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However, variation in hPSCs can compromise cell production protocols and increase unwanted heterogeneity. We hypothesize that intrinsic differences among hPSC lines affect their efficiency and consistency in LSC differentiation. Several hPSC lines were differentiated towards LSCs, and the process was analyzed in different timepoints using real-time qPCR (RT-qPCR) and immunofluorescence (IF) to assess differentiation efficiency. Flow cytometry (FC) was also used to quantify protein expression in the differentiated LSCs. Interestingly, variations in BMP4, LEF1, PAX6, and TGFB1 gene expressions, key factors in corneal epithelium induction, were high even in well-differentiating hPSC lines. Some batches failed to upregulate PAX6 and developed a fibroblastic, mesenchymal morphology, associated with elevated TGFB1 expression. This elevation correlated with fewer cells expressing LSC markers in later differentiation stages. FC results showed similar protein expression variation between lines and batches. 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Based on the Act on the Right in Inventions made at Higher Education Institutions in Finland, all authors employed by Tampere University have given all rights to the University and thus have declared no competing interests. HS is also co-founder and shareholder in StemSight Ltd. The other authors declare no conflicts of interests.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}},{"value":"During preparation of this work, Copilot was used to proofread and improve the fluency of the manuscript text. All text was carefully reviewed and edited as needed by the authors, who take full responsibility of all contents of this publication.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declaration of the use of AI in the writing processof the use of AI in the writing process"}}],"article-number":"9502"}}