Study Of Vitamin C Promoting Mouse Embryonic Stem Cells Maintenance

Vitamin C has recently received a great deal of attention because of its ability to improveinduced pluripotent stem cells (iPSCs) generation and maintain a blastocyst-like state inembryonic stem cells (ESCs). Pioneering studies have linked vitamin C with epigeneticmodification during pluripotency maintenance in ESCs including DNA methylation andhistone modification. Besides, an increasing number of studies have focused on the uniqueexpression pattern of miRNAs in ESCs, indicating the important role of miRNAs inpluripotency maintenance. The miRNAs specific to ESCs enhance the production of inducediPSCs.Here, for the first time we studied the effect of vitamin C on pluripotency geneexpression and characterized the dynamic expression profile of miRNAs with vitamin Ctreatment in mouse ESCs (mESCs). We attempted to delineate the network of vitamin C onmaintaining pluripotency of ESC.1. Vitamin C maintained self-renewal and colony morphology of J1mESCs underfeeder-free conditions. We examined colony morphology and alkaline phosphatase (AP)activity of J1mESCs at48h after vitamin C treatment. We found that J1mESCs grew toform larger colonies after vitamin C treatment, and AP activity was higher compared with thatin the control. Furthermore, the colonies of differentiated J1mESCs induced by retinoic acid(RA) were elongated and had a much lower AP activity. In contrast, addition of vitamin Cnoticeably reduced the number of differentiated cells, maintained better colony morphology,and increased the AP activity compared with that in the RA group. The results indicated thatvitamin C promotes the maintenance of pluripotency and inhibits differentiation.2. Vitamin C facilitated series of pluripotency genes transcription and repressed somesignaling related to proliferation and developmental progression.a) Gene expression profiling revealed that vitamin C promoted pluripotency genesexpression in J1mESCs. We performed microarray anlaysis of J1mESCs with the treatmentof vitamin C or RA to identify divergent gene expression. We found an opposing trend in the variation of gene expression and significant differences between vitamin C and RA groups.Up-regulated genes of vitamin C were mainly associated with the response to oxidative stressand regulation of transcription including series of important pluripotency genes such as Esrrb,Utf1, Klf4, Tcl1, Eras and Nanog, while down-regulated genes were grouped mainly in theregulation of differentiation, development, cell adhesion and biosynthetic processes.b) Vitamin C facilitated key pluripotency genes transcription. We evaluated the promoteractivity of the key pluripotency genes including Oct4, Nanog, Sox2and Klf4by a luciferasereporter assay. The results indicated that vitamin C enhanced Oct4and Nanog promoteractivities directly. Furthermore, real-time quantitative PCR (qPCR) and western blot assaysconfirmed that vitamin C increased the Nanog mRNA level and protein expression.c) Vitamin C regulated signaling pathways in pluripotent stem cells. Pathway profilingvectors were used to screen potential vitamin C signaling. We found that vitamin Cdown-regulated some signaling involved in cell proliferation, the cell cycle anddifferentiation, including NF-κB, JNK and Myc. The results indicated that vitamin C represssome signaling related to proliferation and developmental progression.3. Vitamin C maintained ESC-specific miRNAs expression and vitamin C mediatedmiRNAs regulating gene expression in mESCs, repressing a class of genes linking to ESCdifferentiation and development.a) Vitamin C maintained ESC-specific miRNAs expression level in mESC. The globalmiRNA expression profiling was measured using small RNA sequencing on Illumina HiSeq2000. We found that vitamin C treatment maintained most miRNAs expression levels andinduced a portion miRNAs up-regulating in the ESCs, especially ESC-specific miRNAsclusters including miR-290–295, miR-17–92and miR-106b–25clusters, and Dlk1-Dio3region encoding miRNAs cluster, which was confirmed by miRNA qPCR.b) Vitamin C mediated miRNAs regulating gene expression in mESCs, repressing aclass of genes linking to ESCs differentiation and development. We selected several miRNAtargets predicted in silicon combination TargetScan and RNA22, cloned into dual luciferasereporter vector and performed luciferase reporter detection. We found that vitamin C-inducedmiRNAs commonly targeted a class of genes regulation of transcription, mainly linking toESCs differentiation, development and proliferation. Interestingly, cluster analysis of geneexpression profiling showed that the miRNA targets above were down-regulated by vitaminC widespread. Taken together, these results indicated that vitamin C mediated miRNAsregulating gene expression in mESCs, repressing a class of genes linking to ESCsdifferentiation and development.4. Vitamin C mediated miRNAs promoting DNA demethylation in gene promoters in mESC.a) Vitamin C suppressed Dnmt3a expression by inducing miR-143. miR-143evidentlyenhanced the AP activity and key pluripotency genes expression in J1mESCs and inhibiteddifferentiation induced by RA. We overexpressed and knockdown miR-143using its inhibitorunder vitamin C treatment. Dnmt3a mRNA was detected using qPCR, whereas protein levelwas determined through immunoblotting in J1mESCs. We found that miR-143repressedDnmt3a at post-transcriptional level. Moreover, vitamin C decreased Dnmt3a mRNA proteincompared with that of the control. Nevertheless, the knockdown of miR-143using itsinhibitor resisted the Dnmt3a reduction induced by vitamin C. Taken together with theprevious result that vitamin C increased miR-143expression, these findings indicated thatvitamin C suppressed Dnmt3a expression via miR-143in mESCs.b) Vitamin C promoted DNA demethylation in genes promoters of pluripotency factorsand ESC-specific miRNA clusters. We detected methylation enrichment of promoters ofpluripotency factors, ESC-specially miRNA clusters and intergenic differentially methylatedregion (IG-DMR) of Dlk1-Dio3imprinting region with vitamin C treatment using methylatedDNA immunoprecipitation (MeDIP) assay. We found that the methylation level ofpluripotency gene promoters, including Tbx3, Tcl1, Esrrb, and ESC-specific miRNA clusterpromoters, including miR-290–295and miR-17–92clusters, significantly decreased aftervitamin C treatment. These indicated that vitamin C induced pluripotency factors andESC-specially miRNA expression by promoting DNA demethylation in gene promoters.c) Vitamin C induced Tet1expression and increased hydroxymethylation in IG-DMR ofDlk1-Dio3imprinting region. We detected Tet1mRNA expression by qPCR with vitamin Ctreatment and detected hydroxymethylation enrichment using hMeDIP assay. We found thatvitamin C induced Tet1expression and the hydroxymethylation level of intergenicdifferentially methylated region (IG-DMR) of the Dlk1–Dio3region, evidently increased inresponse to vitamin C. These findings may be attributed to the changes in the Tet1expressioninduced by vitamin C. Thus, vitamin C promotes gene demethylation by regulation of Tet andDnmt3a expression.Overall, our work suggested that vitamin C is capable of maintaining ESCs self-renewaland pluripotency via regulating gene transcriptions and miRNA expression, which provideinsight into the future reearch and application of ESCs and iPSCs.