Non-canonical Wnt signaling via FZD5 and ROR2 contributes to the maintenance of the stemness of human mesenchymal stem cells

We previously showed that the combination of markers LNGFR+ and THY-1+ can be used selectively to isolate human mesenchymal stem cells (hMSCs) in bone marrow and several tissues. However, the molecular mechanisms regulating stemness of hMSCs remain to be elucidated. In this study, we found that Frizzled 5 (FZD5) and Receptor tyrosine kinase-like orphan receptor 2 (ROR2) were highly enriched in LNGFR+THY-1+ derived clones that retain a high potential for proliferation (rapidly expanding clones; RECs). WNT5A, the ligand of FZD5, causes FZD5 and ROR2 to co-localize and activates non-canonical Wnt pathway in RECs. Moreover, FZD5 knockdown attenuated proliferation and caused senescence. In contrast, FZD5 overexpression delayed senescence. These results indicate that intrinsic activation of non-canonical Wnt signaling via FZD5 and ROR2 modulates stemness and senescence of RECs. Thus, control of non-canonical Wnt signaling would enable us to regulate hMSC quality and to enhance the efficacy of cell-replacement therapy using hMSCs. Several human MSC clones that did not have any copy-number abnormality (two REC clones, three MEC clones, and three SEC clones) were cultured, and total RNA was extracted.Next, 300 ng of total RNA was labeled with Cy3 and hybridized to an Agilent human whole-genome chip (4x44K, AMADID=14850, Agilent technologies), which was scanned in a microarray scanner system (Agilent Technologies).These candidate genes were split into four clusters according to K-means (n=63). We performed quantitative PCR to validate the expression levels of genes whose expression levels were over two-fold (Group 1) and one- to two-fold (Group 2) higher in RECs than in MECs/SECs.