Abstract
Purpose :
Photoreceptors could play a crucial role in retinal cell therapy to treat vision loss. The use of retinal organoid has been hailed to be the established in vitro retinal differentiation method to generate photoreceptors from the pluripotent stem cells. However, this approach is evidently limited by its reproducibility and efficiency level during retinal organoidogenesis. Furthermore, the existing retinal organoid based protocols are not chemically defined and xenogen-free, which include the use of non-human animal serum and Matrigel®, a murine Engelbreth-Holm-Swarm sarcoma extract that are not considered clinically safe. Therefore, it is important to generate clinically safe functional photoreceptors that do not compromise patient’s vision conditions.
Methods :
Currently, we have developed a retinal organoid free method to robustly generate photoreceptor progenitors from human embryonic stem cells (hESCs). Our novel alternative approach employs the use of the human recombinant retina-specific laminin isoforms to recapitulate the retinal interphotoreceptor matrix environment. With the support of an analogous retina matrix like surface, the hESCs are being efficiently differentiated towards photoreceptor progenitor-like cells. Unlike retinal organoid, this chemically defined and xenogen-free method does not involve re-plating and manual dissections.
Results :
This laminin based differentiation method consistently generates photoreceptor progenitors in every single batch of differentiation. These hESCs-derived photoreceptors are shown to be positive for PAX6, VSX2, CRX and RCVRN as early as Day 30, based on transcriptome (n = 9, p<0.001) and immunocytochemical analyses (n=9). In contrast, the pluripotency and teratoma transcript markers are shown to be drastically downregulated, suggesting reduced associated risk of teratoma formation.
Conclusions :
We provide an alternative retina laminin based differentiation method that does not require the formation of retinal organoid. This xenogen-free and chemically defined protocol is compatible with GMP (Good Manufacturing Practice) condition and reproducibly promotes hESCs to photoreceptor progenitor lineage. Hence, these results suggest that our method may constitute an important step towards the future use of hESC-derived photoreceptors to treat vision loss.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.