体外扩增脐血间充质干细胞的生物学特性和诱导分化能力的研究

Biological characteristics and induced differentiation ability of in vitro expanded umbilical cord blood mesenchymal stem cells

目的从脐血中分离单个核细胞(MNCs),体外培养扩增间充质干细胞(MSCs)并研究其生物学特性和诱导分化潜能。方法取35份足月顺产新生儿脐血,密度梯度离心法分离出其中的MNCs,采用含胎牛血清的DMEM培养基体外培养扩增MSCs。显微镜下观察MSCs的形态、细胞化学染色,流式细胞仪测定MSCs的细胞免疫表型,体外诱导分化实验检测MSCs分化成骨细胞、脂肪细胞和神经细胞的能力。结果从12份脐血中可培养出MSCs,形态上与从其他来源的MSCs类似,可传至20代而无形态上的变化。细胞化学染色示碱性磷酸酶(ALP)阴性,非特异性酯酶———α丁酸萘酚酯酶(alphanaphtholbutyricacidesterase,NBE)阳性。其表达CD29、CD44和CD105,特别是人类MSCs标记SH2和SH3阳性,而CD3、CD14、CD19、CD34和CD45阴性,说明它们并非来自造血细胞。这些MSCs在适当诱导分化剂的作用下,2周左右可以诱导分化形成骨细胞,20天左右可以诱导分化形成脂肪细胞。脐血MSCs预诱导12h后,胞体发生收缩,细胞边缘有细的突起。正式诱导5h后大多数细胞呈现典型的神经元样。结论胎儿脐血MNCs可分离培养出MSCs。这些MSCs具有与其他来源MSCs类似的表型及分化潜能。

Objective Mesenchymal stem cells (MSCs) from bone marrow are capable of differentiating into cells of different tissue lineages such as bone, cartilage, and adipose tissue and are the best candidates for tissue engineering. It is well accepted that umbilical cord blood (UCB) is a source for hematopoietic stem cells. However, controversy exists as to whether UCB contains MSCs and can serve as a source of MSCs. Therefore, the aim of this study was to explore the biological characteristics and inducing differentiation ability of in vitro expanded UCB MSCs. Methods UCB was collected on normal full term delivery of infants with informed consent (n=35) obtained from the mothers. Mononuclear cells (MNCs) were isolated from UCB by gravity centrifugation and cultured with DMEM including 10% fetal bovine serum. The morphology was observed under microscope per day. Cytochemical staining was carried out and flow cytometry was used to examine the surface antigen phenotype. Fifth passage cells were transferred into a different medium and osteogenic differentiation, adipogenic differentiation, and neurogenic differentiation were assessed. Results MSCs could be isolated and cultured from MNCs of a few UCB sources. These cells displayed fibroblast-like morphology. They withstood over 20 passages without significant structural changes. These MSCs were negative for alkaline phosphatase (ALP) staining and positive for alpha-naphthol butyric acid esterase (NBE) staining. Expression of CD_(29), CD_(44)and CD_(105),especially the human MSCs-specific markers SH-2 and SH-3 were observed, but CD_3, CD_(14), CD_(19), CD_(34) and CD_(45) could not be found, indicating that these cells were not of hematopoietic origin. Exposure of these MSCs to serum-free osteogenic condition, they could differentiate into bone cells and form mineralized matrix as evidenced by Alizarin red staining 2 weeks later. When these UCB-derived MSCs were cultured in adipogenic medium, morphologic changes in cells as well as the formation of neutral lipid vacuoles were noticeable as early as 1 week after induction and visualized by staining with oil-red O. Surprisingly, these MSCs were also able to differentiate into neuroglial-like cells. Morphology of these induced cells resembled that of neurons. Immunocytochemistry showed that they expressed Nestin and neuron-specific enolase (NSE), but not glial fibrillary acidic protein (GFAP). Conclusion UCB does contain MSCs. These MSCs, which are multipotent, could be isolated and cultured from a few UCB sources. UCB might serve as an alternative source of MSCs to bone marrow.