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Defining Reprogramming Checkpoints from Single-Cell Analyses of Induced Pluripotency.

Cell reports (2019-05-09)
Khoa A Tran, Stefan J Pietrzak, Nur Zafirah Zaidan, Alireza Fotuhi Siahpirani, Sunnie Grace McCalla, Amber S Zhou, Gopal Iyer, Sushmita Roy, Rupa Sridharan
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate, ≥95%
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), powder, BioReagent, suitable for cell culture