Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency

Abstract

Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) selfrenewal and differentiation is incomplete. The level of octamerbinding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.human embryonic stem cells; OCT4 isoforms; OCT4A; pluripotency; self-renewal PLURIPOTENT STEM CELLS (PSC) have two unique properties: 1) indefinite self-renewal in culture and 2) the ability to differentiate into tissues from all three embryonic germ layers, or pluripotency (30, 37). Human embryonic stem cells (hESC) and induced pluripotent stem cells, two types of PSC that can be used to model human embryonic development in vitro, represent a promising source of cells for regenerative medicine applications. To realize this potential, a deeper understanding of the genes that regulate self-renewal and differentiation is necessary.Previous studies identified octamer-binding transcription factor 4 (Oct4), sex-determining region Y-box 2 (Sox2), and Nanog as critical transcriptional regulators of early mouse development in vivo and self-renewal and pluripotency of mouse embryonic stem cells (mESC) in vitro (2,6,15,21,22). Oct4, a POU domain transcription factor expressed throughout early mammalian development, is essential for inner cell mass growth and blastocyst survival (22). A 50% increase or decrease in Oct4 expression in mESC promotes differentiation into extraembryonic endoderm and mesoderm, or trophectoderm, respectively, suggesting that the threshold level of Oct...

High OCT4A levels drive tumorigenicity and metastatic potential of medulloblastoma cells

“…Alternative splicing of the POU5F1 primary transcript generates five transcript variants, encoding the isoforms OCT4A, OCT4B-190, OCT4B-265, OCT4B-164 and OCT4B1 [15–17]. OCT4A is the most studied and described isoform, originally reported as a regulator of ESC pluripotency and self-renewal [18], while OCT4B and OCT4B1 functions are still uncertain. There are reports of OCT4B and OCT4B1 involvement with genotoxic stress and anti-apoptotic properties [16,19], but no clear association with stemness [20].…”

RNA-binding proteins in pluripotency, differentiation, and reprogramming

“…In the past few years, studies have been reported on the regulation of AS during differentiation and reprogramming of mouse and human ESCs (Pritsker et al, 2005; Sugnet et al, 2006; Yeo et al, 2007; Kunarso et al, 2008; Salomonis et al, 2009; Salomonis et al, 2010; Wu et al, 2010; Gabut et al, 2011; Ohta et al, 2013b). Pluripotency-associated transcripts such as Dnmt3b, Nanog, Sall4 , and Oct4 have been shown to be themselves subjected to AS (Atlasi et al, 2008; Gopalakrishnan et al, 2009; Rao et al, 2010; Das et al, 2011; Tsai et al, 2014). The importance of the regulation of alternative splicing for the maintenance of the stem cell state and differentiation is exemplified by stage-specific splicing factors such as Rbfox2, Mbnl1, and Son in the pluripotency control process.…”

“…Another Oct4 splicing regulator in hESCs is Tip110, which regulates the Oct4A splicing variant both in vitro and in vivo, and is necessary for hESC maintenance (Liu et al, 2012; Liu et al, 2013). Oct4A has been shown to be the Oct4 variant responsible for maintenance of pluripotency (Tsai et al, 2014). Son and Tip110 regulate AS of pluripotency transcripts in a gene specific manner.…”

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”

“…There may be many reasons for the lack of FGF5 detection in previous studies, but, at the very least, the results presented by Tsai et al (8) suggest that the OCT4A knockdown cells may pass through an FGF5-positive "epiblast-like" stage during differentiation. There may be many reasons for the lack of FGF5 detection in previous studies, but, at the very least, the results presented by Tsai et al (8) suggest that the OCT4A knockdown cells may pass through an FGF5-positive "epiblast-like" stage during differentiation.…”