Lengthened G1 Phase Indicates Differentiation Status in Human Embryonic Stem Cells

Abstract

Human embryonic stem cells (hESC) have potential applications as tools for drug screening to identify small molecule regulators of self-renewal or differentiation. Elucidating the mechanisms governing lineage commitment in hESC will allow for efficient derivation of specified cell types for clinical use. Recognizing the early steps in loss of pluripotency is key to achieving both goals of drug screening and derivation of therapeutically relevant cell types. Here we report the use of a real time cell cycle fluorescent reporter for the first time in hESC that indicates onset of differentiation in a lineage unbiased manner. Pluripotent hESC possess a short cell cycle length, due primarily to a truncated G1 phase. G1 lengthens concomitant with differentiation. Stable hESC lines expressing the live cell cycle reporter exhibit fluorescence only during G1. Due to the short length of pluripotent G1 phase, G1 fluorescence is only weakly and transiently detected, however it is quickly increased to easily detectable levels upon onset of differentiation. We hypothesize that lengthened G1 phase can be used as an indicator of differentiation status of individual human embryonic stem cells. Cells with lengthened G1 are typically negative for pluripotency markers OCT4, Tra-1-60 and SSEA-3 following differentiation. Differentiated cells with lengthened G1 also demonstrate increased levels of lineage-specific differentiation markers at both the protein and mRNA level. Automated image analysis of hESC indicates this mutually exclusive relationship between lengthened G1 and pluripotency exists both on the cellular level and in colonies as a whole. Here we have shown that lengthened G1 indicates both loss of pluripotency and gain of lineage markers.

Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

“…We show here that EndoC-βH2 Fucci derivatives should give additional grips to further experimentally assess these events. In some stem cells/progenitors, such as mouse neural stem cells/progenitors or human embryonic stem cells, an increasing intensity of the orange Fucci indicator reflects ongoing differentiation most probably because both events are linked to G1 lengthening [2], [37], [38]. In the same way, FACS sorting experiments revealed that the mRNA levels of two markers of mature beta cells, INSULIN and IAPP , are higher in the brightest orange Cre-transduced EndoC-βH2 Fucci sub-population than in other orange Cre-transduced cells and, to a much greater extent, than in control (Zeo-transduced) orange cells.…”

Initiation of stem cell differentiation involves cell cycle-dependent regulation of developmental genes by Cyclin D

“…The importance of these mechanisms has been demonstrated in adult stem cells (Oshimori and Fuchs 2012) and also in pluripotent stem cells (Sela et al 2012;Calder et al 2013;Coronado et al 2013;Singh et al 2013Singh et al , 2015. Human embryonic stem cells (hESCs) represent an advantageous model system for investigating the cross-talk between cell cycle and cell fate choice.…”

Atypical heterochromatin organization and replication are rapidly acquired by somatic cells following fusion-mediated reprogramming by mouse ESCs

“…The likely importance of this altered cell cycle structure for maintaining pluripotency is underscored by evidence that pluripotent cells 12 and ESCs lose this unusual profile upon differentiation, [13][14][15][16] and, conversely, that somatic cells regain it when reprogrammed. 17,18 We recently used a biophysical separation method to fractionate mouse ESCs according to size and cell cycle stage.…”