their original claims.

For example one of the statements toward the beginning of the protocol says, “Despite its seeming simplicity, this procedure requires special care in cell handling and culture conditions, as well as in the choice of the starting cell population.” Whereas the original paper, on the first reading at least, seemed to convey that making STAP cells was fairly straightforward, this seems to no longer be the case, if the words of this protocol are taken at face value.

Also, the protocol notes that cultured cells do not work with their protocol. The authors write, “Primary cells should be used. We have found that it is difficult to reprogram mouse embryonic fibroblasts (MEF) that have been expanded in vitro, while fresh MEF are competent.”  This would probably explain inability of several well-regarded stem cell laboratories to recapitulate this work, since the majority of them probably used cultured cells. This, however, seems to contradict claims made in the original paper that multiple, distinct cell types could be converted into STAP cells.

Another clarification that the protocol provides that was not made clear in the original paper is that STAP cells and STAP stem cells are not the same thing. According to the authors, the protocol provided at Nature Protocol Exchange produces STAP cells, which have the capacity to contribute to the embryo and the placenta. On the other hand, STAP stem cells, are made from STAP cells by growing them in ACTH-containing medium on feeder cells, after which the cells are switched to ESC media with 20% Fetal Bovine Serum. STAP stem cells have lost the ability to contribute to extra-embryonic tissues.

Of even greater concern is a point raised by Paul Knoepfler at UC Davis. Knoepfler noticed that the original paper argued that some of their STAP cells were made from mature T cells. T cells rearrange the genes that encode the T cell receptor. If these mature T cells were used to make STAP cells, then they should have rearranged T cell receptor genes. The paper by Vacanti and others shows precisely that in a figure labeled 1i. However, in the protocol, the authors state that their STAP cells were NOT made from T-cells. In Knoepfler’s words: “On a simple level to me this new statement seems like a red flag.”

Other comments from Knoepfler’s blog noted that the protocol does not work on mice older than one week old. Indeed, the protocol itself clearly states that “Cells from mice older than one week showed very poor reprogramming efficiency under the current protocol. Cells from male animals showed higher efficiency than those from female.”  Thus the universe of cells that can be converted into STAP cells seems to have contracted by quite a bit.

From all this it seems very likely that the STAP paper will need to go through several corrections. Some think that the paper should be retracted altogether. I think I agree with Knoepfler and we should take a “wait and see” approach. If some scientists can get this protocol to work, then great. But even then, multiple corrections to the original paper will need to be submitted. Also, the usefulness of these procedure for regenerative medicine seems suspect, at least at the moment. The cells types that can be reprogrammed with this protocol are simply too few for practical use. Also, to date, we only have Vacanti’s word that this protocol works on human cells. Forgive me, but given the gaffes associated with this present paper, that’s not terribly reassuring.

Histones Might Hold the Key to the Generation of Totipotent Stem Cells


Reprogramming adult cells into pluripotent stem cells remains a major challenge to stem cell research. The process remains relatively inefficient and slow and a great deal of effort has been expended to improve the speed, efficiency and safety of the reprogramming procedure.

Researchers from RIKEN in Japan have reported one piece of the reprogramming puzzle that can increase the efficiency of reprogramming. Shunsuke Ishii and his colleagues from RIKEN Tsukuba Institute in Ibaraki, Japan have identified two variant histone proteins that dramatically enhance the efficiency of induced pluripotent stem cell (iPS cell) derivation. These proteins might be the key to generating iPS cells.

Terminally-differentiated adult cells can be reprogrammed into a stem-like pluripotent state either by artificially inducing the expression of four factors called the Yamanaka factors, or as recently shown by shocking them with sublethal stress, such as low pH or pressure. However, attempts to create totipotent stem cells capable of giving rise to a fully formed organism, from differentiated cells, have failed.  However, a paper recently published in the journal Nature has shown that STAP or stimulus-triggered acquisition of pluripotency cells from mouse cells have the capacity to form placenta in culture and therefore, are totipotent.

The study by Shunsuke Ishii and his RIKEN colleagues, which was published in the journal Cell Stem Cell, attempted to identify molecules in mammalian oocytes (eggs) that induce the complete reprograming of the genome and lead to the generation of totipotent embryonic stem cells. This is exactly what happens during normal fertilization, and during cloning by means of the technique known as Somatic-Cell Nuclear Transfer (SCNT). SCNT has been used successfully to clone various species of mammals, but the technique has serious limitations and its use on human cells has been controversial for ethical reasons.

Ishii’s research group focused on two histone variants named TH2A and TH2B, which are known to be specific to the testes where they bind tightly to DNA and influence gene expression.

Histones are proteins that bind to DNA non-specifically and act as little spool around which the DNA winds.  These little wound spools of DNA then assemble into spirals that form thread-like structures.  These threads are then looped around a protein scaffold to form the basic structure of a chromosome.  This compacted form of DNA is called “chromatin,” and the DNA is compacted some 10,000 to 100,000 times.  Histones are the main arbiters of chromatin formation.  In the figure below, you can see that the “beads on a string” consist of histones with DNA wrapped around them.

DNA_to_Chromatin_Formation

There are five “standard” histone proteins: H1, H2A, H2B, H3, and H4.  H2A, H2B, H3 and H4 form the beads and the H1 histone brings the beads together to for the 30nm solenoid.  Variant histones are different histones that assemble into beads that do not wrap the DNA quite as tightly or wrap it differently than the standard histones.  Two variant histones in particular, TH2A and TH2B, tend to allow DNA wrapped into chromatin to form and more loosely packed structure that allows the expression of particular genes.

When members of Ishii’s laboratory added these two variant histone proteins, TH2A/TH2B, to the Yamanaka cocktail (Oct4, c-Myc, Sox2, and Klf4) to reprogram mouse fibroblasts, they increased the efficiency of iPSC cell generation about twenty-fold and the speed of the process two- to threefold. In fact, TH2A and TH2B function as substitutes for two of the Yamanaka factors (Sox2 and c-Myc).

Ishii and other made knockout mice that lacked the genes that encoded TH2A and TH2B. This work demonstrated that TH2A and TH2B function as a pair, and are highly expressed in oocytes and fertilized eggs. Furthermore, these two proteins are needed for the development of the embryo after fertilization, although their levels decrease as the embryo grows.

Graphical Abstract1 [更新済み]

In early embryos, TH2A and TH2B bind to DNA and induce an open chromatin structure in the paternal genome (the genome of sperm cells), which contributes to its activation after fertilization.

These results indicate that TH2A/TH2B might induce reprogramming by regulating a different set of genes than the Yamanaka factors, and that these genes are involved in the generation of totipotent cells in oocyte-based reprogramming as seen in SCNT.

“We believe that TH2A and TH2B in combination enhance reprogramming because they introduce a process that normally operates in the zygote during fertilization and SCNT, and lead to a form of reprogramming that bears more similarity to oocyte-based reprogramming and SCNT” explains Dr. Ishii.

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.

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.

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.

Both Copies of the Nanog Gene Are Expressed in Embryonic Stem Cells


Commonly held ideas are sometimes held because there is a great of evidence to substantiate them. However, other times, an idea is commonly held because simply because it has been repeated over and over and over even though the evidence for it is poor. Thus, when new evidence come to light showing the commonly held believe to be untrue, it becomes incumbent on us to readjust what we think.

When it comes to embryonic stem cells and the genes that keep them pluripotent, the transcription Nanog plays a very critical role in the self-renewal of embryonic stem cells and there is a great deal of evidence for this assertion. However, the expression of the gene that encodes Nanog was thought to follow the same mode of expression as some of the other pluripotency promoting genes. Namely, that only one of the copies of the Nanog gene were thought to be expressed in embryonic stem cells. This turns out to be probably false.

First a little background. In 2007, Ian Chambers and others published a paper in the journal Nature that examined the expression and function of Nanog in embryonic stem cells. Chambers and others found that Nanog expression levels in individual embryonic stem cells from a culture derived from a single cell varied wildly.  The figure from the Chambers et al paper is shown below.

Immunofluorescence of TNG cells for Oct4 and Nanog. Individual signals from 4,6-diamidino-2-phenylindole (DAPI), GFP, anti-Oct4 and anti-Nanog are shown on the left alongside a combined view of GFP with the stainings from anti-Oct4 and anti-Nanog.
Immunofluorescence of TNG cells for Oct4
and Nanog. Individual signals from 4,6-diamidino-2-phenylindole (DAPI),
GFP, anti-Oct4 and anti-Nanog are shown on the left alongside a combined
view of GFP with the stainings from anti-Oct4 and anti-Nanog.

The reason for this fluctuation in Nanog levels was uncertain, but Chambers and others showed that Nanog could be deleted from mouse embryonic stem cells without affecting their ability to contribute to various sundry embryonic tissues during mouse development, even though they do not make functional gametes (eggs and sperm).  In fact, mouse embryonic stem cells can self-renew under particular conditions without a functional copy of the Nanog gene even though they are prone to differentiation.  From this, Chambers and others concluded that Nanog stabilized rather than promoted pluripotency of embryonic stem cells by “resisting or reversing alternative gene expression states.”

Fast forward to 2012 and another Nature paper by Yusuke Miyanari and Maria-Elena Torres-Padilla from the IGBMC in Strasbourg, France, which showed that before mouse embryos implanted into the uterus, only one copy of the Nanog gene was expressed, but after implantation, both copies of the Nanog gene was expressed.  Miyanari and Torres-Padilla also made mouse embryonic stem cells that had copies of the Nanog gene labeled with different glowing proteins.  This ingenious experiment showed confirmed that Nanog levels were variable, but also showed that only one copy of the Nanog gene was expressed in growing embryonic stem cells in culture.

a, Schematic of the Nanog knock-in reporter NGR. A PEST motif in the carboxy terminus of the fluorescent proteins allows monitoring of dynamic Nanog expression. iHyg, internal ribosome entry site (IRES) hygromycin; iNeo, IRES neomycin; mChe, mCherry; NLS, nuclear localization signal; tGFP, TurboGFP. b, Representative image of NGR ES cells cultured with LIF or 2i/LIF. Scale bar, 10 µm. c, The incidence of allelic switching of Nanog expression in ES cells. Cells were classified into four groups: monoallelic (TurboGFP-positive, green), monoallelic (mCherry-positive, red), biallelic (TurboGFP- and mCherry-positive, yellow) and no expression (black). The proportion of cells undergoing a transition between these four groups during a single cell cycle is indicated. Overall, 47% of cells showed a colour change in this period. n, number of cells analysed. d, The asymmetric replication of Nanog in ES cells cultured with LIF changes to symmetric replication upon treatment with 2i. The cell nuclei were classified as single/double (SD), single/single (SS) and double/double (DD) according to DNA-FISH signals5. n, number of nuclei analysed. *, P < 4 × 10−7; **, P < 1.4 × 10−3 (Fisher’s exact test). e, Representative image of DNA-FISH for Nanog (arrowheads) and Oct4 in ES cells cultured with LIF or 2i/LIF. Scale bar, 2 µm. f, Nanog allelic expression is unaffected in the absence of DNA methyltransferase activity. Quantification of RNA-FISH for Nanog in wild-type (WT) ES cells and ES cells lacking all three DNA methyltransferases (TKO) cultured with LIF or 2i/LIF. g, ChIP for H3K4me3, MED12 or NIPBL along the Nanog locus (black line, top) in ES cells cultured with LIF or 2i/LIF. The position of the ChIP amplicons is depicted by the thick boxes below the line, the TSS by an arrow, the first exon by the black box on the line, and the distal enhancer by the blue box on the line. The Oct4 promoter region (Oct4 Pro) and distal enhancer (Oct4 DE) were positive controls (right)20. The mean ± s.d. of three independent biological replicates is shown.
a, Schematic of the Nanog knock-in reporter NGR. A PEST motif in the carboxy terminus of the fluorescent proteins allows monitoring of dynamic Nanog expression. iHyg, internal ribosome entry site (IRES) hygromycin; iNeo, IRES neomycin; mChe, mCherry; NLS, nuclear localization signal; tGFP, TurboGFP. b, Representative image of NGR ES cells cultured with LIF or 2i/LIF. Scale bar, 10 µm. c, The incidence of allelic switching of Nanog expression in ES cells. Cells were classified into four groups: monoallelic (TurboGFP-positive, green), monoallelic (mCherry-positive, red), biallelic (TurboGFP- and mCherry-positive, yellow) and no expression (black). The proportion of cells undergoing a transition between these four groups during a single cell cycle is indicated. Overall, 47% of cells showed a colour change in this period. n, number of cells analysed. d, The asymmetric replication of Nanog in ES cells cultured with LIF changes to symmetric replication upon treatment with 2i. The cell nuclei were classified as single/double (SD), single/single (SS) and double/double (DD) according to DNA-FISH signals5. n, number of nuclei analysed. *, P < 4 × 10−7; **, P < 1.4 × 10−3 (Fisher’s exact test). e, Representative image of DNA-FISH for Nanog (arrowheads) and Oct4 in ES cells cultured with LIF or 2i/LIF. Scale bar, 2 µm. f, Nanog allelic expression is unaffected in the absence of DNA methyltransferase activity. Quantification of RNA-FISH for Nanog in wild-type (WT) ES cells and ES cells lacking all three DNA methyltransferases (TKO) cultured with LIF or 2i/LIF. g, ChIP for H3K4me3, MED12 or NIPBL along the Nanog locus (black line, top) in ES cells cultured with LIF or 2i/LIF. The position of the ChIP amplicons is depicted by the thick boxes below the line, the TSS by an arrow, the first exon by the black box on the line, and the distal enhancer by the blue box on the line. The Oct4 promoter region (Oct4 Pro) and distal enhancer (Oct4 DE) were positive controls (right)20. The mean ± s.d. of three independent biological replicates is shown.

Now if we fast forward one more year and a paper from the journal Cell Stem Cell and a letter to the same edition of this journal, we have an article by Dina Faddah and others from the laboratory of Rudolf Jaenisch at the Whitehead Institute (MIT, Cambridge, MA), and a supporting letter from Adam Filipczyk and others from Germany and Switzerland.  In this article, the authors also double-labeled mouse embryonic stem cells and examined multiple cells and showed that BOTH copies of Nanog were expressed, and that the range of variability of Nanog expression was approximately the same as other pluripotency genes.

Filipczyk and others used a similar approach to examine the expression of Nanog in mouse embryonic stem cells and they came to the same conclusions as those of Faddah and others.

What is the reason for the differences in findings?  Faddah and others did an important experiment to answer this question.  Some of the cells that with labeled copies of the Nanog gene disrupted the production of a functional Nanog protein.  The constructs used in the papers by Faddah and others and by Filipczyk and others did not disrupt Nanog protein production.  When Faddah and others tested these other constructs that disrupted Nanog protein production to determine is the amount of glowing protein tracked with the amount of Nanog protein produced, it was clear that the amount of Nanog protein made by the cells did not reflect the amount of glowing protein produced.  According to Dina Faddah, “The way the reported was inserted into the DNA seems to disrupt the regulation of the alleles so that when the reported said Nanog isn’t being expressed, it actually is.”

Jaenisch sees this as an instructional tale for all stem cell scientists.  He noted: “Clearly, the conclusions for this particular gene need to be reconsidered.  And it raises the question for other genes.  For some genes, there might be similar issues.  For other genes, they might be more resistant to this type of disturbances caused by a reporter.”

Bottom line – read the materials and methods part of the paper carefully because the way these experiments are done can determine if the results are trustworthy.

New Pluripotent Stem Cell Production Protein Identified


Large scale production of stem cells requires an intimate knowledge of the genetic networks that convert adult cells into induced pluripotent stem cells (iPSCs). The original protocol established by Shinya Yamanaka and his colleagues used four genes all clustered on a retrovirus vector, but there are safer, more technically subtle ways to make iPSCs.

Because iPSCs are made from a patient’s own cells, they are less likely to be rejected by the patient’s immune system. They also show tremendous developmental flexibility, they can potentially be differentiated into any adult cell type in the body.  The problem with iPSCs comes from the difficulty of making large quantities of them in a reasonable amount of time.  However, a new research publication from scientists at the University of Toronto, the University for Sick Children and Mount Sinai Hospital, in collaboration with colleagues from the United States and Portugal, identifies specific proteins that play central roles in controlling pluripotency that may mean a potential breakthrough in producing iPSCs.

Researchers discovered these proteins by using something called the “splicing code.”  Benjamin Blencowe discovered the splicing code a few years ago.  “The mechanisms that control embryonic stem cell pluripotency have remained a mystery for some time.  However, what Dr. Blencowe and the research team found is that the proteins identified by our splicing code can activate or deactivate stem cell pluripotency,” said Brendan J. Frey, from the University of Toronto Departments of Electrical Engineering and Medicine, who published with Benjamin Blencowe the paper that deciphered this splicing code (see Nature 2010 465: 53-59).  While a complete recipe for producing iPSCs may not be available yet, it is beginning to look more likely, according to Frey.

In this paper, Blencowe and his collaborators identified two proteins known as muscleblind-like RNA binding proteins, or MBNL1 and MBNL2.  These proteins are conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between embryonic stem cells and other cell types.

RNA splicing occurs in plant, animal, fungal, and protist cells (only very, very rarely in bacteria), and involves the removal of segments of primary RNA transcripts.  When RNA molecules are transcribed in eukaryotic cells, they are engaged by cellular machinery called the RNA spliceosome.  The RNA spliceosome removes segments known as “introns” and the excised introns are degraded and the remaining RNA segments, which are known as “exons, are ligated together to form a mature messenger RNA.

mRNA splicing

Some introns are removed from primary RNA transcripts by all cells, but others are removed in some cells but not others.  This phenomenon is known “alternative splicing” and it is responsible for the differential regulation of particular genes.

alternative_splicing

Alternative splicing is mediated by sequences called splicing enhancers and splicing silencers that are six to either nucleotides long and bind proteins that either induce or repress alternative splicing in those cells that express the proteins that bind these splicing enhancers or silencers.

Alternative RNA splicing mechanism

MBNL is one of these proteins that bind to RNA splicing silencers.  If the quantity of MBNL proteins in differentiated cells is decreased, then these cells switch to an embryonic stem cell-like alternative splicing pattern for approximately half of their genes.  Conversely, overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns.  Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in a protein-coding gene called the forkhead family transcription factor FOXP1.  FOXP1 controls pluripotency, and consistent with a central and negative regulatory role for MBNL proteins in pluripotency, knockdown of MBNL significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.

Thus MBNL proteins should be one of the main targets for the mass production of iPSCs.