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Bar Nachmani; SEAM Organoids Model Iris Muscle Cell Development from the Optic Cup. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4314.
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The differentiation of iris smooth muscle cells is a rare example of neuroectoderm-derived muscle and little is known about how this developmental process occurs. The iris modulates the amount of light entering the eye through the sphincter and dilator muscles. What little is known is increased expression by PAX6 at optic cup lip is necessary for upregulation of aSMA and iris muscle development. Understanding how PAX6 expression drives iris muscle fate specification with what signaling pathways and transcription factors are involved is the aim of this project.
We utilize a previously identified 2-dimentional human embryonic stem cell (hESC)-derived whole-eye development model termed self-formed ectodermal autonomous multi-zone of ocular cells (SEAM) to understand iris muscle development. Previous studies reported the differentiation of optic cup-derived lineages, but iris epithelium and iris muscle cell development has yet to be described. We hypothesized muscle cells of the iris will also be differentiated at a developmentally relevant organ position within the SEAM structures.
After 30 days of differentiation we evaluated the presence of iris muscle cells. We identified a specific lineage in which PAX6 and αSMA are co-expressed by immunofluorescence, suggesting regions of iris muscle development not previously described. These regions often reside near optic cup-derived RPE, similarly to development from the optic cup lip in embryogenesis. We validated this expression pattern in human donor-derived iris tissue. Flow cytometry analysis allows detection of PAX6-aSMA double positive cells. Single cell RNA sequencing of adult human iris confirmed the presence of several iris lineages; a pigmented epithelial lineage expressing DCT and MITF, a neural crest-derived lineage expressing SOX10 and MLANA, and a smooth muscle lineage expressing ACTA2 and TAGLN. We are currently harvesting SEAM-derived iris muscle cells and comparing their expression patterns to our human iris sequencing data.
We successfully generated SEAM whole-eye development organoids that recapitulate iris muscle development in vitro. These iris muscle cells co-express the characteristic PAX6-aSMA pattern as we also demonstrated in human iris tissues. Single cell RNA sequencing of human iris revealed the complexity of the iris tissue and highlights specific expression profiles that can be analyzed in the SEAM differentiation model.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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