Improvement of human embryonic stem cell-derived retinal pigment epithelium cell adhesion, maturation, and function through coating with truncated recombinant human vitronectin
Improvement of human embryonic stem cell-derived retinal pigment epithelium cell adhesion, maturation, and function through coating with truncated recombinant human vitronectin
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通过用截短的重组人玻连蛋白包被来改善人胚胎干细胞来源的视网膜色素上皮细胞的粘附、成熟和功能
DOI：
10.18240/ijo.2021.08.04
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发表时间：
2021-08-18
影响因子：
1.4
通讯作者：
Sun, Xiao-Dong
中科院分区：
文献类型：
--
作者：
Zhu, Xin-Yue;Chen, Yu Hong;Sun, Xiao-Dong
目的：探索一种适合H9人胚胎干细胞源性视网膜色素上皮（hES-RPE）细胞体外培养的无xeno涂层底物，并比较hES-RPE细胞在lamin521 （LN-521）和重组人玻璃体连接蛋白（VTN-N）两种培养底物上的行为和功能。方法：实验采用hES-RPE细胞，采用高含量显像系统比较不同浓度的LN-521和VTN-N对hES-RPE细胞的粘附能力。采用实时定量聚合酶链反应（pcr）检测rpe特异性基因在治疗中期（第10天）和结束时（第20天）的表达水平。通过免疫荧光染色观察RPE的顶端和基部标记物的细胞极性。流式细胞术和免疫荧光法检测hES-RPE细胞的吞噬能力。结果：细胞粘附实验显示，LN-521对hES-RPE细胞的粘附能力呈剂量依赖性。随着涂层浓度的增加，附着在ln -521涂层孔表面的细胞数量增加。相比之下，VTN-N即使在低浓度下也表现出较强的粘附能力。LN-521和VTN-N包被hES-RPE细胞和粘附所需的最佳浓度分别为2和0.25 μ g/cm(2)。此外，LN-521和VTN-N均能促进hES-RPE细胞采用紧密连接的鹅卵石状细胞形态，并显示极性。然而，在VTN-N中培养的hES-RPE细胞具有更强的吞噬能力，并且这些hES-RPE细胞成熟所需的时间更短。结论：VTN-N是培养hES-RPE细胞较合适的包膜底物。
AIM: To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial (hES-RPE) cells in vitro, and compare the behaviors and functions of hES-RPE cells on two culture substrates, laminin521 (LN-521) and truncated recombinant human vitronectin (VTN-N).METHODS: hES-RPE cells were used in the experiment The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system. Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway (day 10) and at the end (day 20) of the time course. Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE. The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence.RESULTS: The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dose dependent. With increasing coating concentration, an increasing number of cells attached to the surface of LN-521-coated wells. In contrast, VTN-N presented a strong adhesive ability even at a low concentration. The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25 mu g/cm(2), respectively. Furthermore, both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells. However, hES-RPE cells cultivated in VTN-N had a greater phagocytic ability, and it took less time for these hES-RPE cells to mature.CONCLUSION: VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.