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Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells.

ABSTRACT: 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.

SUBMITTER: Wang R

PROVIDER: S-EPMC3408772 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells.

Wang Ruoxing R Guo Yan-Lin YL

Experimental cell research 20120613 16

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 ...[more]

PMID: 22705123

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Project description:Successful realization of the enormous potential of pluripotent stem cells in regenerative medicine demands the development of well-defined culture conditions. Maintenance of embryonic stem cells (ESCs) typically requires co-culture with feeder layer cells, generally mouse embryonic fibroblasts (MEFs). Concerns about xenogeneic pathogen contamination and immune reaction to feeder cells underlie the need for ensuring the safety and efficacy of future stem cell-based products through the development of a controlled culture environment. To gain insight into the effectiveness of MEF layers, here we have developed a biomimetic synthetic feeder layer (BSFL) that is acellular and replicates the stiffness and topography of MEFs. The mechanical properties of MEFs were measured using atomic force microscopy. The average Young's modulus of the MEF monolayers was replicated using tunable polyacrylamide (PA) gels. BSFLs replicated topographical features of the MEFs, including cellular, subcellular, and cytoskeletal features. On BSFLs, mouse ESCs adhered and formed compact round colonies; similar to on MEF controls but not on Flat PA. ESCs on BSFLs maintained their pluripotency and self-renewal across passages, formed embryoid bodies and differentiated into progenitors of the three germ layers. This acellular biomimetic synthetic feeder layer supports stem cell culture without requiring co-culture of live xenogeneic feeder cells, and provides a versatile, tailorable platform for investigating stem cell growth.Embryonic stem cells have enormous potential to aid therapeutics, because they can renew themselves and become different cell types. This study addresses a key challenge for ESC use - growing them safely for human patients. ESCs typically grow with a feeder layer of mouse fibroblasts. Since patients have a risk of immune response to feeder layer cells, we have developed a material to mimic the feeder layer and eliminate this risk. We investigated the influence of feeder layer topography and stiffness on mouse ESCs. While the biomimetic synthetic feeder layer contains no live cells, it replicates the stiffness and topography of feeder layer cells. Significantly, ESCs grown on BSFLs retain their abilities to grow and become multiple cell types.

Project description:Due to the limited understanding of self-renewal and pluripotency related signaling in stem cells, extracting information from genome-wide expression data is not only important but also challenging. With the combined use of two methods, we analyzed a set of microarray data at 11 time points from three mouse embryonic stem cell lines cultivated with and without leukemia inhibitory factor (LIF) for 14 days. Albeit the expression of individual genes in signaling pathways was not noticeably different between cells cultivated with and without LIF, at gene-set level the expression of ERK/MAPK (but not JAK/STAT) and cell cycle related genes was found significantly enriched in cells cultivated with LIF. This indicates that the Ras/Raf/ERK pathway, in addition to JAK/STAT, may also be a key player to carry on external LIF signal into mouse embryonic stem cells to promote self-renewal. When data at the first 7 time points were compared with data at the last 4 time points, the expression of several cell cycle related gene sets was apparently enriched in all three cell lines, indicating the active cell proliferation in the first 2 days. Compared with the slight decay of Oct4/Nanog/Sox2 during the 14 days, the expression of cell differentiation genes such as Gata4/6 underwent a drastic increase, which indicates that the upregulated expression of cell differentiation genes may better reflect the loss of self renewal than the down regulated expression of the stemness indicators Oct4, Sox2 and Nanog. Apart from differential expression and gene set enrichment analyses, a clustering algorithm was also used to classify genes into co-expression clusters. The possible regulation of two clusters, whose expression was most changed during cell culture from very low to very high, was explored. The drastic changes of these genes, including Slc39a8 which was a potential indicator of cell differentiation, in contrast the slight changes of self-renewal genes, imply that differentiation may be the default fate of stem cells and self-renewal may rely on a maintenance mechanism. When that mechanism weakens, cell differentiation begins.

Project description:Self-renewal of embryonic stem cells (ESCs) is orchestrated by a vast number of genes at the transcriptional and translational levels. However, the molecular mechanisms of post-translational regulatory factors in ESC self-renewal remain unclear. Histidine phosphorylation, also known as hidden phosphorylation, cannot be detected by conventional experimental methods. A recent study defined phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) as a histidine phosphatase, which regulates various biological behaviors in cells via histidine dephosphorylation. In this study, the doxycycline (DOX)-induced hLHPP-overexpressing mouse ESCs and mouse LHPP silenced mESCs were constructed. Quantitative polymerase chain reaction (qPCR), western blotting analysis, immunofluorescence, Flow cytometry, colony formation assays, alkaline phosphatase (AP) and bromodeoxyuridine (Brdu) staining were performed. We found that the histidine phosphorylation level was strikingly reduced following LHPP overexpression. Besides, the expression of Oct4 and Lefty1, indispensable genes in the process of ESCs self-renewal, was significantly down-regulated, while markers related to the differentiation were markedly elevated. Moreover, LHPP-mediated histidine dephosphorylation induced G0/G1 phase arrest in mESCs, suggesting LHPP was implicated in cell proliferation and cell cycle. Conversely, silencing of Lhpp promoted the self-renewal of mESCs and reversed the RA induced increased expression of genes associated with differentiation. Mechanistically, our findings suggested that the enzymatic active site of LHPP was the cysteine residue at position 226, not 53. LHPP-mediated histidine dephosphorylation lowered the expression levels of β-catenin and the cell cycle-related genes CDK4 and CyclinD1, while it up-regulated the cell cycle suppressor genes P21 and P27. Taken together, our findings reveal that LHPP-mediated histidine dephosphorylation plays a role in the self-renewal of ESCs. LHPP-mediated histidine dephosphorylation inhibited the self-renewal of ESCs by negatively regulating the Wnt/β-catenin pathway and downstream cell cycle-related genes, providing a new perspective and regulatory target for ESCs self-renewal.

Project description:BackgroundSelf-renewal and pluripotency are considered as unwavering features of embryonic stem cells (ESCs). How ESCs regulate the self-renewal and differentiation is a central question in development and regenerative medicine research. Epidermal growth factor receptor (EGFR) was identified as a critical regulator in embryonic development, but its role in the maintenance of ESCs is poorly understood.MethodsHere, EGFR was disrupted by its specific inhibitor AG1478 in mouse ESCs (mESCs), and its self-renewal and pluripotency were characterized according to their proliferation, expression of pluripotency markers, embryoid body (EB) formation, and mRNA expression patterns. We also used another EGFR inhibitor (gefitinib) and RNA interference assay to rule out the possibility of non-specific effects of AG1478.ResultsEGFR inhibition by AG1478 treatment in mESCs markedly reduced cell proliferation, caused cell cycle arrest at G0/G1 phase, and altered protein expressions of the cell cycle regulatory genes (CDK2 (decreased 11.3%) and proliferating cell nuclear antigen (decreased 25.2%)). The immunoreactivities and protein expression of pluripotency factors (OCT4 (decreased 26.9%)) also dramatically decreased, while the differentiation related genes (GATA4 (increased 1.6-fold)) were up-regulated in mESCs after EGFR inhibition. Meanwhile, EGFR inhibition in mESCs disrupted EB formation, indicating its impaired pluripotency. Additionally, the effects observed by EGFR inhibition with another inhibitor gefitinib and siRNA were consistent with those observed by AG1478 treatment in mESCs. These effects were manifested in the decreased expression of proliferative and pluripotency-related genes and the increased expression of genes involved in differentiation. Moreover, RNA-seq analysis displayed that transcript profiling was changed significantly after EGFR inhibition by AG1478. A large number of differentially expressed genes were involved in cell cycle, apoptotic process, epigenetic modification, and metabolic process, which were related to self-renewal and pluripotency, confirming that EGFR deficiency impaired self-renewal and pluripotency in mESCs.ConclusionsTaken together, our results demonstrated the importance of EGFR in guarding the stemness of mESCs.

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Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells.

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Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells.

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