Sequential Expression of Pluripotency Markers during Direct Reprogramming of Mouse Somatic Cells

Abstract

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming.

Two-Phase Analysis of Molecular Pathways Underlying Induced Pluripotent Stem Cell Induction

“…Using an elegant transgenic system with doxycycline-inducible expression of the four Yamanaka factors in MEFs, several studies have defined the sequential expression of pluripotency markers during iPSC induction [28][29][30] regulated by Sox2 or regulated by Sox2 in cooperation with other transcription factors. In support of this view, chromatin immunoprecipitation-seq studies show that Sox2 can bind directly to the promoter of Dnmt3b [50,51], and a dramatic decrease of Zfp459 expression is observed after Sox2 knockout [52].…”

“…This characteristic leads to heterogeneous cell populations composed of individual cells at different reprogramming phases for any given time point after the initial induction [28][29][30]. In this study, we have firmly established a stable phase during the reprogramming process through the overexpression of only Oct4, Klf4, and c-Myc (OKM).…”

Klf4 Interacts Directly with Oct4 and Sox2 to Promote Reprogramming

“…Further analysis shows that three of these genes, Oct4, Sox2, and Klf4, are critical to the process and that c-Myc functions to enhance reprogramming efficiency [4][5][6]. Additionally, inducible systems have been developed to better control transgene expression [7]. Novel methods requiring no viral integration have also been developed [8][9][10][11][12], as have strategies using small molecules to promote reprogramming efficiency [13][14][15].…”

Overcoming barriers to the clinical utilization of iPSCs: reprogramming efficiency, safety and quality

“…The production of iPSCs has so far been achieved through nucleic-acid-based delivery of the reprogramming factors (Maherali and Hochedlinger, 2008). Initial generations of mouse and human iPSCs employed retroviral vectors (Takahashi and Yamanaka, 2006;Takahashi et al, 2007) and constitutive lentiviruses (Blelloch et al, 2007;Yu et al, 2007), while later generations were produced using inducible lentiviruses (Brambrink et al, 2008;Stadtfeld et al, 2008b). Residual expression of integrated copies of reprogramming factors in iPSCs can affect the gene expression and potentially biological properties of the resulting iPSC derivatives.…”

“…There are several drawbacks for the genome integration system for example their infectivity is limited to dividing cells, thus restricting the range of cell types that can be reprogrammed; silencing occurs gradually during the course of iPSC induction, resulting in a lowered efficiency of conversion compared to nonsilencing viral methods and iPSCs made with retroviruses often maintain viral gene ex- Yamanaka, 2006 Blelloch et al, 2007;Yu et al, 2007;Brambrink et al, 2008Brambrink et al, 2008Stadtfeld et al, 2008b Free of genetic modification No genetic modification, still requires at least one factor to be transduced Okita et al, 2008Chang et al, 2009Soldner et al, 2009Stadtfeld et al, 2008cFusaki et al, 2009;Gonzalez et al, 2009Kaji et al, 2009Woltjen et al, 2009Bosnali and Edenhofer, 2008Huangfu et al, 2008aShi et al, 2008b;Ichida et al, 2009;Lyssiotis et al, 2009 pression thus limiting their utility (Maherali and Hochedlinger, 2008). Although iPSCs can be generated by constitutive lentiviruses, their poor silencing within pluripotent cells make them less suitable for direct reprogramming attempts (Blelloch et al, 2007;Yu et al, 2007;Brambrink et al, 2008). Drug-inducible lentiviruses have provided a more attractive approach, as they permit temporal control over factor expression.…”