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Jun 1, 2012 (Vol. 32, No. 11)

Reprogramming Alters Cells’ Fate

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    Researchers have been able to reprogram a patient’s own cells to produce stem cells that exhibit few DNA structural mutations. [Arvid Emtegren/Istockphoto.com]

    The electrifying possibility of reprogramming human cells to different fates has huge ramifications for human therapeutics. The technology is gaining momentum as scientists make headway to better understand the molecular and cellular aspects of reprogramming.

    Some of these new and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.

    Pioneer transcription factors help untangle and pry open targeted chromatin sites. Equally important is delineating how to remove inhibitory mechanisms. Understanding the complex maze of reprogramming may ultimately provide new targeted therapies for a host of human diseases.

    The intrinsic cellular programming that primordial germ cells (PGCs) undergo can be difficult to study. PGCs represent the common origins of spermatozoa and oocytes, which are specified from the ectoderm (epiblast) of human embryos in the second week of development. They eventually migrate into the gonads where sexual differentiation begins about six weeks after conception, ending with development of egg or sperm.

    The fate of a cell is considered to be “specific” when it is capable of following an autonomous program following closure of other options. Specification of PGCs is linked with extensive epigenetic reprogramming that is critical for establishing totipotency, the ability to give rise to a whole new organism.

    M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. He said his group focused on establishing a cell culture-based system able to provide mechanistic insights into how PGCs induce reprogramming in germ cells.

    “We use murine-derived epiblast stem cells (epiSCs) to investigate aspects of epigenetic reprogramming and the roles of key genes in early germ cells. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.”

    Dr. Surani and colleagues developed two critical reporter epiSC lines. “There are two major attributes of epiSCs that can be exploited to generate reporter lines for exploring epigenetic reprogramming. One is monitoring the re-activation of the X chromosome.

    “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”

    The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer.

    “Using these two systems, we found that transcriptional regulators Prdm14 and Klf2 synergized to accelerate and enhance X chromosome reactivation and DNA demethylation, which are among the most important reprogramming events seen in early PGCs. While neither alone was sufficient to enhance this epigenetic programming, together they provided a potent combination for reprogramming. Further, the same combination provided a fast and efficient enhancement of Oct4 activity.”

    Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.

  • Reprogramming Expressway

    A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others.

    The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.

    One example Dr. Schöler cites is that Oct4 is downregulated in all three germ layers during gastrulation of the embryo.

    “This suggests that Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.

    “These studies indicate that Oct4 is not simply a reprogramming factor,” continued Dr. Schöler. “Instead it is becoming evident that it is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. It is likely these results will be soon extended to include many other lineages, making Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention.”

    Dr. Schöler is now concentrating on reprogramming cells for multipotency, the potential to give rise to a defined set of cell lineages.

    “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity. This approach demonstrates that differentiated cells can be directly programmed into specific types of somatic stem cells. The next step will be to push back cells to stem cells of the same tissue.”


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