Promotion of Reprogramming to Ground State Pluripotency by Signal Inhibition

Abstract

Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency—including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras—were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1–2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon.

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

“…This unexpected finding was consistent with our hypothesis that a stable intermediate phase between fibroblasts and iPSCs could be achieved through the expression of only three reprogramming factors (Oct4, Klf4, and c-Myc). In fact, similar colonies were present in the original discovery [24,32], although subsequently identified differences in potential for reprogramming set these apart [21][22][23].…”

“…The same genome-wide mapping method has been applied to study the function of Yamanaka factors among ESCs, iPSCs, and what have been termed ''partially reprogrammed cells.'' The last group of cells have ES-like morphology without ES-like pluripotency, following transduction of all four Yamanaka factors [21][22][23]. Examination of these cells can enhance our understanding of reprogramming barriers, but identified molecular mechanisms from comparison of these partially reprogrammed Author Contributions: Z.L.…”

Crucial Role of C-Myc in the Generation of Induced Pluripotent Stem Cells

“…The generation of iPSCs and subsequent real-time polymerase chain reaction verification were performed as described previously [17]. For immunocytochemical staining, anti-Nanog (1:50, ReproCELL, Yokohama, Japan), anti-Oct3/4 h-134 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), and anti Sox-2 Y-17 (1:200, Santa Cruz) antibodies were used [18].…”

Mechanism and methods to induce pluripotency

“…Signaling pathways have significant influence on the state of pluripotency. Silva and colleagues showed that neural stem cells could be rapidly converted to iPSCs with only two factors, Oct4 and Klf4, while application of the ERK inhibitor PD0325901 and GSK3 inhibitor CHIR99021 promoted these iPS cells to enter a more authentic ES state, reflected by the reactivation of both X chromosomes and ability to contribute to the germline (Silva et al, 2008).…”