Direct cell reprogramming is a stochastic process amenable to acceleration

Abstract

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming is a continuous stochastic process where almost all donor cells eventually give rise to iPSCs upon continued growth and transcription factor expression. Additional inhibition the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPSC formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell division rate independent manner. Quantitative analyses define distinct cell division rate dependent and independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.

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).…”

Induction of pluripotency by defined factors

“…In the initiation of reprogramming, OSKM induce the stochastic changes in gene expression. 50 53) overexpressing Lin28 49) or Lin41 54) promote the reprogramming efficiency. And then, in deterministic phase, they are committed into pluripotent cell fate with expressing predictive reprogramming markers such as Utf1, Esrrb, Dppa2 and Lin28 in mouse case.…”

Concise Review: Induced Pluripotent Stem Cells Versus Embryonic Stem Cells: Close Enough or Yet Too Far Apart?

“…When looking at the total population of cells expressing reprogramming factors in a limited time frame, the reprogramming process is highly inefficient. It depends on poorly understood stochastic events in the cells and requires cell division [3,4]. The final result is an iPSC colony with newly restructured epigenetic marks driving expression of the endogenous transcription factors and chromatin regulators that further sustain and balance the achieved pluripotent state.…”