Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells
Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive # 5018, Hattiesburg, MS 39406, USA. Experimental Cell Research
(Impact Factor: 3.25).
06/2012; 318(16):2094-104. DOI: 10.1016/j.yexcr.2012.05.017
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.
Available from: liebertonline.com
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ABSTRACT: Previously, we have shown that increasing the intracellular cGMP concentration enhances the sonic hedgehog (Shh) response in neural plate cells. The use of two mouse embryonic stem (ES) cell lines allowed a highly sensitive and reproducible quantification of the Shh response in neuralized embryoid bodies. Here we demonstrate that the specific, membrane-permeable cGMP-dependent protein kinase G-Ialpha (PKG-Ialpha) inhibitor DT-2 prevents an efficient Shh response, indicating that the effects of cGMP on the Shh response are mediated via PKG. We also demonstrate that the PKG acts upon the Shh response upstream of the Ptc1 promoter, which is up-regulated invariably and early in response to Shh, significantly limiting the targets for PKG phosphorylation to molecules involved in the early steps of the Shh response. These effects of cGMP and PKG are antagonistic to those of cAMP and PKA, and thus provide a mechanism by which the sensitivity of cells to the effects of Shh can be regulated, by modulating the intracellular cyclic nucleotide concentration.
Stem Cells and Development 11/2006; 15(5):647-54. DOI:10.1089/scd.2006.15.647 · 3.73 Impact Factor
Available from: Emmanuel S Tzanakakis
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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
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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.
Journal of Biological Chemistry 04/2013; 288(22). DOI:10.1074/jbc.M112.421438 · 4.57 Impact Factor
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