Defining Reprogramming Checkpoints from Single-Cell Analyses of Induced Pluripotency

Abstract

SUMMARY Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.

Dot1L methyltransferase activity is a barrier to acquisition of pluripotency but not transdifferentiation

“…Since d4 Dot1Li has a greater number of upregulated genes, we looked for known pluripotency factors. In a previously reported single cell transcriptomic analysis from our lab, we found that the co-expression of a quartet of four genes Nanog, Sall4, Tdgf1 and Epcam within the same cell predicted a more homogenous transition to an iPSC state (Tran et al, 2019).…”

“…Mesenchymal to epithelial transition is an early phase of reprogramming when starting with MEFs (Apostolou and Hochedlinger, 2013). From our co-expression analysis of genes in single cells, we have found that mesenchymal gene downregulation and Cdh1 upregulation are regulated independent of each other such that cells simultaneously express genes from both programs (Tran et al, 2019). Given that overexpression of Cdh1 alone could not replace Dot1Li function (Fig 4I), we assessed how reduction of H3K79me affects reprogramming of cells that are already epithelial.…”

Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity

“…To establish whether the Tet enzymes function at different phases of reprogramming, we first determined their expression patterns. Since reprogramming is a low‐efficiency process where expression patterns can be obscured by bulk RNA sequencing, we analyzed our single‐cell transcription data during a reprogramming time course (Fig A; Tran et al , ). We found that Tet2 is upregulated earlier in reprogramming similar to the activation of Cdh1 by day 6 (Fig B).…”

“… tSNE of Tet1, Tet2, Nanog, and Cdh1 expression in single‐cell RNA sequencing of MEF undergoing OSKM reprogramming from Tran et al (). MEF, reprogramming (REPROG), and mESCs cells are highlighted in red dotted lines.…”

“…(GSE108222) using MEF reprogramming populations (days 3, 6, 9, and 12) and ESCs (See “Datasets used in the study” Table ). Data were processed as in Tran et al (). Briefly, FASTQ files were aligned to mm10 genome using Spliced Transcripts Alignment to a Reference (STAR) and single cells were identified using Illumina BaseSpace SureCell RNA Single‐Cell Application.…”