Isolation and Characterization of Human Amniotic Mesenchymal Stem Cells and Their Chondrogenic Differentiation
Isolation and Characterization of Human Amniotic Mesenchymal Stem Cells and Their Chondrogenic Differentiation
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DOI：
10.1097/tp.0b013e3182529b76
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发表时间：
2012-06-27
期刊：
影响因子：
6.2
通讯作者：
Nikaido, Toshio
中科院分区：
文献类型：
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作者：
Nogami, Makiko;Tsuno, Hiroaki;Nikaido, Toshio
背景新鲜分离的人羊膜间充质（fHAM）细胞含有具有增殖能力和多能性的体干细胞，包括软骨形成谱系。然而，由于fHAM细胞在体外培养条件下很难存活并长时间扩增，因此对fHAM来源的间充质干细胞（MSCs）的生物学特性知之甚少。本研究通过分离fHAM细胞，接种于骨髓基质细胞中，分离骨髓间充质干细胞，并对其特性进行分析。通过颗粒培养法筛选出合适的软骨生长因子，并将其移植到兔膝关节内，观察其在异种环境中的活性。我们成功地纯化了fHAM细胞的增殖亚群，其可以继续增殖超过50个累积群体倍增水平，并将其命名为HAM α细胞。流式细胞术分析显示，他们是阳性的MSC标志物（CD 44，CD 73，CD 90和CD 105）和阴性的造血细胞标志物（CD 34，CD 14和CD 45）和主要组织相容性复合物II类抗原（人白细胞抗原-DR）。还通过免疫细胞化学染色证实了各种干细胞标志物如OCT 3/4、C-MYC、SOX 2、NANOG、CD 44、SSEA-3和SSEA-4的表达。使用补充有转化生长因子β 3、转化生长因子β 3加骨形态发生蛋白（BMP）-2或BMP-2的软骨形成培养基的颗粒培养暗示单独补充BMP-2最有效地诱导体外软骨形成。HAM α细胞的异种移植实现了8周的体内存活。这些结果表明，HAM α细胞对应于高度增殖和多能的MSC。它们的软骨形成潜力和低免疫原性表明HAM α细胞可能是用于软骨修复的同种异体移植细胞资源。
Background. Freshly isolated human amniotic mesenchymal (fHAM) cells contain somatic stem cells possessing proliferative ability and pluripotency, including a chondrogenic lineage. However, little is known about the biology of amnion-derived mesenchymal stemcells (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 HAM alpha cells. Flow cytometry analysis revealed that they were positive for MSC markers (CD44, CD73, CD90, and CD105) and negative for hematopoietic cell markers (CD34, CD14, 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 beta 3, transforming growth factor beta 3 plus bone morphogenetic protein (BMP)-2, or BMP-2 implied that supplementation of BMP-2 alone most effectively induced chondrogenesis in vitro. Xenotransplantation of HAM alpha cells achieved 8-week survival in vivo.Conclusions. These results suggest that HAM alpha cells correspond to MSCs that are highly proliferative and multipotent. Their chondrogenic potential and low immunogenicity indicate that HAM alpha cells could be an allotransplantable cell resource for cartilage repair.