人脐带间充质干细胞分离培养条件的优化及其生物学特性

背景：由于大量获取骨髓细胞存在困难，并且骨髓间充质干细胞随年龄的增加出现数量及分化潜能下降，因此需要寻找其他间充质干细胞来源。 目的：优化人脐带间充质干细胞的分离培养条件，进一步明确其生物学特性。 设计、时间及地点：细胞学体外观察，于2006-09/2008-03在中国医学科学院血液学研究所国家重点实验室完成。 材料：17份脐带标本取自健康足月新生儿，由天津市中心妇产医院提供。 方法：脐带采集后在6 h内分别用植块法、胶原酶消化法、胶原酶与胰酶联合消化法进行分离培养，于培养第14天计数集落数，超过50个细胞计为集落。当细胞达70%~80%融合时，胰蛋白酶-乙二胺四乙酸混合消化，以(2.5~5.0)×103/cm2密度传代培养。 主要观察指标：比较不同培养方法所获贴壁细胞的形态学特征、细胞产率，应用流式细胞技术分析细胞增殖活性、免疫表型，特异性染色方法鉴定细胞的多向分化潜能。 结果：植块法培养6~10 d可见成纤维样细胞从植块边缘爬出，形态与骨髓间充质干细胞相似，散在分布或形成小克隆，原代可获得(5.2±1.7)×105个贴壁细胞/0.5 cm脐带组织，至少可稳定传15代，平均每代扩增6.2倍，在原代细胞克隆数、细胞产率、传代时间及扩增倍数上与胶原酶消化法比较存在明显差异(P < 0.05)；而胶原酶与胰酶联合消化法接种后未见明显贴壁细胞。人脐带间充质干细胞的倍增时间为45 h，72.09%细胞处于G0/G1期，不表达造血细胞及内皮细胞标记，高表达整合素和黏附分子CD44，CD90，CD95以及CD73，CD105，在特定诱导条件下，人脐带间充质干细胞能够向成骨及成脂肪细胞方向分化。 结论：应用植块法可以从人脐带组织中简便高效地分离出一群具有高增殖活性及多向分化潜能的间充质干细胞，其免疫表型类似于骨髓间充质干细胞。

Optimization of culture condition and biological characteristics of human umbilical cord-derived mesenchymal stem cells

BACKGROUND: It is necessary to find other sources of mesenchymal stem cells (MSCs) due to the difficulty in obtaining enough bone marrow derived cells as well as the decreased number and differentiation potential of bone marrow derived-MSCs (BM-MSCs) with increasing age. OBJECTIVE: To optimize the isolation and expansion system of umbilical cord-derived mesenchymal stem cells (UC-MSCs), and to explore the biological characteristics of UC-MSCs. DESIGN, TIME AND SETTING: The in vitro cytology experiments were performed at the State Key Laboratory of Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union of Medical College from September 2006 to March 2008. MATERIALS: Seventeen samples of human umbilical cords were obtained from healthy full-term neonates from Tianjin Central Hospital of Gynaecology and Obstetrics. METHODS: Human umbilical cords were processes within 6 hours with explantation technique, collagenase digestion and enzymes combination digestion after harvested, and followed by counted colony numbers. A colony contains more than 50 cells. When the cells reached 70%-80% confluency, cells were recovered using trypsin-EDTA and were passaged at a density of (2.5-5.0)×103/cm2. MAIN OUTCOME MEASURES: Cell morphological feature and cell yields with different isolation and expansion system were compared. Then the proliferation capability and immunophenotype of UC-MSCs were analyzed by flow cytometry. The differentiation potential of UC-MSCs was studied by specific staining methods. RESULTS: At 6-10 days after explantation, the fibroblast-like cells were sprawled out from the edge of the little cubes as scattered or formed little clones, and morphology of which was similar to that of BM-derived MSCs, an 0.5 cm umbilical cord tissues contained (5.2±1.7) ×105 adhesive cells were harvested at the primary culture, which could steady passage for 15 passages, with 6.2 folds amplication in per passage. The cell yields, passage time and doubling times in explantation technique group were higher than those in collagenase digestion group (P < 0.05). While there was no adhesive cells could be found in enzymes combination digestion group. The population doubling time of UC-MSCs was 45 hours, with 72.09% cells in G0/G1 phase. The UC-MSCs did not express hematopoietic specific antigens; however, they were high expressed integrins/adhesion molecules, such as CD44, CD73, CD90, CD95 and CD105. UC-MSCs could differentiate to osteoblasts or adipocytes under appropriate experimental conditions. CONCLUSION: With explantation technique, UC-MSCs with potent expansion capacity and multiple differentiations potential can be isolated easily and effectively. The phenotype of these cells is similar to that of BM-MSCs.