Generation of lung epithelial-like tissue from human embryonic stem cells
Generation of lung epithelial-like tissue from human embryonic stem cells
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DOI：
10.1186/1465-9921-10-105
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发表时间：
2009-11-05
影响因子：
5.8
通讯作者：
De Rycke, Martine
中科院分区：
文献类型：
--
作者：
Van Haute, Lindsey;De Block, Gert;De Rycke, Martine
背景：人类胚胎干细胞(HESC)在体内和体外均能分化为三种生殖系的细胞。本研究旨在探讨特定培养条件对人胚胎干细胞向肺上皮细胞分化的影响。方法：将未分化的人胚胎干细胞在人胚胎干细胞的多孔膜上培养4天后，转入分化培养基中培养4天，然后在气液界面条件下培养20天。结果：CC16和Nkx2.1的表达在分化第10天分别增加1000倍和10,000倍。其他肺标志物如SP-C和Aquaporin 5在培养20d后表达最高，纤毛细胞标志物FOXJ1和微管蛋白IV表达最强。免疫组织化学证实了qRT-PCR的结果。选择抗CC16、SP-A和SP-C抗体作为Clara细胞和肺泡II型细胞的特异性标志物。通过酶免疫分析法检测培养液中CC16的分泌来检测其功能。结论：利用我们的新的培养方案，hESC可以分化为肺上皮组织的主要细胞类型。
Background: Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells.Methods: Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls.Results: Expression of CC16 and NKX2.1 showed a 1,000- and 10,000-fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and -tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay.Conclusion: These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.