Gain of 20q11.21 in human pluripotent stem cells enhances differentiation to retinal pigment epithelium

TL;DR

This study shows that a genetic change in stem cells improves their ability to become retinal pigment epithelium cells without causing harmful effects.

Contribution

The study reveals that the 20q11.21 copy number variant enhances RPE differentiation and identifies BCL-XL as a key factor.

Findings

20q11.21 clones differentiate into RPE cells faster and with higher yield.

BCL-XL activity is essential for the enhanced differentiation of 20q11.21 clones.

20q11.21 RPE cells are phenotypically mature and non-tumorigenic.

Abstract

Cell therapies based on human pluripotent stem cells (hPSCs) are in clinical trials with the aim of restoring vision in people with age-related macular degeneration. The final cell therapy product consists of retinal pigment epithelium (RPE) cells differentiated from hPSCs. However, hPSCs recurrently acquire genetic abnormalities that give them an advantage in culture with unknown effects to the clinically-relevant cell progeny. One of the most common genetic abnormalities in hPSCs is the sub-karyotype 20q11.21 copy number variant, known to carry oncogenes. Understanding the impact of this variant on RPE differentiation and its potential for malignant transformation is crucial for the development of safe and effective cell therapies.

We monitored the RPE differentiation efficiency of hPSCs with or without the 20q11.21 variant. We then phenotyped the purified RPE cells for functionality, purity and tumorigenicity potential.

We observed that 20q11.21 clones exhibited an enhanced differentiation capacity, developing pigmented foci at a higher rate and yield compared to normal clones. Gene expression analysis confirmed the upregulation of key RPE markers in 20q11.21 clones. The enhanced differentiation capacity of 20q11.21 clones was found to be dependent on the activity of BCL-XL, located within the amplicon. Furthermore, we demonstrated that 20q11.21-containing RPE cells displayed a mature phenotype, maintained long-term stability, and exhibited no malignant transformation capacity in vitro.

We demonstrated that gain of 20q11.21 enhances the speed and yield of RPE differentiation without compromising the phenotype of the derivatives. Finally, we discovered that 20q11.21-localised BCL-XL is important for RPE differentiation with potential non-canonical roles in retinal biology.

The online version contains supplementary material available at 10.1186/s13287-025-04196-7.

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