Cell cycle dynamics of human pluripotent stem cells primed for differentiation

Abstract

Understanding the molecular properties of the cell cycle of human pluripotent stem cells (hPSCs) is critical for effectively promoting differentiation. Here, we use the Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) system adapted into hPSCs and perform RNA-sequencing on cell cycle sorted hPSCs primed and unprimed for differentiation. Gene expression patterns of signaling factors and developmental regulators change in a cell cycle-specific manner in cells primed for differentiation without altering genes associated with pluripotency. Furthermore, we identify an important role for PI3K signaling in regulating the early transitory states of hPSCs towards differentiation. INTRODUCTIONDespite recent advances in generating specialized cell types from human pluripotent stem cells (hPSCs), many studies have noted that pluripotent stem cell lines often have an inherent inability to differentiate even when stimulated with a proper set of signals 1-5 . The cell cycle, particularly the G1 phase, may play an important role in enhancing the differentiation potential of PSCs 2,6-9 . However, simply lengthening the G1 phase in embryonic stem cells is not sufficient to facilitate differentiation 10 , suggesting that an improved understanding of the molecular properties of the embryonic cell cycle is needed.In a prior study, we demonstrated that transiently treating hPSCs with dimethylsulfoxide (DMSO) for 24h prior to directed differentiation significantly increases the propensity for differentiation across all germ layers. This technique is now used by multiple laboratories to improve differentiation across species (including mouse, rabbit, primate, and human) into more than a dozen lineages, ranging from neurons and cortical spheroids to smooth muscle cells to hepatocytes [11][12][13][14][15][16][17][18][19][20][21][22][23] . While the DMSO treatment activates the retinoblastoma protein (Rb) and increases the percentage of hPSCs in the G1 phase of the cell cycle 2 , 24 , it remains unknown whether the DMSO treatment simply enriches cells in G1 or whether there are intrinsic changes to the cell cycle following the DMSO treatment that may potentiate differentiation.Here, we use Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology to systematically track and understand cell division in hPSCs primed and unprimed for differentiation. The FUCCI system fuses red-and green-emitting fluorescent proteins to the cell cycle ubiquitination oscillators, Cdt1 and Geminin, whereby cdt1 tagged with RFP is present only when cells are in G1 and geminin tagged with GFP is only present when cells reside in the S/G2/M phases 25 . By performing RNA-sequencing on hPSCs sorted from the early G1, late G1, and SG2M phases of the cell cycle, we show that gene expression patterns of signaling factors and developmental regulators change in a cell cycle-specific manner in cells primed for differentiation following a 24h DMSO treatment. Changes in signaling pathways controlling cell proliferation, differentiation, and apoptosis, particularly th...