| dc.description.abstract | Human induced pluripotent stem cells (hiPSCs) have significant potential in therapeutic applications for many diseases because they have the specific ability to differentiate into all types of human somatic cells. However, the tentative clinical potential of hiPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The feeder-free cultures using synthetic biomaterials having nanosegments as stem cell culture materials offer more reproducible culture conditions and lower the cost of production without introducing xenogenic contaminants. These improvements will increase the potential clinical applications of differentiated hiPSCs. Here we report that hiPSCs can be successively generated with usage of a feeder layer of MEFs during generation of hiPSCs by transfection of retrovirus containing pluripotent genes into human adipose-derived stem cells (hADSCs). hiPSC colonies were clearly observed for the cells cultured on MEFs at day 8 after transfection The number of colonies generated on MEFs was 484 per 10 cm dishes, when 105 hADSCs were seeded on the dishes. The efficiency of hiPSC generation on MEFs was 0.484%. Furthermore, the hiPSC colony showed alkali phosphatase activity clearly, and immunofluorescence suggested that the hiPSCs were generated on MEFs expressing pluripotent protein of Oct4, Sox2 and SSEA-4.
At same time, polyvinylalcohol-co-itaconic acid (PVA-IA) films grafted with nanosegment (KGGPQVTRGDVFTMP [cell-binding domain derived from vitronectin, oligoVN] was established for cultivation of human pluripotent stem cells. In this study, with increase the concentration of oligo-VN, hPSCs shown the higher colony attachment ratio, colony expansion fold and lower differentiation ratio. Furthermore, the elasticity of PVA-IA films grafted with oligo-VN was regulated from 10.3 kPa to 30.4kPa by control of crosslinking time of PVA-IA. hESCs (WA09) cultured on PVA-IA culture system with stiffer elasticity of surface (25.3kPa) shown the better performance than the soft one (15.8kPa). Moreover, hESCs (WA09) cultured on PVA-IA films grafted with 500μg/ml oligo-VN with 25.3kPa elasticity of surface for 5 passage shown alkali phosphatase activity and pluripotent protein expression such as Oct4, Sox2, Nanog, SSEA-4, Tra-1-60 and Tra-1-81. This result indicates that hPSCs could be cultured on our PVA-IA culture system and maintain pluripotency of hPSCs. In the future, the PVA-IA culture system could be used to generation of hiPSCs from primary human tissue cells on xenogenic-free and feeder-free conditions.
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