Xeno- and Feeder-Free Differentiation of Human iPSCs to Trabecular Meshwork-Like Cells by Recombinant Cytokines.
Xeno- and Feeder-Free Differentiation of Human iPSCs to Trabecular Meshwork-Like Cells by Recombinant Cytokines.
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DOI：
10.1167/tvst.10.6.27
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发表时间：
2021-05-03
影响因子：
3
通讯作者：
Zhu W
中科院分区：
文献类型：
--
作者：
Wang W;Miao Y;Sui S;Wang Y;Wu S;Cao Q;Duan H;Qi X;Zhou Q;Pan X;Zhang J;Chen X;Han Y;Wang N;Kuehn MH;Zhu W
基于干细胞的治疗有可能成为再生青光眼中受损的小梁网（TM）的一种方法。诱导多能干细胞（iPSC）与人TM细胞的共培养已经是产生自体TM样细胞的成功方法。然而，使用这种方法产生的分化细胞对于临床使用仍然是有问题的。本研究旨在开发一种临床适用的策略，用于从iPSC产生TM样细胞。通过AutoSOME、Gene Ontology和逆转录聚合酶链反应（RT-PCR）分析鉴定iPSC分化期间高表达的受体。与这些受体结合的重组细胞因子用于产生新的分化方案。所得到的TM样细胞的特征在于形态学，荧光化学，和转录。我们首先确定了iPSC分化的两个阶段，并鉴定了与每个阶段分化相关的高表达受体。通过RT-PCR分析进一步证实了这些受体的表达。暴露于与这些受体结合的重组细胞因子，包括转化生长因子β 1、神经生长因子β、促红细胞生成素、前列腺素F2 α和表皮生长因子，可以有效地将iPSC分化为TM样细胞，其表达TM生物标志物并可以形成地塞米松诱导的CLAN。我们成功地产生了一种无异种和饲养层的分化方案，用重组细胞因子从人iPSC产生TM祖细胞和TM样细胞。这种新方法最大限度地减少了污染的风险，并提高了分化效率和一致性，这对于干细胞在青光眼治疗中的临床应用尤为重要。
Stem cell-based therapy has the potential to become one approach to regenerate the damaged trabecular meshwork (TM) in glaucoma. Co-culture of induced pluripotent stem cells (iPSCs) with human TM cells has been a successful approach to generate autologous TM resembling cells. However, the differentiated cells generated using this approach are still problematic for clinical usage. This study aimed to develop a clinically applicable strategy for generating TM-like cells from iPSCs. Highly expressed receptors during iPSC differentiation were identified by AutoSOME, Gene Ontology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. The recombinant cytokines that bind to these receptors were used to generate a new differentiation protocol. The resultant TM-like cells were characterized morphologically, immunohistochemically, and transcriptionally. We first determined two stages of iPSC differentiation and identified highly expressed receptors associated with the differentiation at each stage. The expression of these receptors was further confirmed by RT-PCR analysis. Exposure to the recombinant cytokines that bind to these receptors, including transforming growth factor beta 1, nerve growth factor beta, erythropoietin, prostaglandin F2 alpha, and epidermal growth factor, can efficiently differentiate iPSCs into TM-like cells, which express TM biomarkers and can form dexamethasone-inducible CLANs. We successfully generated a xeno- and feeder-free differentiation protocol with recombinant cytokines to generate the TM progenitor and TM-like cells from human iPSCs. The new approach minimizes the risks from contamination and also improves the differentiation efficiency and consistency, which are particularly crucial for clinical use of stem cells in glaucoma treatment.