A recipe for pluripotency: the correct sp(l)ices make all the difference. Focus on “Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency”

Abstract

HUMAN PLURIPOTENT STEM CELLS (PSCs), either directly isolated from embryos or induced by exogenous factors, have not only opened up the possibility of deriving genetically normal cell types in large numbers, but they also offer a window into normally inaccessible human embryogenesis. The understanding and control of human PSC self-renewal and differentiation rely on a thorough molecular understanding of control of the stem cell state: how cells exit the pluripotent state, and how cells are specified to particular fates. Much of the understanding of PSC biology has come from studies in the mouse. However, it has recently become apparent that PSCs from mouse and human, previously thought of as equivalent types, are, in fact, quite different entities. Mouse embryonic stem cells (ESCs) can be grown on feeders with leukemia inhibitory factor or by restriction of differentiation and, thus, heterogeneity using chemical inhibitors (2i dual-inhibition conditions) (4). PSCs have also been derived from postimplantation mouse embryos; such cells are referred to as epiblast stem cells (EpiSCs). These cells share many of the characteristics of human ESCs (hESCs), including basic gene expression and growth factor requirements. However, there are some differences between human and mouse EpiSCs, such as the tendency to express markers of the primitive streak stage of development (6). To complicate the picture further, hESCs appear to be complex mixtures of cell states and seem to be more heterogeneous than mouse primed or naive ESCs (2a).Much research has been conducted over the past decade into the mechanisms by which pluripotency is controlled. Significant insights into the regulation of PSCs have been obtained from the investigation of transcription factors and their associated networks. Most of these studies have treated the mouse as a paradigm for the human system, which is likely to be a misleading assumption. Three transcription factors, octamerbinding transcription factor 4 [OCT4, POU domain class 5 transcription factor 1 (POU5F1)], sex-determining region Ybox 2 (SOX2), and NANOG, have emerged as central players in the control of pluripotency (3). These studies, however, have tended to use mixtures of cells, and, given the apparent heterogeneity in human PSC (hPSC) cultures, further refinement of the gene regulatory networks is required.The realization that splice variation may play a role in the control of the pluripotent phenotype in humans adds a further layer of complexity (1, 11). In the human system, OCT4 (POU5F1) can be expressed as different splice variants: OCT4A, OCT4B, and OCT4B1 (9). OCT4A is the direct ortholog of the mouse Oct4 transcript and is critical for self-renewal of hPSCs (9). The roles of OCT4B and OCT4B1 are much less well understood, despite their expression at the transcript level in hPSCs. Previous observations of subcellular location are suggestive of functional differences between OCT4A and OCT4B (2), but proper functional analysis is lacking. The presence of six transcribed pseudogenes makes ...