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.

CAF1

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.

chromatin+structure

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,

Yale Scientists Find Marker for High-Quality Induced Pluripotent Stem Cells


Pluripotent stem cells can be made by genetically engineering adult cells into less mature cells that have pluripotency. These induced pluripotent stem cells or iPSCs can potentially differentiate into any cell type in the adult body and because they are made from the patient’s own cells, they have a lower risk of being rejected by the patient’s immune system.

However, iPSCs suffer from an increased mutation rate when they are made and these increased mutation rate increases their risk of causing tumors and being rejected by the patient’s immune system. Having said that, not all iPSCs are created equal, and the safety of iPSCs seems to be very line-specific. Thus, how do you know a good stem cell from a bad one?

Yale Stem Cell Center researchers led by Andrew Xiao Yale have published a report in the Sept. 4 issue of Cell Stem Cell in which they describe an indicator that seems to predict which batch of personalized stem cells will differentiate into patient-specific tissue types and which will develop into unusable placental or tumor-like tissues.

Xiao’s group identified a variant histone protein called H2A.X that seems to predict the developmental path of iPSC cells in mice. Histone proteins assemble into tiny spools around which DNA winds. This DNA spooling allows cells to tightly package their DNA into a tight, compact structure that is easily stored called “chromatin.” Histones that are commonly used include histones H2A, H2B, H3 and H4.

Core histones

Two copies of each of these proteins assemble into a globular structure called a core histone and the DNA of the cell winds around this core histone to form a “nucleosome.” Then linker histones (H1 or H5) take these nucleosomes package them into spiraled coils.

DNA solenoids

H2A.X is a variant version of histone H2A is modified when DNA damage occurs. Modified H2A.X signals to the DNA repair machinery to fix the broken DNA (see TT Paull, and others, Curr. Biol. 10(15):886–95).

nrm3659-f3

According to the data from Xiao’s research team, in pluripotent stem cells, H2A.X is specifically targeted to those genes typically expressed in cells used to make the placenta, and it helps suppress differentiation of pluripotent stem cells into cells of the placental lineage. Given this distribution in mouse embryonic stem cells, H2A.X deposition pattern is a functional marker of the quality of iPSCs. Conversely, defective H2A.X deposition predisposes iPSCs toward differentiating into placental-type cells and tumors.

“The trend is to raise the standards and quality very high, so we can think about using these cells in clinic,” Xiao said. “With our assay, we have a reliable molecular marker that can tell what is a good cell and what is a bad one.”

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.

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.