Rajendran Arun Kumar, Arisaka Yoshinori, Iseki Sachiko, Yui Nobuhiko
Section of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8549, Japan.
Department of Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan.
ACS Biomater Sci Eng. 2019 Nov 11;5(11):5652-5659. doi: 10.1021/acsbiomaterials.8b01343. Epub 2019 Mar 26.
Human mesenchymal stem cells (hMSCs) are prone to senescence and lose their differentiation potential when expanded under nonfavorable conditions. This leads to the underutilization of hMSCs in clinical situations such as bone regeneration. The use of growth factors and small molecules as supplements and changing the physical properties of the cell culture surface have been explored to maintain the self-renewal and differentiation potential of hMSCs during the in vitro expansion phase. Here, we have explored the effect of polyrotaxanes (PRXs) with different molecular mobilities along with either soluble or immobilized fibroblast growth factor 2 (FGF2) in the maintenance of the osteogenic differentiation potential of hMSCs during in vitro expansion. We found that a less expanded shape of the hMSCs was associated with highly mobile PRX surfaces, and less mobile PRX surfaces led to flattened cell morphology. The presence of FGF2 induced further expansion of the cell shape and size. The immobilization of FGF2 helped to improve the yield of hMSCs on highly mobile surfaces by promoting cell attachment to the surfaces. hMSCs cultured on highly mobile PRX surfaces exhibited poor actin cytoskeletal organization and retention of the transcriptional regulator, yes-associated protein (YAP), in cytoplasm in contrast to the hMSCs on less mobile PRX surfaces. When the hMSCs that proliferated under these conditions were collected and subjected to osteogenic differentiation on tissue culture polystyrene (TCPS) surfaces, we found that only the hMSCs cultured on highly mobile PRXs with FGF2 in both soluble and immobilized forms showed mineralization indicative of osteogenic differentiation. Further, we found that hMSCs cultured on highly mobile PRX surfaces expressed higher levels of stemness marker genes, and . These results indicate that culturing hMSCs on PRX surfaces with different molecular mobilities even for a short period of time (4 days) was sufficient to cause a drastic change in the osteogenic potential. From these results, it is suggested that apart from the use of supplements such as FGF2 in its freely soluble or immobilized form, the consideration of proper molecular mobility of the substrates could enable us to design better culture conditions for the hMSCs with osteogenic potential.
人间充质干细胞（hMSCs）在不利条件下扩增时容易衰老并失去分化潜能。这导致hMSCs在骨再生等临床应用中未得到充分利用。人们已经探索使用生长因子和小分子作为补充剂，并改变细胞培养表面的物理性质，以在体外扩增阶段维持hMSCs的自我更新和分化潜能。在此，我们研究了具有不同分子迁移率的聚轮烷（PRXs）以及可溶性或固定化成纤维细胞生长因子2（FGF2）在体外扩增过程中对hMSCs成骨分化潜能维持的影响。我们发现，hMSCs的形状扩张程度较小与高迁移率PRX表面相关，而低迁移率PRX表面导致细胞形态扁平。FGF2的存在诱导细胞形状和大小进一步扩张。FGF2的固定化通过促进细胞附着于表面，有助于提高hMSCs在高迁移率表面上的产量。与在低迁移率PRX表面上的hMSCs相比，在高迁移率PRX表面上培养的hMSCs肌动蛋白细胞骨架组织较差，转录调节因子Yes相关蛋白（YAP）保留在细胞质中。当收集在这些条件下增殖的hMSCs并在组织培养聚苯乙烯（TCPS）表面进行成骨分化时，我们发现只有在高迁移率PRXs上培养且同时含有可溶性和固定化形式FGF2的hMSCs显示出矿化，表明有成骨分化。此外，我们发现，在高迁移率PRX表面上培养的hMSCs表达更高水平的干性标记基因 和 。这些结果表明，即使在短时间内（4天）将hMSCs培养在具有不同分子迁移率的PRX表面上，也足以导致成骨潜能发生剧烈变化。从这些结果表明，除了使用可溶性或固定化形式的FGF2等补充剂外，考虑底物的适当分子迁移率可以使我们为具有成骨潜能的hMSCs设计更好的培养条件。
