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Role of circadian gene Clock during differentiation of mouse pluripotent stem cells.

ABSTRACT: Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.

SUBMITTER: Lu C

PROVIDER: S-EPMC5084156 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Role of circadian gene Clock during differentiation of mouse pluripotent stem cells.

Lu Chao C Yang Yang Y Zhao Ran R Hua Bingxuan B Xu Chen C Yan Zuoqin Z Sun Ning N Qian Ruizhe R

Protein & cell 20160923 11

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadi ...[more]

PMID: 27664156

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Project description:Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and are, therefore, expected to be useful for bone regenerative medicine; however, the characteristics of iPSC-derived osteogenic cells remain unclear. Here, we provide a direct in vitro comparison of the osteogenic differentiation process in mesenchymal stem cells (MSCs) and iPSCs from adult C57BL/6J mice. After 30 days of culture in osteogenic medium, both MSCs and iPSCs produced robustly mineralized bone nodules that contained abundant calcium phosphate with hydroxyapatite crystal formation. Mineral deposition was significantly higher in iPSC cultures than in MSC cultures. Scanning electron microscopy revealed budding matrix vesicles in early osteogenic iPSCs; subsequently, the vesicles propagated to exhibit robust mineralization without rich fibrous structures. Early osteogenic MSCs showed deposition of many matrix vesicles in abundant collagen fibrils that became solid mineralized structures. Both cell types demonstrated increased expression of osteogenic marker genes, such as runx2, osterix, dlx5, bone sialoprotein (BSP), and osteocalcin, during osteogenesis; however, real-time reverse transcription-polymerase chain reaction array analysis revealed that osteogenesis-related genes encoding mineralization-associated molecules, bone morphogenetic proteins, and extracellular matrix collagens were differentially expressed between iPSCs and MSCs. These data suggest that iPSCs are capable of differentiation into mature osteoblasts whose associated hydroxyapatite has a crystal structure similar to that of MSC-associated hydroxyapatite; however, the transcriptional differences between iPSCs and MSCs could result in differences in the mineral and matrix environments of the bone nodules. Determining the biological mechanisms underlying cell-specific differences in mineralization during in vitro iPSC osteogenesis may facilitate the development of clinically effective engineered bone.

Project description:BackgroundUnderstanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1-4 EBs.ResultsAn initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile.ConclusionES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results. EBs cultured in the same dish vary widely in terms of their gene expression (and hence, undoubtedly, in their future differentiation potential). This may explain some of the inherent variability in differentiation protocols that use EBs.

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Role of circadian gene Clock during differentiation of mouse pluripotent stem cells.

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Role of circadian gene Clock during differentiation of mouse pluripotent stem cells.

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