Cellular trajectories and molecular mechanisms of iPSC reprogramming

Abstract

The discovery of induced pluripotent stem cells (iPSCs) has solidified the concept of transcription factors as major players in controlling cell identity and provided a tractable tool to study how somatic cell identity can be dismantled and pluripotency established. A number of landmark studies have established hallmarks and roadmaps of iPSC formation by describing relative kinetics of transcriptional, protein and epigenetic changes, including alterations in DNA methylation and histone modifications. Recently, technological advancements such as single-cell analyses, high-resolution genome-wide chromatin assays and more efficient reprogramming systems have been used to challenge and refine our understanding of the reprogramming process. Here, we will outline novel insights into the molecular mechanisms underlying iPSC formation, focusing on how the core reprogramming factors OCT4, KLF4, SOX2 and MYC (OKSM) drive changes in gene expression, chromatin state and 3D genome topology. In addition, we will discuss unexpected consequences of reprogramming factor expression in in vitro and in vivo systems that may point towards new applications of iPSC technology.

Figure 1. Developmental fates triggered by reprogramming factors

A) In cultured fibroblasts, OKSM leads to initiation of senescence or controlled cell death in the majority of cells. A small subset of cells gives rise to unstable reprogramming intermediates that still require exogenous reprogramming factors before giving rise to nascent iPSCs upon factor withdrawal and to established iPSCs upon passaging. Fibroblasts expressing OKSM can also give rise to alternative cell fates, which was most convincingly shown for extraembryonic endoderm, but evidence suggest that intermediates and nascent iPSCs might also be more prone to differentiate into other cell lineages than established iPSCs. Nascent murine iPSCs have less pronounced self-renewal capacity than established iPSCs, but both can efficiently give rise to all somatic cell types upon blastocyst injection. Established iPSC can be endowed with the ability to give rise to extraembryonic (ExE) tissues by culture in two alternative approaches targeting indicated molecules. B) Prolonged OKSM expression in adult transgenic mice yields intermediates as well as senescent cells that support reprogramming by secretion of factors such as IL-6. Culture of circulation-derived intermediates and ex vivo culture yields iPSCs that not only can give rise to extraembryonic and embryonic tissues. C) An intermediate interval of OKSM expression in vivo triggers partial epigenetic remodeling and in tissue such as kidney the emergence of transplantable tumors with molecular features of tissue-specific embryonic progenitor cells. D) Repeated short intervals of OKSM expression in vivo lead to reversal of molecular features of aging and functional restoration of tissue function in aged animals.

Figure 2. Mechanisms and molecular consequences of OKSM binding

OKSM impact on the silencing of the somatic program, activation of pluripotency program and topological reorganization of the chromatin are illustrated. Direct mechanisms assume that OSK(M) binding is sufficient to induce transcriptional or topological changes in cis by recruiting the necessary cofactors that are already available in the nuclear milieu. Indirect mechanisms rely on epigenetic and transcriptional changes that occur at different genomic sites in an OKSM-dependent or independent manner and result in the activation of critical co-factors (co-activators or co-repressors) that mediate the silencing or activation of OKSM target genes.