Isolation and Characterization of Human Amniotic Mesenchymal Stem Cells and Their Chondrogenic Differentiation

摘要

Background. Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing pro-liferative ability and pluripotency, including a chondrogenic lineage. However, little is known about the biology of amnion-derived mesenchymal stem cells (MSCs) because fHAM cells can barely survive to expand under culture conditions in vitro for a long time. Methods. In this study, we separated fHAM cells and seeded them to isolate MSCs and analyze its character. In addition, suitable chondrogenic growth factor was determined by pellet culture, and their viability under xenogenic environment was examined by transplantation into rabbit knee joints. Results. We succeeded in purifying proliferative subpopulations of fHAM cells, which could continue to proliferate more than 50 cumulative population doubling levels, and designated them as HAMa cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD 14, and CD45) and major histocompatibility complex class II antigen (human leukocyte antigen-DR). The expression of various stem-cell markers such as OCT3/4, C-MYC, SOX2, NANOG, CD44, SSEA-3, and SSEA-4 was also proved by immunocytochemical staining. Pellet culture using chondrogenic medium supplemented with transforming growth factor (33, transforming growth factor (33 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAMa cells achieved 8-week survival in vivo. Conclusions. These results suggest that HAMa cells correspond to MSCs that are highly proliferative and multipotent. Their chondrogenic potential and low immunogenicity indicate that HAMa cells could be an allotransplantable cell resource for cartilage repair.