Understanding the Molecular Details of Pluripotency

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Areas of investigation
The goal of our laboratory is to generate pluripotent stem cells from human somatic cells. Somatic cells can be reprogrammed either by nuclear transfer into oocytes or by fusion with embryonic stem (ES) cells. These results suggest that oocytes and ES cells contain factors that induce reprogramming. By identifying these factors, it should be possible to induce pluripotency in somatic cells without using embryos or oocytes.

Significance
ES cells, derived from the inner cell mass of mammalian blastocysts, can grow indefinitely while maintaining pluripotency. These properties have led to expectations that ES cells might be useful to treat a host of degenerative diseases, such as Parkinson’s disease and diabetes, as well as injuries, such as spinal cord injury. However, clinical application of human ES cells raises issues about the ethical use of human embryos and problems with tissue rejection after implantation. By generating pluripotent cells directly from somatic cells, we can circumvent these issues. Once established, these cells may be used in regenerative medicine and also to elucidate disease mechanisms and to screen drugs.

Approaches
We hypothesized that factors with important roles in maintaining the pluripotency of ES cells may also be critical for inducing pluripotency. The long-term maintenance of pluripotency in ES cells requires transcription factors that are specifically expressed in pluripotent cells (e.g., Oct3/4, Sox2) and activation of tumor-related genes (e.g., Stat3, c-Myc, b-catenin).

We recently showed that, in addition to these factors, the transcription factors Nanog and Klf4 and the Ras-like protein ERas play important roles in the self-renewal of mouse ES cells. Thus, these factors are candidates for pluripotency-inducing factors.

To evaluate these candidates, we developed systems in which the induction of pluripotency can be detected by expression of a marker gene. We genetically modified mice by inserting reporter cassettes into genes specifically expressed in ES cells and early embryos, such as Fbx15 and Nanog. Somatic cells derived from the mice do not express the marker genes, but when reprogrammed, the cells should become positive for these markers.

Contributions
We showed that pluripotent stem cells can be generated from adult mouse tail tip fibroblasts and adult human fibroblasts by the retrovirus-mediated transfection of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4. We designated these cells as induced pluripotent stem (iPS) cells. Mouse iPS cells are indistinguishable from ES cells in morphology, proliferation, gene expression, and teratoma formation. When transplanted into blastocysts, mouse iPS cells derived from mouse embryonic fibroblasts can give rise to adult chimeras, which are competent for germline transmission. These results are proof-of-principle that pluripotent stem cells can be generated from somatic cells by the combination of a small number of factors.

Some questions addressed in ongoing studies

1. Which types of somatic cells are ideal as a source for iPS cell induction?

2. What are the molecular mechanisms underlying reprogramming by the four factors?

3. Can the use of retrovirus, which may result in tumorigenesis, be replaced by other methods?

4. Can small molecules, instead of genes, induce pluripotency in somatic cells?