Prospective Isolation of Poised iPSC Intermediates Reveals Principles of Cellular Reprogramming

Abstract

Cellular reprogramming converts differentiated cells into induced pluripotent stem cells (iPSCs). However, this process is typically very inefficient, complicating mechanistic studies. We identified and molecularly characterized rare, early intermediates poised to reprogram with up to 95% efficiency, without perturbing additional genes or pathways, during iPSC generation from mouse embryonic fibroblasts. Analysis of these cells uncovered transcription factors (e.g., Tfap2c and Bex2) that are important for reprogramming but dispensable for pluripotency maintenance. Additionally, we observed striking patterns of chromatin hyperaccessibility at pluripotency loci, which preceded gene expression in poised intermediates. Finally, inspection of these hyperaccessible regions revealed an early wave of DNA demethylation that is uncoupled from de novo methylation of somatic regions late in reprogramming. Our study underscores the importance of investigating rare intermediates poised to produce iPSCs, provides insights into reprogramming mechanisms, and offers a valuable resource for the dissection of transcriptional and epigenetic dynamics intrinsic to cell fate change.

Multi-Omic Profiling Reveals Dynamics of the Phased Progression of Pluripotency

“…In summary, our studies provide a comprehensive molecular description of the phased progression of pluripotency. Our data, together with the complementary data describing sequence of molecular events inherent to reprogramming somatic cells into iPSCs (Cacchiarelli et al, 2015;Chronis et al, 2017;Polo et al, 2012;Schwarz et al, 2018), provide a foundation for investigating mechanisms that regulate pluripotent state transitions. The general framework we employed to gain insights into the multi-layered control of pluripotent cell fate transitions is a paradigm that can readily be used to investigate any differentiation process.…”

High Throughput Mechanobiological Screens Enable Mechanical Priming of Pluripotency in Mouse Fibroblasts

“…To further confirm the expression of pluripotency related markers in the MEFs, we treated MEFs with a subset of conditions from our high throughput screen and then further analyzed these cells for the expression of pluripotency related transcription factors and gene expression for markers related to different stages of pluripotency (Schwarz et al, 2018). We found significant increases in SOX2 and NANOG with the brachial waveform and kinase inhibitors by immunostaining ( Fig.…”

Dynamic Erasure of Random X-Chromosome Inactivation during iPSC Reprogramming

“…To define the kinetics of XCR with allele-resolution, we isolated, at different time points, reprogramming intermediates marked by reactivation of the cell surface marker SSEA1 ( Figure S1C). SSEA1 has been shown to mark cells poised for successful acquisition of the pluripotency program and XCR (38)(39)(40). The first SSEA1 positive (+) cells isolated did not show significant GFP fluorescence, then gradually reactivated GFP, while fully reprogrammed iPSCs were mostly GFP+ ( Figure S1C).…”

“…Additionally, the initiation of XCR during reprogramming is challenging to capture due to the degree of heterogeneity associated with factor-induced iPSC reprogramming. To minimize the variability, our study provides information from SSEA1 sorted cells that have been shown as a robust marker of cells poised to reprogram successfully (38). Nevertheless, single cell allele-resolution approaches would allow to pinpoint most upstream events of XCR.…”