Embryoid body-based differentiation of human-induced pluripotent stem cells into cells with a corneal stromal keratocyte phenotype.

IF 2 Q2 OPHTHALMOLOGY BMJ Open Ophthalmology Pub Date : 2024-11-28 DOI:10.1136/bmjophth-2024-001828
Jie Chen, Qingjian Ou, Yifan Liu, Tingting Cui, Huimin Yang, Jiancen Tang, Lixia Lu, Guotong Xu, Hongping Cui, Caixia Jin, Qian Li
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Abstract

Objective: The transparency of the cornea is determined by the extracellular matrix, which is secreted by corneal stromal keratocytes (CSKs). Human-induced pluripotent stem cell (hiPSC)-derived keratocytes (hiPSC-CSKs) can be used in cell-based therapy for treating corneal blindness. Our goal was to develop an effective small molecule-based technique for differentiating hiPSCs into keratocytes.

Methods and analysis: hiPSCs were cultured in chemically defined medium, and embryoid bodies (EBs) were generated; these EBs were induced into CSKs using keratocyte-differentiated medium. The expression of keratocyte-specific markers was assessed using quantitative RT-PCR, immunostaining and Western blotting.

Results: We found that the expression of genes encoding keratocyte markers, including aldehyde dehydrogenase 1 family member A1 (ALDH1A1), lumican and keratocan, was upregulated. Immunostaining showed positive staining for ALDH1A1 and keratocan in the hiPSC-CSK samples. Similarly, western blot analysis indicated that ALDH1A1 and keratocan expression levels were significantly greater in the hiPSC-CSKs than in the control cells. In addition, hiPSC-CSKs were not transformed into fibroblasts or myofibroblasts.

Conclusion: We established an innovative and effective method to generate CSKs via the EB-based differentiation of hiPSCs, which might be employed for cell-based therapy of corneal stromal opacities.

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基于胚胎体的人诱导多能干细胞分化为具有角膜基质角质细胞表型的细胞。
目的:角膜基质角质细胞(csk)分泌的细胞外基质决定角膜透明度。人诱导多能干细胞(hiPSC)衍生的角质细胞(hiPSC- csks)可用于角膜失明的细胞治疗。我们的目标是开发一种有效的基于小分子的技术,将hipsc分化为角化细胞。方法与分析:hiPSCs在化学定义的培养基中培养,产生胚状体(EBs);用角化细胞分化培养基将这些EBs诱导成CSKs。采用定量RT-PCR、免疫染色和Western blotting检测角化细胞特异性标志物的表达。结果:我们发现编码角化细胞标记的基因,包括醛脱氢酶1家族成员A1 (ALDH1A1)、lumican和keratocan的表达上调。免疫染色显示hiPSC-CSK样品中ALDH1A1和角蛋白阳性染色。同样,western blot分析显示,与对照细胞相比,hiPSC-CSKs中ALDH1A1和角化蛋白的表达水平显著升高。此外,hiPSC-CSKs不会转化成纤维母细胞或肌成纤维细胞。结论:我们建立了一种创新、有效的方法,通过hiPSCs的EB-based分化生成CSKs,可用于角膜基质混浊的细胞治疗。
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来源期刊
BMJ Open Ophthalmology
BMJ Open Ophthalmology OPHTHALMOLOGY-
CiteScore
3.40
自引率
4.20%
发文量
104
审稿时长
20 weeks
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