Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs.
[Show abstract][Hide abstract] ABSTRACT: Heterogeneity is an often unappreciated characteristic of stem cell populations yet its importance in fate determination is becoming increasingly evident. Although gene expression noise has received greater attention as a source of non-genetic heterogeneity, the effects of stochastic partitioning of cellular material during mitosis on population variability have not been researched to date. We examined self-renewing human embryonic stem cells (hESCs), which typically exhibit a dispersed distribution of the pluripotency marker NANOG. In conjunction with our experiments, a multiscale cell population balance equation (PBE) model was constructed accounting for transcriptional noise and stochastic partitioning at division as sources of population heterogeneity. Cultured hESCs maintained time-invariant profiles of size and NANOG expression and the data were utilized for parameter estimation. Contributions from both sources considered in this study were significant on the NANOG profile, although elimination of the gene expression noise resulted in greater changes in the dispersion of the NANOG distribution. Moreover, blocking of division by treating hESCs with nocodazole or colcemid led to a 39% increase in the average NANOG content and over 68% of the cells had higher NANOG level than the mean NANOG expression of untreated cells. Model predictions, which were in excellent agreement with these findings, revealed that stochastic partitioning accounted for 17% of the total noise in the NANOG profile of self-renewing hESCs. The computational framework developed in this study will aid in gaining a deeper understanding of how pluripotent stem/progenitor cells orchestrate processes such as gene expression and proliferation for maintaining their pluripotency or differentiating along particular lineages. Such models will be essential in designing and optimizing efficient differentiation strategies and bioprocesses for the production of therapeutically suitable stem cell progeny.
PLoS ONE 11/2012; 7(11):e50715. DOI:10.1371/journal.pone.0050715 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem cells (ESCs) are considered to be a promising cell source for regenerative medicine because of their unlimited capacity for self-renewal and differentiation. However, little is known about the innate immunity in ESCs and ESC-derived cells. We investigated the responses of mESCs to three types of live viruses; La Crosse virus (LACV), West Nile virus (WNV), and Sendai virus (SeV). Our results demonstrated mESCs were susceptible to viral infection, but they were unable to express type I interferons (IFNα and IFNβ,IFNα/β which differ from fibroblasts (10T1/2 cells) that robustly express IFNα/β upon viral infections. The failure of mESCs to express IFNα/β was further demonstrated by treatment with polyIC (polyinosinic-polycytidylic), a synthetic viral dsRNA analog that strongly induced IFNα/β in 10T1/2 cells. Although polyIC transiently inhibited the transcription of pluripotency markers, the stem cell morphology was not significantly affected. However, polyIC can induce dsRNA-activated protein kinase (PKR) in mESCs and this activation resulted in a strong inhibition of cell proliferation. We conclude that the cytosolic receptor PKR is functional, but the mechanisms that mediate type I IFN expression are deficient in mESCs. This conclusion is further supported by the findings that the major viral RNA receptors are either expressed at very low levels (TLR3 and MDA5) or may not be active (RIG-I) in mESCs.
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem (ES) cells are originated from the inner cell mass of a blastocyst stage embryo. They can proliferate indefinitely, maintain an undifferentiated state (self-renewal), and differentiate into any cell type (pluripotency). ES cells have an unusual cell cycle structure, consists mainly of S phase cells, a short G1 phase and absence of G1/S checkpoint. Cell division and cell cycle progression are controlled by mechanisms ensuring the accurate transmission of genetic information from generation to generation. Therefore, control of cell cycle is a complicated process, involving several signaling pathways. Although great progress has been made on the molecular mechanisms involved in the regulation of ES cell cycle, many regulatory mechanisms remain unknown. This review summarizes the current knowledge about the molecular mechanisms regulating the cell cycle of ES cells and describes the relationship existing between cell cycle progression and the self-renewal.
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