Department of Anatomy and Cell Biology, Faculty of Medicine, Technion, Haifa, Israel,
Cytotechnology. 2006 Oct;52(2):125-37. doi: 10.1007/s10616-007-9043-x. Epub 2007 Feb 2.
The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (PCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in alphaMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue(R) assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45(+), CD105(+), CD14(+), CD49a(+), CD49f(+), CD44(+) and CD34(-). The highest incidence of PCs was observed on fibronectin and polystyrene. Passages were CD45(-), CD14(-), CD34(-) and weakly CD105(+). Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing Vit D(3) and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.
本研究旨在探索从脐血中分离间充质祖细胞（MPC）的最佳条件，并确定其成骨分化的潜能。采用菲可（Ficoll）-泛影葡胺密度梯度离心法分离单个核细胞（MNC），并种植于预先涂有人或牛Ⅰ型胶原、人Ⅳ型胶原、纤维连接蛋白或基质胶的 48 孔培养板上。细胞在含胎牛血清的α-MEM 中孵育。在培养 2、3 和 4 周后，通过 alamarBlue（R）检测确定贴壁细胞的存活率。4 周后，细胞用胰蛋白酶消化并重新铺板。原代培养通过组织化学进行分析，第三代细胞通过流式细胞术进行分析。分离的成纤维细胞样细胞在成骨因子存在的情况下培养，并通过茜素红 S 染色、RT-PCR 和电子能谱（EDS）确定分化情况。MNC 可以贴附于所有基质，在纤维连接蛋白上的贴附率最高。这些细胞 CD45（+）、CD105（+）、CD14（+）、CD49a（+）、CD49f（+）、CD44（+）和 CD34（-）。在纤维连接蛋白和聚苯乙烯上观察到最高比例的 PCs。传代后细胞 CD45（-）、CD14（-）、CD34（-），且 CD105（+）弱阳性。无论在纤维连接蛋白还是聚苯乙烯上培养，原代培养物均表达内皮/巨噬细胞 RNA 标志物，传代后这些标志物减少。在含有 Vit D（3）和 FGF9 的成骨培养基中培养的 MPC 表现出最佳的成骨分化。这些细胞表达与骨相关的 mRNA、Ⅰ型胶原、核心结合因子Ⅰ（Cbfa I）、骨钙素和骨桥蛋白。这些细胞产生的沉积物的 EDS 分析显示钙/磷比值与羟磷灰石平行。综上所述，纤维连接蛋白增加了脐血间充质祖细胞的贴附率和分离潜能。
