Transdifferentiation of Rat Keratinocyte Progenitors to Corneal Epithelial Cells by Limbal Niche via the STAT3/PI3K/AKT signaling pathway

PURPOSE. To develop a method for enriching keratinocyte progenitor cells (KPCs) and establish a limbal niche (LN)-mediated transdifferentiation protocol to induce KPCs transdifferentiation into corneal epithelial cells. METHODS. Limbal niche cells (LNCs) were isolated from limbal tissues through enzymatic digestion and characterized. Conditioned medium from LNCs cultures was collected. KPCs were enriched by rapid adhesion of Matrigel and subsequently cultured in either an LNCs-conditioned medium supplemented with KSFM (LN-KS) or SHEM (LN-SH) for 14 days. Corneal-specific marker expression was assessed to evaluate transdifferentiation efficiency. Key transcription factors and signaling pathways involved in the transdifferentiation process were identified through single-cell and RNA sequencing, and validated using western blot and quantitative real-time PCR. RESULTS. Both LN-KS and LN-SH protocols successfully induced corneal epithelial cell transdifferentiation from KPCs, with LN-KS demonstrating higher efficiency in generating CK12+ and p63+ cells (p<0.001). RNA sequencing analysis and western blot have revealed significant activation of STAT3 and PI3K/AKT signaling pathways. Inhibition of STAT3 blocked PI3K/AKT activation and impaired corneal epithelial cell transdifferentiation. CONCLUSIONS. This study demonstrates the ability of LN to promote KPCs transdifferentiation into corneal epithelial cells in vitro, a process partially mediated by the STAT3 and PI3K/AKT signaling pathways.【研究目的】 本研究旨在开发一种富集角质形成细胞祖细胞（keratinocyte progenitor cells, KPCs）的方法，并建立一套由角膜缘微环境（limbal niche, LN）介导的转分化方案，以诱导角质形成细胞祖细胞向角膜上皮细胞转分化。 【研究方法】 通过酶消化法从角膜缘组织中分离角膜缘微环境细胞（limbal niche cells, LNCs）并完成鉴定。收集角膜缘微环境细胞培养的条件培养液。采用基质胶快速黏附法富集角质形成细胞祖细胞，随后将其分别置于添加了角质形成细胞无血清培养基（KSFM）的角膜缘微环境细胞条件培养液（记为LN-KS组），以及添加SHEM的角膜缘微环境细胞条件培养液（记为LN-SH组）中培养14天。通过检测角膜特异性标志物的表达水平，评估转分化效率。通过单细胞测序与RNA测序鉴定参与转分化过程的关键转录因子与信号通路，并采用蛋白质印迹法（western blot）与实时定量聚合酶链反应（qRT-PCR）对上述结果进行验证。 【研究结果】 LN-KS与LN-SH两种方案均成功诱导角质形成细胞祖细胞向角膜上皮细胞转分化，其中LN-KS组在生成CK12阳性与p63阳性细胞方面表现出更优的诱导效率（p<0.001）。RNA测序与蛋白质印迹分析显示，STAT3与PI3K/AKT信号通路被显著激活。抑制STAT3可阻断PI3K/AKT通路的激活，并削弱角膜上皮细胞的转分化过程。 【研究结论】 本研究证实，角膜缘微环境可在体外促进角质形成细胞祖细胞向角膜上皮细胞转分化，该过程部分由STAT3与PI3K/AKT信号通路介导。