Retina-Specific Laminin 523 Drives Differentiation of Human Pluripotent Stem Cells to Photoreceptors [bulk_RNAseq]

Inherited retinal diseases and aged related macular degeneration are main causes of blindness that involves irreversible photoreceptor loss. Gene therapy approaches do not prevent photoreceptor degeneration during disease progression, and to date protocols for generating photoreceptors from pluripotent stem cells do not exist. Therefore, transplantation of photoreceptors containing retinal organoids is considered a potential option. However, in vitro formation of organoids requires animal-derived materials and they vary considerably in cell composition between batches, which strongly limits their applications in therapy. Here, we show that human recombinant retina-specific laminin isoform LN523, normally present in the extra cellular matrix ECM surrounding photoreceptors, supports differentiation of pluripotent embryonic stem cells to photoreceptor progenitors in vitro. Using a rabbit macular degeneration model, the transplanted and engrafted cells mature in vivo and form synaptic connectivity with the host retina. Furthermore, addition of a rod-derived cone viability factor increased the formation of cone photoreceptors. These results may pave the way for cell therapy treatment of macular degeneration. Human embryonic stem cells H1 were cultured and differentiated in wells with three types of laminin coating: LN-323 alone, LN-323 + LN-521, and LN-323 + LN-523. CKI-7 and SB431542 was added at Day 2 to inititate differentiation towards neuroectodermal lineage. This medium is replaced by photoreceptor differentiation media (PRDM) on Day 7 for specification towards photoreceptor lineage. The photoreceptor medium consists of 2% B27- without vitamin A supplement, 1% N2 supplement (CTS), 10ng/ml human BDNF (Brain-derived Neurotrophic Factor, PEPROTECH), 10ng/ml human CNTF (Ciliary Neurotrophic Factor, PROSPEC-TANY TECHNOGENE), 0.5uM retinoid acid (Tocris Bioscience) and 10uM DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester, Selleckchem). All media are changed every other day until D32. RNA was extracted from wells at Day 2, 22 and 32. Note: D0, D20, D30 correspond to D2, D22, D32 in the publication.