Retina-specific laminin-based generation of photoreceptor progenitors from human pluripotent stem cells under xeno-free and chemically defined conditions

Photoreceptor cell replacement therapy for retinal degenerative diseases is a promising approach. Presently, most protocols aimed at generating clinically safe and functional cells for retinal diseases face challenges such as low efficiency, poor reproducibility, and time-consuming and complex procedures. These could be due to the dependency on animal-derived components in cell culture media and substrates that support the cell differentiation process. Such conditions are poorly defined chemically, which could affect the robustness of the method and hinder clinical translation of cell therapy in retinal diseases. Here, we describe a simple protocol that is xenogen free and chemically defined to differentiate human embryonic stem cells to photoreceptor progenitors. Human recombinant extracellular matrix laminin 523 and 521 isoforms were used to mimic the inter-photoreceptor matrix niche environment to promote the retinal cell differentiation process. This was also accomplished by the unique combination of two cell differentiation media that recapitulates the retinal development signaling processes. In comparison to other protocols, our protocol does not require any mechanical dissection, which can be technically subjective and tedious. Our directed differentiation method generates photoreceptor progenitors that express similar to 17% cone-rod homeobox (CRX) transcript based on single-cell transcriptomic analyses by day 32. These day 32 photoreceptor progenitors can be cryopreserved and still maintain high cell viability after thawing for cell transplantation. This protocol can be easily reproduced and performed by researchers with basic cell culture experience, which is particularly important for retinal research progress and clinical cell manufacturing in a Good Manufacturing Practice facility.