# Co-differentiation and enrichment of corneal endothelial cells and keratocytes from human pluripotent stem cells

## TL;DR

This study shows how to grow corneal cells from human stem cells using specific lab conditions, which could help address donor shortages for corneal transplants.

## Contribution

A new method for co-differentiating and enriching corneal endothelial cells and keratocytes from human pluripotent stem cells using defined culture conditions.

## Key findings

- Differentiated cells expressed keratocyte markers like KERA, LUM, and ACTA2, with proteoglycan expression increasing over time.
- The En protocol maintained a stable corneal keratocyte phenotype.
- Xeno-free conditions enabled successful differentiation and purification of both cell types.

## Abstract

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.

The online version contains supplementary material available at 10.1038/s41598-025-03509-3.