Selection of human iPS cell-derived photoreceptors by targeting of cell surface antigen CD73

Abstract

Purpose : For retinal cell therapy based on human induced pluripotent stem (hiPSCs), one of the major challenges is to identify surface antigens as specific markers of hiPSC-derived photoreceptors that could be used for the separation of transplantation-competent cell population. Here, we focused on the cell surface antigen CD73 during retinal differentiation of hiPSCs

Methods : Retinal organoids containing photoreceptor precursors were generated according to our protocol (Reichman et al. 2014). At different time points of differentiation, expression of CD73 in retinal organoids was assessed by RT-qPCR and immunostaining. For cell separation, retinal organoids were dissociated into single cells, immunostained with a CD73-PE antibody and Magnetic-Activated Cell Sorting (MACS) was performed by using anti-PE Microbeads. Cells from dissociated organoids (unsorted, CD73+ and CD73- fractions) were analyzed and characterized by flow cytometry, RT-qPCR and immunohistochemistry

Results : Analysis of CD73 expression in hiPSC-derived retinal organoids indicated that CD73 is specific of cells committed into the photoreceptor lineage, as all of the CD73+ cells co-localized with well-established photoreceptor markers (RECOVERIN, CONE ARRESTIN and OPSIN). Flow cytometry analysis indicated that the percentage of CD73+ cells in dissociated retinal organoids increased with maturation, with CD73+ cells representing more than 60% of cells at day 180 of differentiation. Dissociated retinal cells expressing CD73 could be sorted by Magnetic-Activated Cell Sorting (MACS), leading to enrichment to 90% of CD73+ cells in the positive sorted fraction. RT-qPCR analysis on sorted CD73+ cells showed over-expression of the most significant photoreceptor-specific genes compared to dissociated retinal cells before CD73 MACS. Re-plating of the CD73-sorted cells demonstrated the cell viability, even several days after the separation process, and expected expression of CRX and RECOVERIN. Interestingly, MACS of CD73+ cells could be performed on freeze-thawed dissociated retinal organoids, with equivalent results to MACS on not frozen organoids

Conclusions : Our results support the use of CD73 as a marker of hiPSC-derived photoreceptors. MACS of CD73+ cells resulted in a significant enrichment of photoreceptor precursors from both fresh and freeze-thawed retinal organoids, which will be of a great utility for future clinical translation

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.