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,

Identifying Barriers to Cell Reprogramming


A new study from the laboratory of Miguel Ramalho-Santos, associate professor of obstetrics, gynecology and reproductive sciences at the University of California, San Francisco (UCSF), might lead to a faster way to derive stem cells that can be used for regenerative therapies.

Induced pluripotent stem cells or iPSCs, which are made from adult cells by means of genetic engineering and cell culture techniques, behave much like embryonic stem cells. These adult cell-derived stem cells are pluripotent and can be differentiated into heart, liver, nerve and muscle cells. This present work by Ramalho-Santos and his colleagues builds upon the reprogramming protocols that have been developed to de-differentiate mature adults cells into iPSCs.

Ramalho-Santos and his co-workers have been interested in understanding the reprogramming process more completely in order to increase the efficiency and safety of this process. In particular, the Ramalho-Santos laboratory has been examining the cellular barriers that prevent adult cells from being reprogrammed in order to circumvent them and increase the efficiency of stem-cell production. In this present work, Ramalho-Santos’ group identified many of these cellular barriers to reprogramming.

“Our new work has important implications for both regenerative medicine and cancer research,” said Ramalho-Santos, who is also a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.

In 2012, Shinya Yamanaka from Kyoto University won the Nobel Prize in Physiology or Medicine for his discovery of iPSCs. Yamanaka discovered ways to turn back the clock on adult cells, but the protocol that he developed and others have used for years is inefficient, slow, and tedious. The percentage of adult cells successfully converted to iPS cells is usually rather low, and the resultant cells often retain traces of their earlier lives as mature, fully-differentiated cells.

To make iPSCs, researchers force the expression of pluripotency-inducing genes in adult cells. These four genes (Oct4, Klf4, Sox2, cMyc) have become known as the so-called “Yamanaka factors” and they work to turn back the clock on cellular maturation. However, as Ramalho-Santos explained: “From the time of the discovery of iPS cells, it was appreciated that the specialized cells from which they are derived are not a blank slate. They express their own genes that may resist or counter reprogramming.”

So what are those barriers? Ramalho-Santos continued: “Now, by genetically removing multiple barriers to reprogramming, we have found that the efficiency of generation of iPS cells can be greatly increased.” This discovery will contribute to accelerating the production of safe and efficient iPSCs and other types of other reprogrammed cells, according to Ramalho-Santos.

Instead of identifying individual genes that act as barriers to reprogramming, Ramalho-Santos and others discovered that sets of genes acted in combination to establish barriers to reprogramming. “At practically every level of a cell’s functions there are genes that act in an intricately coordinated fashion to antagonize reprogramming,” Ramalho-Santos explained. These existing mechanisms probably help mature, adult cells maintain their identities and functional roles. Ramalho-Santos explained it this way: “Much like the Red Queen running constantly to remain in the same place in Lewis Carroll’s ‘Through the Looking-Glass,’ adult cells appear to put a lot of effort into remaining in the same state.” Ramalho-Santos also added that apart from maintaining the integrity of our adult tissues, the barrier genes probably serve important roles in other diseases, including in the prevention of certain cancers

To identify these barriers, Ramalho-Santos and his team had to employ cutting-edge genetic, cellular and bioinformatics technologies. They collaborated with other UCSF labs headed by Jun Song, assistant professor of epidemiology and biostatistics, and Michael McManus, associate professor of microbiology and immunology.

They conducted genome-wide RNAi screens that revealed known and novel barriers to human cell reprogramming. Of these, a protein called ADAM29 antagonizes reprogramming as does clathrin-mediated endocytosis, which antagonizes reprogramming by enhancing TGF-β signaling. Also it became apparent that different barrier pathways have a combined effect on reprogramming efficiency. Additionally, genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport, and cell adhesion also act as barriers to reprogramming.

Barriers to reprogramming

The hopes are that this knowledge will produce iPSCs faster that are safer to use and differentiate more completely.