| Objective:Hair follicles are an easily accessible rich source of autologous stem cells,exhibiting tremendous advantages over other cell sources in various clinical applications.Meanwhile,hHF-MSC can be reprogrammed to generate iPSC by lentiviral transduction with SOMK,and these HF-iPSC could be used as alternative cellular tools for regenerative medicine.But,the production of hHF-MSC and HF-iPSC is inefficient that cannot to keep up with the clinical demand.Thus,Maintenance of hHF-MSC self-renewal ability and enhancement of iPSC generation are essential for applications in stem cell-based regenerative medicine.Methods and Results:The roles of PBX1 in proliferation were investigated by CCK8 and flow cytometry.RT-qPCR and dual luciferase reporter assays were performed to evaluate pluripotent gene expression.Western blotting was performed to identify molecules downstream of PBX1 involved in proliferation and reprogramming,aiming to explore the molecular mechanisms of PBX1 enhances hHF-MSC proliferation and somatic cell reprogramming.The results are as follows(a)Hair follicle-derived fibroblast-like cells exhibited surface markers of mesenchymal stem cells and display trilineage differentiation potentials toward osteoblasts and adipocytes.Accordingly,these cells were designated hHF-MSC.(b)hHF-MSC were transduced with a lentiviral vector encoding PBX1.CCK-8 assays showed that overexpression of PBX1 significantly increased the rate of HF-MSC proliferation and cell cycle analyses showed that overexpression of PBX1 induced the entry of HF-MSC from G0/G1 into S phases.PBX1 increased the levels of phospho-AKT,phospho-GSK3?,and cyclin D1 and promoted ?-catenin translocation from the cytoplasm to the nucleus.Moreover,PBX1 expression decreased p16 and p21 expression.Moreover,PBX1 knockdown significantly reduced the percentage of hHF-MSC in the S phase and suppressed AKT/GSK3? pathway.HF-MSC-PBX1 were treated with LY294002 which inhibitory abolished the effects of PBX1 overexpression.Suggesting that PBX1 increased hHF-MSC proliferation through activation of the AKT/GSK3? signaling pathway.(c)SOMKP transduction significantly increased HF-iPSC colony formation.In contrast,knockdown of PBX1 significantly decreased SOMK-induced HF-iPSC colony formation.Compared with SOMK transduction,SOMKP transduction increased the expression levels of the endogenous OCT4,LIN28,SOX2,and NANOG genes.SOMKP transduction significantly increased NANOG promoter activities and increased NANOG expression during reprogramming of hHF-MSC into HF-iPSC.Western blotting showed that SOMKP transduction significantly increased the phosphorylation of AKT and GSK3?,promoted the nuclear translocation of ?-catenin,and downregulated p53 and p21 expression during reprogramming.During the late stage of reprogramming,NANOG activated by PBX1 can further promote the nuclear translocation of ?-catenin.Meanwhile,activated AKT mediated by PBX1 can further enhance the expression of NANOG.(d)Activation of AKT/GSK3? induced by PBX1 can reduce mitochondrial membrane permeabilization mediates apoptosis.SOMKP transduction significantly downregulated caspase3 and cleaved PARP1 expression,and decreased caspase3 activity during early stage of reprogramming.Meabwhile inhibition of endogenous PBX1 expression promoted p53 mediates apoptosis during reprogramming.The inhibitory activity of the AKT/GSK3? pathway accounted for the effects of overexpression of PBX1 on protection of HF-MSC against apoptosis and reprogramming.Conclusion:1.We identified PBX1 enhancing hHF-MSC proliferation by increasing AKT phosphorylation and ?-catenin nuclear translocation.2.PBX1 upregulated NANOG expression by activating the NANOG promoter and AKT/GSK3? signaling pathway in late stage of somatic cell reprogramming.3.PBX1 activated the AKT/GSK3? signaling pathway,inhibited mitochondrion-mediated apoptosis during the early stage of somatic cell reprogramming and enhance reprogramming efficiency. |
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