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Reprogramming MEFs (mouse iPSCs)

ABSTRACT: Proteome of reprogramming mouse embryonic fibroblasts (MEFs) with or without Akt activation

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Tohru Kimura

PROVIDER: PXD014856 | JPOST Repository | Fri Jul 31 00:00:00 BST 2020

REPOSITORIES: jPOST

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			Action	DRS
	Multiconsensus%20from%206%20Reports.xlsx	Xlsx
	Qskt001_001_mouse_iPS-40HT_1_0o05ug_5uL_T160-01.msf	Msf
	Qskt001_001_mouse_iPS-40HT_1_0o05ug_5uL_T160.mgf	Mgf
	Qskt001_001_mouse_iPS-40HT_1_0o05ug_5uL_T160.raw	Raw
	Qskt001_002_mouse_iPS-40HT_2_0o06ug_5uL_T160-01.msf	Msf

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Project description:In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1-11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming the cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed that the transcriptional regulators Nr0b1 and Etv5 are specifically expressed in this early reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study shows an ordered sequence of transitions during the earliest steps of iPS cell reprogramming that deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. Samples for poised (CD73+ or CD49d+) and non-poised (CD73-) reprogramming samples were FACS sorted 6 and 9 days after induction of Klf4, Oct4, Sox2 and cMyc in Rosa-rtTA +/- mouse embryonic fibroblasts (MEFs). 'Total' populations are expression analyses for unsorted populations analyzed at the same time points. Control populations were also sampled: mouse embryonic fibroblasts (MEFs), partially reprogrammed cells (SC4) and mouse embryonic stem cell (ESC).

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Reprogramming MEFs (mouse iPSCs)

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