Shandong First Medical University (Shandong Academy of Medical Sciences), Jinan, 250000, China; Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao, 266071, China.
Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao, 266071, China; Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University (Shandong Eye Hospital), Jinan, 250021, China.
Exp Cell Res. 2024 Oct 1;442(2):114227. doi: 10.1016/j.yexcr.2024.114227. Epub 2024 Aug 30.
Severe conjunctival damage can lead to extensive ocular cicatrisation, fornix shortening, and even ocular surface failure, resulting in significant vision impairment. Conjunctival reconstruction is the primary therapeutic strategy for these clinical conjunctival diseases. However, there have been limited studies on induced differentiation of conjunctival epithelial cells derived from stem cells. In this study, we established a chemical defined differentiation protocol from human embryonic stem cells (hESCs) into conjunctival epithelial cells. hES cell line H1 was used for differentiation, and RT-qPCR, immunofluorescence staining, Periodic-acid-Schiff staining (PAS), and transcriptome analysis were employed to identify the differentiated cells. Here, to imitate the development of the vertebrate conjunctiva, hESCs were induced using a three-step process involving first chetomin was used to induce ocular surface ectoderm, then nicotinamide was used to induce ocular surface epithelial progenitor cells, and finally epidermal growth factor, keratinocyte growth factor and other factors were used to differentiate mature conjunctival epithelial cells. hESC-derived conjunctival epithelial cells expressed mature conjunctival epithelial lineage markers (including PAX6, P63, K13). The presence of goblet cells was confirmed by positive PAS. Transcriptome analysis revealed that hESC-derived conjunctival epithelial cells possessed a more naïve phenotype, and exhibited greater proliferation capacity compared to mature human conjunctival epithelial cells, suggesting their potential as alternative seed cells for conjunctival reconstruction.
严重的结膜损伤可导致广泛的眼瘢痕形成、穹窿缩短，甚至眼表面衰竭，从而导致显著的视力损害。结膜重建是这些临床结膜疾病的主要治疗策略。然而，对于来源于干细胞的结膜上皮细胞的诱导分化研究有限。在这项研究中，我们建立了一个从人胚胎干细胞（hESC）分化为结膜上皮细胞的化学定义分化方案。使用 hESC 细胞系 H1 进行分化，并通过 RT-qPCR、免疫荧光染色、过碘酸希夫染色（PAS）和转录组分析来鉴定分化细胞。在这里，为了模拟脊椎动物结膜的发育，我们使用三步法诱导 hESC，首先使用 chetomin 诱导眼表外胚层，然后使用烟酰胺诱导眼表上皮祖细胞，最后使用表皮生长因子、角质细胞生长因子等因子分化成熟的结膜上皮细胞。hESC 来源的结膜上皮细胞表达成熟的结膜上皮谱系标志物（包括 PAX6、P63、K13）。阳性 PAS 证实存在杯状细胞。转录组分析显示，hESC 来源的结膜上皮细胞具有更原始的表型，与成熟的人结膜上皮细胞相比，具有更强的增殖能力，这表明它们可能作为结膜重建的替代种子细胞。
