Reprogramming Pluripotent Cells into Totipotent Cells

With the advent of the Nobel Prize-winning research of Shinya Yamanaka, scientists are presently able to reprogram mature, adult cells into pluripotent cells. These “induced pluripotent stem cells” or iPSCs are made by genetically engineering adult cells and culturing them in special conditions. These iPSCs can also be differentiated, potentially, into every cell type in the adult human body. Now Maria-Elena Torres-Padilla‘s research team is trying to push the limits of stem cell science even further.

Torres-Padilla and her coworker successfully made “totipotent” cells, which have the same characteristics as those of the earliest embryonic stages, from pluripotent stem cells. This work was the result of a fruitful collaboration between Juanma Vaquerizas from the Max Planck Institute for Molecular Biomedicine (Münster, Germany), and Maria-Elena Torres-Padilla and her colleagues at the Institute of Genetics and Molecular and Cellular Biology (IGBMC) at Illkirch, France. This work has been published in the journal Nature Structural & Molecular Biology.

Soon after fertilization, the embryo begins it first rounds of cell division, which is known as the “early cleavage” stages. At this early stage of development, when the embryo is composed of only 1 or 2 cells, the “blastomeres” or cells of the embryo are “totipotent.” Totipotent means that these blastomeres can produce an entire embryo, or the placenta and umbilical cord that accompany it.

Early Cleavage Stages

After the 12-16-cell stage, the embryo undergoes a complex process called “compaction,” in which the cells become very tightly bound together and two populations of cells become apparent.

Post-Compaction Embryo
Post-Compaction Embryo

The cells on the inside, which give rise to the cells of the “inner cell mass” (ICM), and outside cells, which give rise to the “trophectoderm” that produce the placenta. Trophectoderm cells express a specific set of genes; Yap1, Tead4, Gata3, Cdx2, Eomes and Elf5. The ICM generates the embryo proper and the ICM cells express a cadre of genes specific to these cells; Oct4, FGF4, Sall4, Sox2 and Nanog. These ICM cells are no longer totipotent, but have become “pluripotent,” can differentiate into any tissue, but they cannot alone produce the placenta or a whole embryo. As development progresses, these pluripotent cells continue to specialize and form the various tissues of the body through the process of cellular differentiation.

While it is possible to make pluripotent cells from mature, adult cells, Torres-Padilla and her coworkers have studied the characteristics of totipotent cells of the embryo and discovered factors capable of inducing a totipotent-like state.

When they cultured pluripotent stem cells in culture, a small percentage of totipotent cells appear spontaneously. Such cells are called “2C-like cells,” after their resemblance to the 2-cell stage embryo. Torres-Padilla and her team compared these 2C-like cells to totipotent cells in early embryos in order to determine their common characteristics and the features that distinguish them from pluripotent cells. In particular, Torres-Padilla and her collaborators found that the chromosomes of totipotent cells were less condensed and that the amount of the protein complex CAF1 was diminished. CAF1 (Chromatin Assembly Factor 1) is a protein complex that helps assemble histone proteins onto DNA.


Histones act as tiny spools around which DNA is wound. Because DNA is negatively charged, and histones are positively charged, the two have a natural affinity for each other. CAF1 binds to histones and regulates the association of histones with DNA in order to ensure that the assembly of histones on DNA is and orderly process. Histones wind DNA into a tight structure called “chromatin.” CAF1, as it turns out, is responsible for maintaining the pluripotent state by ensuring that the DNA remains properly wound around histones.


As an extension of this hypothesis, Torres-Padilla and her crew were able to induce a totipotent state by inactivating the expression of the CAF1 complex. CAF1 inactivation caused the chromatin of pluripotent cells to reform into a less condensed state, and this less condensed state was conducive to totipotency.

These data provide new avenues for understanding the nature of pluripotency, and could increase the efficiency of reprogramming somatic cells to be used for applications in regenerative medicine,

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.

Forming Induced Pluripotent Stem Cells Inside a Living Organism

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

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

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

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

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

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

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

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

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

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

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

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

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