# Exploring the impact of human pluripotent stem cell heterogeneity on corneal limbal stem cell differentiation outcomes

## TL;DR

This study shows that differences between human pluripotent stem cell lines affect how well they can be turned into corneal limbal stem cells, which are important for treating eye diseases.

## Contribution

The study reveals that intrinsic hPSC line variability impacts LSC differentiation efficiency and consistency.

## Key findings

- Variations in BMP4, LEF1, PAX6, and TGFB1 gene expression were observed even in well-differentiating hPSC lines.
- Elevated TGFB1 expression correlated with fewer LSC markers and a mesenchymal morphology in some batches.
- Protein expression variation between hPSC lines and batches was confirmed using flow cytometry.

## Abstract

Differentiation of limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) shows promise for treating bilateral limbal stem cell deficiency. 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. These findings highlight the need for further optimization and quality control to ensure reliable production of homogeneous starting material for LSC differentiation.

The online version contains supplementary material available at 10.1038/s41598-026-40503-9.