Kethiri Abhinav Reddy, Grönroos Pyry, Nagaraj Ajai Suwaraj Chinnaiah, Skottman Heli
Eye Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.
Sci Rep. 2025 May 28;15(1):18750. doi: 10.1038/s41598-025-03509-3.
Corneal keratocytes (CK) and endothelial cells (CEnC) maintain corneal stromal transparency. Damage to these cell types can lead to visual impairment. Although corneal transplantation is effective, donor shortages limit its availability. Human pluripotent stem cells (hPSC) offer a promising alternative for generating corneal cell types. While hPSC-derived epithelial cells and CEnC are well-studied, attempts to differentiate CK from hPSC have received less attention. This study investigates the differentiation of hPSC-CK using defined culture conditions and approaches to enrich both CK and CEnC. hPSC were cultured on laminin 521 (LN-521) coatings and differentiated using transforming growth factor β (TGFβ) and glycogen synthase kinase-3 (GSK-3) inhibitors, along with retinoic acid (RA) with (mEn protocol) or without (En protocol) fibroblast growth factor-2 (FGF2). Differentiated cells were characterized using qPCR and immunofluorescence on days (D) 8, 10, and 13. CK enrichment utilized collagen-1-coated plates with keratocyte-specific media, while CEnC were purified through metabolic starvation. Results showed the formation of heterogenous cultures containing both CK and CEnC. CK-like cells expressed keratocan (KERA), lumican (LUM), paired box 6 (PAX6) and actin α2 (ACTA2) genes, with proteoglycan expression (lumican (LUM) and decorin (DCN)) increasing over time. En protocol however maintained a stable CK phenotype. Furthermore, enriched hPSC-CK were LUM/DCN/PAX6/CD166 and hPSC-CEnC were CD166/ZO-1. Using xeno-free, defined conditions, we differentiated both CK and CEnC from hPSC using a single protocol with further optimized enrichment methods for hPSC-CK and hPSC-CEnC purification, advancing cell differentiation techniques.
角膜基质细胞（CK）和内皮细胞（CEnC）维持角膜基质的透明度。这些细胞类型受损会导致视力障碍。尽管角膜移植有效，但供体短缺限制了其应用。人多能干细胞（hPSC）为生成角膜细胞类型提供了一种有前景的替代方法。虽然hPSC来源的上皮细胞和CEnC已得到充分研究，但从hPSC分化出CK的尝试受到的关注较少。本研究使用特定的培养条件和方法来富集CK和CEnC，从而研究hPSC-CK的分化。hPSC在层粘连蛋白521（LN-521）包被上培养，并使用转化生长因子β（TGFβ）和糖原合酶激酶-3（GSK-3）抑制剂，以及视黄酸（RA），在有（mEn方案）或无（En方案）成纤维细胞生长因子-2（FGF2）的情况下进行分化。在第8、10和13天，使用qPCR和免疫荧光对分化细胞进行表征。CK富集使用涂有胶原蛋白-1的培养板和角膜基质细胞特异性培养基，而CEnC则通过代谢饥饿进行纯化。结果显示形成了同时包含CK和CEnC的异质培养物。CK样细胞表达角蛋白聚糖（KERA）、光蛋白聚糖（LUM）、配对盒6（PAX6）和肌动蛋白α2（ACTA2）基因，蛋白聚糖表达（光蛋白聚糖（LUM）和核心蛋白聚糖（DCN））随时间增加。然而，En方案维持了稳定的CK表型。此外，富集的hPSC-CK为LUM/DCN/PAX6/CD166，hPSC-CEnC为CD166/ZO-1。使用无动物源的特定条件，我们通过单一方案从hPSC中分化出CK和CEnC，并对hPSC-CK和hPSC-CEnC的纯化采用了进一步优化的富集方法，推动了细胞分化技术的发展。
