GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS BY PROTEIN TRANSDUCTION.

Nemes, Csilla and Varga, Eszter and Polgár, Zsuzsanna and Klincumhom, Nuttha and Pirity, Melinda K. and Dinnyés, András (2013) GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS BY PROTEIN TRANSDUCTION. TISSUE ENGINEERING PART C METHODS, 2013. pp. 1-10. ISSN 1937-3384

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Abstract

Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2 and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in frame to the GST tag for affinity purification and to the TAT-NLS polypeptide to facilitate membrane penetration and nuclear localization. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-hour intervals, and cultured on mitomycin C treated MEF (mouse embryonic fibroblast) cells in complete embryonic stem cell medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to embryonic stem (ESC) cells and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by RT-PCR. The protein iPSCs (piPSCs) formed EBs and subsequently differentiated towards all three germ layer lineages. Importantly the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming mouse embryonic fibroblasts to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESC cells: form round colonies with defined boundaries; have a tendency to attach together with high nuclear/cytoplasmic ratio; express key pluripotency markers; and are capable of in vitro differentiation into ecto-, endo-, and mesoderm, and in vivo chimera formation.

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