Title:
An epithelial program driven by KLF4 stoichiometry determines hallmarks of somatic cell reprogramming.

 

Knut Woltjen
Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Japan
Hakubi Center for Advanced Research, Kyoto University, Japan

 

Abstract:
Through the expression of four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Yet, as the molecular hallmarks are often inconsistent between reprogramming systems, the exact mechanisms remain to be elucidated. Variation in construct design was suspected to be an underlying cause for these differences. We therefore employed a standardized piggyBac transposon vector system to assess the cellular and molecular hallmarks of mouse somatic cell reprogramming with various published constructs. We revealed a KLF4 N-terminal variance amongst the construct panel that ultimately affected KLF4 protein levels. As a result, high-KLF4 generated few colonies, yet induced a fully pluripotent state indicated by activation of a Nanog-GFP reporter. In contrast, low-KLF4 gave rise to an increased number of colonies that failed to activate Nanog-GFP expression, indicating partially reprogrammed cells.
Using global gene expression analysis at intermediate reprogramming stages, we observed an expression profile associated with epithelialization that dominated the high-KLF4 condition. We identified enriched cell surface markers and confirmed their presentation by FACS. During low-KLF4 reprogramming, these markers were either delayed in their presentation or absent. Using CRISPR/Cas9, we performed a focused screen for modulators of the mesenchymal-to-epithelial transition (MET) by gene knockout. With the intention of identifying intermediate cell populations that contribute to “true” reprogramming, we are now employing these markers to fractionate cells, in order to examine their reprogramming potential. Beyond reprogramming, I will discuss how our findings have initiated an active branch of research into human epidermal development and genodermatoses.

 

References:
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