Human corneal endothelial cell precursors isolated by sphere-forming assay
Human corneal endothelial cell precursors isolated by sphere-forming assay
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DOI：
10.1167/iovs.04-1263
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发表时间：
2005-05-01
影响因子：
4.4
通讯作者：
Amano, S
中科院分区：
文献类型：
--
作者：
Yokoo, S;Yamagami, S;Amano, S
目的。体外分离人角膜内皮细胞（HCEC）前体细胞。方法。对 HCEC 进行球体形成测定，其中球体漂浮在含有生长因子的无血清培养基中。为了促进分化，将分离的球集落铺在涂有聚-L-赖氨酸（PLL）/层粘连蛋白或胎牛内皮细胞外基质的培养皿中。通过免疫细胞化学和/或逆转录-聚合酶链反应（RT-PCR）检查球集落及其后代中神经细胞和间充质细胞的标记表达。对来自球集落的贴壁分化细胞进行形态学和功能评估。结果。体外球形成试验显示，HCEC 形成初级和次级球形集落。免疫细胞化学显示，集落表达巢蛋白、β3-微管蛋白、胶质纤维酸性蛋白和α-平滑肌肌动蛋白。后代在源自牛角膜内皮的细胞外基质上增殖，但不在 PLL/层粘连蛋白包被和非包被的培养皿上增殖，表达巢蛋白和 β3-微管蛋白。这些标记通过 RT-PCR 得到证实。来自球集落的贴壁分化细胞具有类似 HCEC 的六边形形状和令人满意的转运活性，这对于 HCEC 至关重要。结论。这些发现表明 HCEC 含有具有分化为 HCEC 倾向的前体细胞，并且这些细胞还可以产生神经元和间充质细胞蛋白。
PURPOSE. To isolate precursors of human corneal endothelial cells (HCECs) in vitro.METHODS. HCECs were subjected to a sphere-forming assay in which spheres floated in serum-free medium containing growth factors. To promote differentiation, the isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/ laminin or fetal bovine endothelium extracellular matrix. Marker expression of neural and mesenchymal cells was examined in the sphere colonies and their progenies by immunocytochemistry and/or reverse transcription - polymerase chain reaction (RT-PCR). Adherent differentiated cells from the sphere colonies were evaluated morphologically and functionally.RESULTS. HCECs formed primary and secondary spherical colonies, as shown by sphere-forming assay in vitro. The colonies expressed nestin, beta 3-tublin, glial fibrillary acidic protein, and alpha-smooth muscle actin on immunocytochemistry. The progeny, proliferating on extracellular matrix derived from bovine corneal endothelium, but not on PLL/laminin-coated and noncoated dishes, expressed nestin and beta 3-tublin. These markers were confirmed by RT-PCR. Adherent differentiated cells from the sphere colonies had an HCEC-like hexagonal shape and satisfactory transport activity that is essential in HCECs.CONCLUSIONS. These findings indicate that the HCEC contains precursor cells with a propensity to differentiate into HCECs and that these cells can also produce neuronal and mesenchymal cell proteins.