In Vitro Reprogramming of Somatic Cells into Pluripotent ES-like Cells

Whitehead Institute For Biomedical Res, Cambridge MA

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Abstract

A major focus of research for the next years will be to understand on a more detailed level the molecular mechanisms driving the reprogramming of somatic cells to pluripotent IPS cells. 1. Single-cell analysis of gene expression during cellular reprogramming An unresolved issue is whether activation of specific genes can predict early in the reprogramming process whether a given cell will develop into an iPS cell. Single RNA molecule detection methods will be Used to: a. assess whether a hierarchical program of gene expression leads to IPS cell formation or whether the process is entirely stochastic as suggested by previous observations. b. define markers that at early stages of reprogramming allow the prospective identification of cells that will generate IPS cells. For this, GFP will be inserted into candidate genes to give a marker for prospective isolation of IPS precursors. 2. Stoichiometry of reprogramming factors and quality of iPS cells mRNA mediated reprogramming will be used to systematically titrate the various factors for investigating the effect of factor stoichiometry on the biological properties of the IPS cells. This will allow the optimization of reprogramming with the goal of generating high quality genetically unmodified iPS cells. 3. Transdifferentiation of somatic cells to cells of different lineages Different somatic donor cells such as liver cells and skin keratinocytes will be used for direct conversion into neural precursors and neurons. Stringent reporters will allow the retrospective confirmation of the endodermal arid ectodermal donor cell type.