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.


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.

Human STAP cells – Troubling Possibilities

Soon after the publication of this paper that adult mouse cells could be reprogrammed into embryonic-like stem cells simply by exposing them to acidic environments or other stresses , Charles Vacanti at Harvard Medical School has reported that he and his colleagues have demonstrated that this procedure works with human cells.

STAP cells or stimulus-triggered acquisition of pluripotency cells were derived by Vacanti and his Japanese collaborators last year. These new findings show that adult cells can be reprogrammed into embryonic-like stem cells without genetic engineering. However, this technique worked well in mouse cells, but it was not clear that it would work with human adult cells.

Vacanti and others shocked the world when they published their paper in the journal Nature earlier this year when they announced that adult cells in mice could be reprogrammed through exposure to stresses and proper culture conditions.

Now Vacanti has made good on his promise to test his protocol on human adult cells. In the photo below, provided by Vacanti, human adult cells were reprogrammed to a pluripotent state by exposing them to stresses, followed by growth in culture under specific conditions.

Human STAP cells
Human STAP cells

“If they can do this in human cells, it changes everything, said Robert Lanza of Advanced Cell Technologies in Marlborough, Massachusetts. Such a procedure promises cheaper, faster, and potentially more flexible cells for regenerative medicine, cancer therapy and cell and tissue cloning.

Vacanti and his colleagues say they have taken human fibroblast cells and tested several environmental stressors on them to recreate human STAP cells. He will not presently disclose which particular stressors were applied, he says the resulting cells appear similar in form to the mouse STAP cells. His team is in the process of testing to see just how stem-cell-like these cells are.

According to Vacanti, the human cells took about a week to resemble STAP cells, and formed spherical clusters just like their mouse counterparts. Vacanti and his Harvard colleague Koji Kojima emphasized that these results are only preliminary and further analysis and validation is required.

Bioethical problems potentially emerge with STAP cells despite their obvious potential. The mouse cells that were derived and characterized by Vacanti’s group and his collaborators were capable of making placenta as well as adult cell types. This is different from embryonic stem cells, which can potentially form all adult cell types, but typically do not form placenta. Embryonic stem cells, therefore, are pluripotent, which means that they can form all adult cell types. However, the mouse STAP cells can form all embryonic and adult cell types and are, therefore, totipotent. Mouse STAP cells could form an entirely new mouse. While it is now clear if human STAP cells, if they in fact exist, have this capability, but if they do, they could potentially lead to human cloning.

Sally Cowley, who heads the James Martin Stem Cell Facility at the University of Oxford, said of Vacanti’s present experiments: “Even if these are STAP cells they may not necessarily have the same potential as mouse ones – they may not have the totipotency – which is one of the most interesting features of the mouse cells.”

However the only cells known to be naturally totipotent are in embryos that have only undergone the first couple of cell divisions immediately after fertilization. According to Cowley, any research that utilizes totipotent cells would have to be under very strict regulatory surveillance. “It would actually be ideal if the human cells could be pluripotent and not totipotent – it would make everyone’s life a lot easier,” she opined.

Cowley continued: “However, the whole idea that adult cells are so plastic is incredibly fascinating,” she says. “Using stem cells has been technically incredibly challenging up to now and if this is feasible in human cells it would make working with them cheaper, faster and technically a lot more feasible.”

This is all true, but Robert Lanza from Advanced Cell Technology in Marlborough, Massachusetts, a scientist with whom I have often deeply disagreed, noted: “The word totipotent brings up all kinds of issues,” says Robert Lanza of Advanced Cell Technology in Marlborough, Massachusetts. “If these cells are truly totipotent, and they are reproducible in humans then they can implant in a uterus and have the potential to be turned into a human being. At that point you’re entering into a right-to-life quagmire”

A quagmire indeed, for Vacanti has already talked about using these STAP cells to clone human embryos. Think of it: the creation of very young human beings just for the purpose of ripping them apart and using their cells for research or medicine. Would we allow this if the embryo were older; say the age of a toddler? No we would rightly condemn it as murder, but because the embryo is very young, that somehow counts against it. This is little more than morally grading the embryo according to astrology.

Therefore, whole Vacanti’s experiments are exciting and novel, they hold chilling possibilities. Lanza is right, and it is doubtful that scientists would show the same deference or sensitivities to the moral exigencies he has shown.

A Gene that Prevents Induced Pluripotent Stem Cell Formation Linked to Cancer Severity

A Mount Sinai research team has published some remarkable observations in the journal Nature Communications. Emily Bernstein, PhD, and her team at Mount Sinai have discovered a particular protein that prevents normal cells from being reprogrammed into induced pluripotent stem cells (iPSCs). Since iPSCs resemble embryonic stem cells, these data might provide significant insights into how cells lose their plasticity during normal development, which has wide-reaching implications for how cells change during both normal and disease development.

Previously, Bernstein and others showed that during the formation of particular tumors known as melanomas in mice and human patients, the loss of a specific histone variant called macroH2A (a protein that helps package DNA) correlated rather strongly to the growth and metastasis of the tumor. In this current study, Bernstein and her team determined if macroH2A acted as a barrier to cellular reprogramming during the derivation of iPSCs (see Costanzi C, Pehrson JR (1998). “Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals”. Nature 393 (6685): 599–601).

In collaboration with researchers at the University of Pennsylvania, Bernstein evaluated mice that had been genetically engineered to lack macroH2A. When skin cells were used from macroH2A(-) mice were used to make iPSCs and compared with skin cells from macroH2A(+) mice, the cells from macroH2A(-) mice that lacked macroH2A were much more plastic and were much more easily reprogrammed into iPSCs compared to the wild-type or macroH2A(+) mice. This indicates that macroH2A may block cellular reprogramming by silencing genes required for plasticity.

Bernstein, who is an Assistant Professor of Oncological Sciences and Dermatology at the Graduate School of Biomedical Sciences at Mount Sinai, and corresponding author of the study, said: “This is the first evidence of the involvement of a histone variant protein as an epigenetic barrier to induced pluripotency (iPS) reprogramming.” She continued: “These findings help us to understand the progression of different cancers and how macroH2A might be acting as a barrier to tumor development.”

In their next group of experiments, Bernstein and her team plan to create cancer cells in a culture dish by inducing mutations in genes that are commonly abnormal in particular types of cancer cells and then couple those mutations to the removal of macroH2A to examine whether the cells are capable of forming tumors.

Making Preneurons from White Blood Cells for ALS Patients

ALS or amyotrophic lateral sclerosis is a disease that results in he death of motor neurons. Motor neurons enable skeletal muscles to contract, which drives movement. The death of motor neurons robs the patient of the ability to move and ALS patients suffer a relentless, progressive, and sad decline that culminates in death from asphyxiation. Treatments are largely palliative, but stem cells treatments might delay the onset of the disease, or even regenerate the dead neurons.

To this end a Mexican group from Monterrey has used a protocol to isolate white blood cells from the circulating blood of ALS patients, and differentiate a specific population of stem cells from peripheral blood into preneurons. Although these cells were not used to treat the patients in this study, such cells do show neuroprotective features and using them in a clinical study does seem to be the next step.

In this study, CD133 cells were isolated from peripheral blood and subjected to a special culture system called a neuroinduction system. After 2-48 hours in this system, the cells showed many features that were similar to those of neurons. The cells express a cadre of neural genes (beta-tubulin III, Oligo 2, Islet-2, Nkx6.1, and Hb9). Some of the ells also grew extensions that resemble the axons of true neurons.

Interestingly, the conversion of the CD133 cells into preneurons showed similar efficiency regardless of the age, sex, or health of the individual. Even those patients with more advanced ALS had CD133 cells that differentiated into preneurons with efficiencies equal to those of their healthier counterparts. While each patient showed variation with regards to the efficiency at which their CD133 cells differentiated into preneurons, these variations could not be correlated with the age, health or sex of the patient.

The fact that these preneurons expressed Oligo2, suggests that they could differentiate into motor neurons. Therefore, even though this study was small (13 patients), it certainly shows that cells that might provide treatment possibilities for ALS patients can be made from the patient’s own blood cells.

See Maria Teresa Gonzalez-Garza et al., Differentiation of CD133+ Stem Cells from Amyotrophic Lateral Sclerosis Patients into Preneuron Cells. Stem Cells Translational Medicine 2013;2:129-35.

Leprosy Bacterium Reprograms Adult Cells into Stem Cells

Hansen’s disease is another name for the modern known as leprosy. Leprosy is known from old documents, for example, the Bible, but what is described in the Old Testament as leprosy seems to be a combination of various conditions. Plague psoriasis, for example, could fit the biblical description of leprosy. Also, in the Old Testament, a house or fabrics could get leprosy (Leviticus 13-14, which suggests that mildew, or something like it, was regarded as leprosy. Thus leprosy in the Old Testament seems to refer to a broad category of diseases. However, in the New Testament, when leprosy is described, it might be a variant of the modern Hansen’s disease.

Hansen's disease

Hansen’s disease is caused by a microorganism called Mycobacterium leprae. It causes skin lesions, loss of sensation, muscle weakness, and numbness in the hands, arms, feet and legs. The skin lesions are lighter than normal skin color, which have decreased sensation to touch, heat, or pain. These lesions do not heal after several weeks to months.

Leprosy is not very contagious and it has a long incubation period (time before symptoms appear). This makes it rather difficult to know where or when someone caught the disease. Children are more likely than adults to get leprosy.

There are two common forms of leprosy, tuberculoid and lepromatous leprosy. Both forms produce sores on the skin, but the lepromatous form is most severe. It causes large lumps and bumps. Leprosy is common in many countries worldwide, but it is also found in temperate, tropical, and subtropical climates. There are about 100 cases diagnosed per year in the United States, and most are in the South, California, and Hawaii.

Mycobacterium leprae (M. leprae) attacks, among other things, the peripheral nerves. The organism causes the insulating myelin sheath that surrounds the nerve to unravel, thus leaving the nerves without their insulating layer, which causes nerve malfunction. However, recent work has shown that M. leprae unravels the myelin sheath by reprogramming the cells that make the myelin sheath. These myelin-making cells are known as Schwann cells, and M. leprae, reprograms Schwann cells to revert to a stem-cell-like state, which causes them to leave the nerves, leaving the nerves in the lurch.

schwann cells

These finding were published in the prestigious international journal Cell.

Scientists from the laboratory of Anura Rambukkana, who holds a dual appointment at the Rockefeller University in New York and The MRC Centre for Regenerative Medicine in Edinburgh, Scotland, discovered this remarkable finding while examining how leprosy spreads around the body.

The initial target of M. leprae is Schwann cells. To understand how the organism affects Schwann cells, Rambukkana and co-workers isolated Schwann cells from mice and infected them with M. leprae. Once infected with M. leprae, the infecting bacteria reprogrammed the cells into a stem-like state. It turned off genes associated with mature Schwann cells and turned on genes associated with embryonic stages or other developmental stages.

M. leprae seemed to trigger Schwann cells’ plasticity. Plasticity refers to the ability of cells to revert to an immature state and differentiate into new types of cells. In fact, healthy Schwann cells do exactly that in order to help nerves recover and regenerate after an injury.

Rambukkana notes that however the bacteria are reprogramming the Schwann cells, they seem to be employing a “very sophisticated mechanism — it seems that the bacterium knows the mechanistic interaction of the Schwann cell better than we do.”

Upon being reprogrammed, the stem cells can migrate to different locations in the body with the bacterium housed inside then. Once the infected cells reach another tissue, such as skeletal muscle, the stem cells integrate with that tissue’s cells, thus spreading the bacteria. The infected stem cells also attract immune cells by secreting summoning proteins called chemokines. This brings more potential carriers to the bacteria’s doorstep.

What do the bacteria do to trigger a reprogramming event? At this point these researchers do not know, but they suspect that the mechanism could exist in other infectious diseases.

According to Sheng Ding, a stem cell biologist at the Gladstone Institute of Cardiovascular Disease in San Francisco, CA, “Cellular plasticity may represent an underlying mechanism of disease, as other cellular reprogramming events have been shown in cancers and metabolic diseases.”

By understanding these precise mechanisms, scientists could devise precise ways to improve treatment and earlier diagnosis of leprosy itself. These latest findings also provide vital clues about how leprosy spreads throughout the body, and how to catch the disease before it spreads rapidly.

In the future, bacteria or products made by the bacteria could be used to change adult tissue cells into stem cells in the laboratory, thus potentially leading to new regenerative treatments for diseases such as diabetes and Alzheimer’s.

See Masaki, T. et al. Cell 152, 51–67 (2013).