Hou Guang-hui, Ye Nan, Wu Jing, Xu Jin-tang, Shi Wen-jian, Chen Yi, Liu Xiao-hao, Zhang Ze-hua
Department of Ophthalmology, the People's Hospital of Zhuhai city, Zhuhai 519000, Guangdong 519000, China.
Zhonghua Yan Ke Za Zhi. 2010 Aug;46(8):719-24.
To investigate the differentiation potential of human bone marrow mesenchymal stem cells (hMSC) inducing into epithelial-like cells, even corneal epithelial-like cells, and to discuss the plasticity that make hMSC the seed cells used in corneal tissue engineering.
hMSC were isolated and purified by density gradient centrifugation combined with an attachment culture method and passaged in vitro. hMSC were identified by flow cytometry. The passaged hMSC were planted on fresh pig corneal Bowman's membrane. The expression of CK12, ABCG2 and CK19 in hMSC was identified by immunofluorescence staining. We used in vitro method to obtain a multilayer culture of hMSC. When hMSC formed a monolayer, the cells were inserted to Millicell culture and grew into multilayers by using the air-lifting cultivation methodology. Four weeks later, after fixed and dehydrated, the hMSC were observed under the light microscope after hemotoxylin and eosin (HE) and immunohistochemistry staining.
hMSC could be cultured, expanded in vitro, and showed great potential of proliferation. The result of flow cytometry showed that the positive staining percentage was 0.06% for CD45, 0.41% for CD34, 86.43% for CD44, 85.72% for CD29 and 90.72% for CD105. This indicated that hMSC expressed CD44, CD29, CD105 but not CD45 and CD34. After four weeks induction, part of hMSC expressed CK12 and CK19 but not ABCG2. In the in vitro stratification, HE and immunohistochemical staining showed that there were one or two layers epithelial-like cells, even corneal epithelial-like cells after using the air-lifting cultivation.
This study suggests that hMSC have the potential to differentiate into epithelial cells, even corneal epithelial cells. hMSC could be the option of cells used to reconstruct the corneal epithelium by tissue engineering technology.
研究人骨髓间充质干细胞（hMSC）诱导分化为上皮样细胞甚至角膜上皮样细胞的潜能，并探讨使hMSC成为角膜组织工程种子细胞的可塑性。
采用密度梯度离心结合贴壁培养法分离纯化hMSC，并进行体外传代培养。通过流式细胞术对hMSC进行鉴定。将传代后的hMSC接种于新鲜猪角膜前弹力层上。采用免疫荧光染色法鉴定hMSC中细胞角蛋白12（CK12）、ATP结合盒转运体G2（ABCG2）和细胞角蛋白19（CK19）的表达。采用体外方法获得hMSC多层培养物。当hMSC形成单层时，将细胞接种到Millicell培养板中，采用气升式培养方法使其生长为多层。4周后，固定脱水后，苏木精-伊红（HE）染色和免疫组化染色后在光学显微镜下观察hMSC。
hMSC能够在体外培养、扩增，具有很强的增殖潜能。流式细胞术结果显示，CD45阳性染色率为0.06%，CD34阳性染色率为0.41%，CD44阳性染色率为86.43%，CD29阳性染色率为85.72%，CD105阳性染色率为90.72%。这表明hMSC表达CD44、CD29、CD105，而不表达CD45和CD34。诱导4周后，部分hMSC表达CK12和CK19，但不表达ABCG2。在体外分层培养中，HE染色和免疫组化染色显示，采用气升式培养后有一层或两层上皮样细胞，甚至角膜上皮样细胞。
本研究提示hMSC有分化为上皮细胞甚至角膜上皮细胞的潜能。hMSC可作为组织工程技术重建角膜上皮的细胞选择。
