人脐带血和骨髓来源间充质干细胞的体外分离、培养、分化及

背景：间充质干细胞由于具有自我更新能力且在适宜微环境下具有多向分化潜能，近年来已成为细胞领域的研究热点。 目的：对比观察脐带血与骨髓来源的间充质干细胞其体外分离、纯化及培养条件，并对其进行生物学特性比较。 设计：体外细胞学对比观察。 材料：新鲜脐带血在征得产妇同意后采集。骨髓由健康的成年志愿者捐赠。 方法：取脐带血和成人骨髓分离、培养并纯化间充质干细胞，对获得的间充质干细胞进行形态学观察。 主要观察指标：两种来源间充质干细胞的形态和生长特性；流式细胞仪检测脐带血间充质干细胞表面抗原的表达情况；对脐带血间充质干细胞多向分化的能力进行鉴定。 结果：两种来源的单个核细胞经体外培养贴壁后均出现间充质样细胞，原代骨髓间充质干细胞的贴壁时间和培养时间均短于脐带血间充质干细胞，两种细胞传代生长呈共同特性：传代培养潜伏期为24~36 h，传代培养对数增殖期为接种后3～7 d，接种后八九天，生长进入平台期；脐带血来源的间充质干细胞表达相关抗原CD29、CD44、CD105；但不表达造血细胞抗原CD34、CD45；脐带血间充质干细胞可诱导分化为脂肪细胞、成骨细胞。 结论：人脐带血和骨髓来源间充质干细胞均可在体外分离培养、扩增；脐带血的间充质干细胞原代贴壁时间、形成细胞克隆的时间、集落交错融合的时间均较骨髓间充质干细胞晚，传代培养的细胞形态，生长速度均无明显差异；两种来源的间充质干细胞具有相同的表面标志物；脐带血间充质干细胞有多项分化潜能，可诱导为脂肪细胞、成骨细胞。

Isolation, culture, differentiation and biological characteristics of mesenchymal stem cells derived from human umbilical cord blood versus bone marrow in vitro

BACKGROUND: Mesenchymal stem cells (MSCs) have the potential of self-renewal and multi-directional differentiation under a suitable microenvironment. Recently, MSCs have been a hot focus in cell research. OBJECTIVE: To compare in vitro isolation, purification and culture condition of MSCs from umbilical cord blood and bone marrow, and to compare their biological characteristics. DESIGN: Controlled study. MATERIALS: Fresh umbilical cord blood was collected following the puerperants signed the informed consent. Bone marrow was gifted by healthy adult volunteers. METHODS: Umbilical cord blood and adult bone marrow were obtained to isolate, purify and culture MSCs. Morphology of MSCs was observed. MAIN OUTCOME MEASURES: MSC morphology and growth characteristics were measured. Flow cytometry was used to detect the expression of surface antigen of umbilical cord blood MSCs. The potential of multi-directional differentiation of umbilical cord blood MSCs was assessed. RESULTS: Following in vitro culture and adherence, mononuclear cells presented mesenchyme-like cells. Adherent time and culture time of primary cultured bone marrow MSCs were shorter compared with the umbilical cord blood MSCs. Passage growth showed identical characteristics. Latency of passage culture was 24-36 hours. Proliferative phase was from 3-7 days following incubation. Platform phase was from 8-9 days following incubation. Umbilical cord blood derived MSCs were positive for CD29, CD44, CD105, but negative for CD34, CD45. Umbilical cord blood derived MSCs could differentiate into adipocytes and osteoblasts. CONCLUSION: Human umbilical cord blood and bone marrow derived MSCs can be isolated, cultured and amplified in vitro. Adherent time, cell clone forming time and colony overlapping confluence time are later in umbilical cord blood derived MSCs compared with bone marrow MSCs. No significant difference in cell morphology and growth speed is detected between the two kinds of cells. The two cells have the same surface marker. Umbilical cord blood derived MSCs have the potential of multi-directional differentiation and can differentiate into adipocytes and osteoblasts.