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.

Defining reprogramming checkpoints from single-cell analysis of induced pluripotency

“…These studies have led to an understanding of reprogramming trajectories taken by the majority of the cells. Here, by applying single-cell transcriptional sequencing, we find that there is overlapping expression of genes that were thought to be temporally activated (Apostolou and Stadtfeld, 2018;Brambrink et al, 2008;Stadtfeld and Hochedlinger, 2010) (Fig 7). Since most studies have focused on MEFs as the starting cell type, an important early event is the mesenchymal to epithelial transition, a process amenable to acceleration (Liang et al, 2012;Zhou et al, 2017).…”

“…From bulk sequencing experiments, it is thought that downregulation of somatic cell gene expression, including the mesenchymal genes, are early events in reprogramming (Apostolou and Hochedlinger, 2013;Apostolou and Stadtfeld, 2018;R. Li et al, 2010;Samavarchi-Tehrani et al, 2010).…”

“…Expression of several collagens, Egr1, and Twist 1 (group C), was retained in an even higher proportion of cells than group B ( Fig 3A). Thus, there are three different trends for populations to lose mesenchymal gene expression with a large majority of cells in FBS reprogramming still retaining MEF-like gene expression even at later timepoints The mesenchymal MEFs have to transition to an epithelial state indicated by the upregulation of E-cadherin (Cdh1) (Apostolou and Hochedlinger, 2013;Apostolou and Stadtfeld, 2018;R. Li et al, 2010;Samavarchi-Tehrani et al, 2010).…”

“…Transcriptional profiling of bulk reprogramming populations over time has led to the description of a temporal series of events with early downregulation of somatic cell expression followed by metabolic and cell cycle changes that culminates in the activation of the pluripotency gene regulatory network (Apostolou and Hochedlinger, 2013;Apostolou and Stadtfeld, 2018). Mouse embryonic fibroblasts (MEFs) undergo a mesenchymal to epithelial transition (MET) before pluripotency gene activation during reprogramming (Hussein et al, 2014;R.…”

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

“…Notwithstanding the highly justified initial enthusiasm associated with the laudable discoveries that led to the development of the above method(s), it does appear today that the associated risks and complexities, as well as the time-consuming nature of the methods, make it necessary for us to search for a more efficient, economical, risk-free and ethically acceptable method of inducing either pluripotency or multi-potency (Brouwer et al, 2016;Borgohain et al, 2018;Kogut et al, 2018;Apostolou and Stadtfeld 2018;Badieyan and Evans;2019;Shivashankar 2019;Gagliano et al, 2019;Haake et al, 2019).…”