DIFFERENTIAL PLURIPOTENT REGULATION DEPENDENT UPON DEFINED FACTORS IN HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract

<p>Human pluripotent stem cells (hPSCs) exist as a heterogeneous population within a dynamic niche, which governs their ability to self-renew and differentiate. Evidence modeled after mouse embryonic stem cells (mESCs) reveals the existence of a developmentally primitive, or homogeneous, state through chemically defined culture methods that is modulated by NANOG, a core pluripotent regulator. However, the differentiation potential and transcription factor control of the homogeneous state in human pluripotent stem cells remains elusive. Previous work suggests that bFGF/ACTIVIN extrinsic regulation provides the heterogeneous nature of hiPSCs with ability to differentiate into several multilineage lineage progenitors. Here, we illustrate that altering the extrinsic environment of hiPSCs with LIF and inhibitors of GSK3b and MAPK/ERK1/2 pathways (LIF/2i), rewires the intrinsic pluripotent regulation of OCT4 and NANOG, which ultimately prevents the <em>in vitro</em> hematopoietic differentiation potential. Upon conversion of hiPSCs to a primitive state of pluripotency with LIF/2i, this study reveals that prolonged culture of hiPSCs with LIF/2i erases the hematopoietic differentiation potential through retained expression of the POU domain pluripotent transcription factor, OCT4. Interestingly, shRNA mediated knockdown of <em>OCT4</em> recovers the restricted differentiation potential in LIF/2i cultured hiPSCs, while knockdown of <em>NANOG</em>, does not. This study identifies a distorted differentiation potential of hPSCs cultured in mouse ESC conditions, despite comparable gene expression profiles and signaling pathway dependence. In efforts to simplify culture methods of human pluripotent stem cells, we identify that alteration of the extrinsic environment highlights explicit differences between human and mouse intrinsic pluripotent regulation, which ultimately controls differentiation efficiency.</p>