| dc.description.abstract | Graft-versus-host disease (GVHD) is one of the major limiting factors of organ transplantation and hematopoietic stem cell transplantation. The transplantation of human mesenchymal stem cells (hMSCs) has shown great effects to protect transplanted tissues from GVHD. However, hMSC treatment has the main barrier because of limited passage numbers of hMSCs and painful extraction procedures from bone marrow. Human pluripotent stem cells (hPSCs)-derived hMSCs (hPSC-MSCs) should be a promising and limitless source of hMSCs for patient treatment of GVHD. hMSCs were generated from differentiation of hPSCs using the modified protocol reported by Xu et al. (Int. J. Biol. Sci., 14 (2018) 1901) where Matrigel used in the original protocol was changed to xeno-free biomaterials. hPSCs were differentiated into hMSCs by treating bone morphogenetic protein 4 (BMP-4) and A83-01 inhibitor. The xeno-free protocol for hMSC differentiation was developed using different human extracellular matrix proteins (hECMs)-coated dishes, including recombinant vitronectin, laminin-511, laminin-521, fibronectin, and collagen I. The flow cytometry was used to evaluate the expression of MSC markers (CD44, CD73, CD90, and CD105) on hPSC-MSCs, and the differentiation abilities of hPSC-MSCs into osteoblasts, chondrocytes, and adipocytes were evaluated. Laminin-521 and Collagen type I were found to be the top two best cell culture biomaterials for hPSCs differentiation into hMSCs. Although hPSCs differentiated on all ECM protein-coated dishes showed similar hMSC characteristics such as cell morphology, the cells cultured on Laminin-521-coated and Collagen type I-coated dishes showed good (short) doubling time and the highest MSC marker expression in this study. Therefore, Laminin-521 coated surface is considered to be the most reliable and xeno-free cell culture biomaterial for hPSC-MSCs preparation. Besides, in previous studies in our laboratory, hPSCs were cultured on polyvinyl alcohol-co-itaconic acid (PVA-IA) hydrogels, which were conjugated with oligo-vitronectin (KGGPQVTRGDVFTMP) and laminin beta 4-derived peptide (PMQKMRGDVFSP). They found that insertion of the positive joint segment (KGG) on the oligopeptides could improve the hPSC cultivation. Several molecular designs of oligopeptide-grafted hydrogels having an optimal number of positive joint segments were developed in this study, which supported the proliferation of hESCs while hESCs maintained their pluripotency and differentiation ability into hMSCs under xeno-free cell culture conditions. Especially, new oligopeptides of laminin-derived peptides (PMQKMRGDVFSP) with different numbers of positive joint segments were grafted on PVA-IA hydrogels. PVA-IA hydrogels grafted with these synthetic peptides were investigated to support long-term hESCs cultivation and differentiation into hMSCs. PVA-IA hydrogels grafted with KLBC2K (adding the positive amino acid in front of the linker) and LB2CKKK (which has more positive joint segments between the linker and the main chain) were stable for hESC long-term cultivation and can promote differentiation of hESCs into hMSC. After hESC cultivation on those synthetic peptides-grafted PVA_IA hydrogels, hESCs were evaluated for the expression level of four pluripotent protein (Oct4, Sox2, and Nanog) expression. Furthermore, hESC-MSCs cultured on PVA-IA hydrogels grafted with LB2CK and KLB2CK showed better doubling time and higher MSC marker expression than those cultured on PVA-IA hydrogels grafted with another design of peptides in this study. Therefore, the KLB2CK grafted on the PVA-IA hydrogels surface is a reliable new design of oligopeptide for hPSC-MSCs differentiation. Live and dead staining and secretion of inflammatory cytokines were also evaluated after hPSC-MSCs were treated with allogeneic mononuclear cells to evaluate the protective effect of hPSC-MSCs from GVHD. It is expected that the present hPSC-MSCs can be widely applied for regenerative medicine in the future. | en_US |