Histone methylation and microRNA mediated regulation of the multipotential state of Flk1+ mesenchymal stem cells

Stem cells hold great potential for regenerative medicine. We firstly isolated a type of mesenchymal stem cells named Flk1 + ‐MSCs (Flk1 + CD44 + CD29 + CD105 + CD166 + CD 34 − CD31 − Lin − ) from a variety of human fetal and adult tissues and found they have mutilineage differentiatiation potential. Flk1 + ‐MSCs could give rise to cells of endothelial, hepatic epithelial, neural, hematopoietic, adipogenic and osteogenic lineages. To explore the mechanisms underlying their multilineage state, we performed a genome‐wide analysis on H3K4me3 and H3K27me3 profiles in Flk1 + ‐MSCs by ChIP‐seq, and compared these results with those obtained in ESCs, HSCs and HPCs. Pluripotent‐associated genes Klf4 were modified by H3K4me3, Sall4 , Sox2, and Foxd3 were marked by bivalent, and Oct4(Pou5f1) and Nanog exhibited repressive state or no modification in Flk1 + ‐MSCs, whereas all the abovementioned genes were marked by H3K4me3 in ESCs; either modified by H3K27me3 or carried no modification in HSCs and HPCs. We speculate that distinct histone modifications at pluripotency‐associated genes might be partly responsible for the phenomenon that only ESCs give rise to teratomas in vivo among the four stem cell types. We further evaluated the histone methylation status of genes associated with lineage specification. Neural differentiation associated genes such as Zic1 , Hes1 , Neurog2 , Dlx1 , Msi1 and Nes , were modified by H3K4me3 alone or by bivalent in Flk1 + ‐MSCs, consistent with their modification pattern in ESCs. Critical regulators of early hepatic epithelial differentiation such as Sox17, Gsc, Hhex, Onecut1, Prox1 and Mixl1 , also have the activated modification pattern both in ESCs and Flk1 + ‐MSCs. Whereas in HSC and HPC, most neural and early hepatic epithelial differentiation related genes were modified by H3K27m3 or no modification. We then analyzed histone methylation of hematopoiesis‐related genes and found that Egr1 , Tcf3 and Tal1 were marked by H3K4me3 in all four cell types. Zfpm1 and Ikzf1 were marked by bivalent in ESCs and Flk1 + ‐MSCs, whereas these genes carried H3K4me3 marks in HSCs and HPCs. Gata1 was marked by H3K27me3 or unmarked in ESCs and Flk1 + ‐MSCs, whereas in HSCs and HPCs, it was marked by H3K4me3. In summary, Flk1 + ‐MSCs share similar histone modification patterns of multilineage differentiation‐associated genes with ESCs. As our analysis moved from ESCs to AD‐MSCs, HSCs, and finally, to HPCs, there is an increasing frequency of active modifications on hematopoietic lineage‐related genes and a decreasing frequency on genes related to other lineages. As microRNAs play a major role in stem cell differentiation, we further obtained the miRNA profiles of Flk1 + ‐MSCs and ESCs. Expression of miRNAs associated with pluripotency was higher in ESCs than Flk1 + ‐MSCs, while expression levels of most miRNAs associated with multilineage differentiation were similar between ESCs and Flk1 + ‐MSCs. Overall, the miRNA expression profile correlated well with histone modification patterns of genes associated with pluripotency and lineage specification in ESCs and Flk1 + ‐MSCs. Here we firstly bridge a gap between various factors governing multipotency of Flk1 + ‐MSCs such as histone methylation and microRNAs. These findings will broaden current understanding of stem cell multipotency. This work was supported by grants from the “863 Projects” of Ministry of Science and Technology of PR China (no.2011AA020100) and the National Key Scientific Program of China (No. 2011CB964901).