Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan.
Biomaterials. 2011 Oct;32(29):6929-45. doi: 10.1016/j.biomaterials.2011.05.092. Epub 2011 Jul 16.
Stem cells can lose their primitive properties during in vitro culture. The culture substrate may affect the behavior of stem cells as a result of cell-substrate interaction. The maintenance of self-renewal for adult human mesenchymal stem cells (MSCs) by a biomaterial substrate, however, has not been reported in literature. In this study, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional spheroids that kept attached on the membranes. Spheroid formation was associated with the increased MMP-2 expression. Cells on chitosan-HA formed spheroids more quickly and the size of spheroids were larger than on chitosan alone. The expression of stemness marker genes (Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. The maintenance of stemness marker gene expression was especially remarkable in hPDMC spheroids (vs. hADAS spheroids). Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. Our study suggested that the formation of adhered spheroids on chitosan and chitosan-HA membranes may sustain the expression of stemness marker genes of MSCs and increase their chondrogenic differentiation capacity. The Rho/Rho-associated kinase (ROCK) signaling pathway may be involved in spheroid formation.
干细胞在体外培养过程中可能会失去其原始特性。由于细胞-基质相互作用，培养基质可能会影响干细胞的行为。然而，文献中尚未报道生物材料基质对成人骨髓间充质干细胞（MSCs）自我更新的维持作用。在这项研究中，从人脂肪（hADAS）和胎盘（hPDMC）中分离的 MSC 分别在壳聚糖膜和进一步用透明质酸（壳聚糖-HA）修饰的膜上培养。结果观察到，源自任何来源的 MSC 均形成了附着在膜上的三维球体。球体形成与 MMP-2 表达的增加有关。壳聚糖-HA 上的细胞更快地形成球体，并且球体的大小大于单独的壳聚糖。通过实时 RT-PCR 分析了材料上 MSC 的干性标记基因（Oct4、Sox2 和 Nanog）的表达。结果发现，与在聚苯乙烯培养皿中培养细胞相比，壳聚糖和壳聚糖-HA 膜上球体的形成有助于维持 MSC 的干性标记基因的表达。hPDMC 球体（与 hADAS 球体相比）中干性标记基因表达的维持尤其明显。用抗体阻断 CD44 可阻止球体形成并适度降低干性基因表达；而 Y-27632 化合物处理抑制球体形成并显著降低干性基因表达。软骨诱导后，MSC 球体表现出更高水平的 Sox9、聚集蛋白聚糖和 II 型胶原基因表达，并对糖胺聚糖和 II 型胶原呈阳性染色。诱导后 hPDMC 比 hADAS 具有更好的软骨分化潜能。我们的研究表明，壳聚糖和壳聚糖-HA 膜上附着球体的形成可能维持 MSC 干性标记基因的表达并增加其软骨分化能力。Rho/Rho 相关激酶（ROCK）信号通路可能参与球体形成。
