Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids

Abstract

SummaryPhotoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.

Resolvin D1 Modulates the Intracellular VEGF-Related miRNAs of Retinal Photoreceptors Challenged With High Glucose

“…Primary retinal cells were suspended in FACS Buffer, containing 0.5% bovine serum albumin (BSA), 2 mM EDTA-2H 2 O in FluoroBrite DMEM (Thermo Fisher Scientific, Japan), and were incubated with the anti-mouse CD73 Monoclonal Antibody (Thermo Fisher Scientific, Cat# MA5-15537, RRID: AB_10981671) for 1 h on ice as marker of retinal photoreceptor (Gagliardi et al, 2018). Cells were then washed with FACS Buffer for three times and centrifuged at 400 g for 5 min.…”

Improved Ocular Tissue Models and Eye-On-A-Chip Technologies Will Facilitate Ophthalmic Drug Development

“…2,3 In addition, the time course of expression of developmental markers and mature cell type markers closely mirrored the development of retinal tissue in vivo. [4][5][6][7] This demonstrates that the vision research field now has a powerful tool to aid in the understanding of how retina tissue develops and matures.…”

“…6 When organoids were cultured individually in 96-well plates, these showed a marked increase in the percentage of organoids that exhibited light sensitivity by electrophysiological testing; retina organoids cultured in large groups in 6-well plates were sensitive to light *25% of the time, but *37% of organoids cultured individually were light sensitive. 5 These increases in light sensitivity correlated with increased expression of MITF, a factor important in differentiation of the retinal pigmented epithelium (RPE); TFAP-2a, a factor closely linked to retinal development; and MATH5 and RBPMS, both of which are critical for retinal ganglion cell differentiation. [10][11][12][13][14] Also associated with increased expression of these gene markers was an increased incidence of spontaneously generated RPE in individual organoid cultures.…”

The challenge of developing human 3D organoids into medicines

“…To achieve this, GMP covers all aspects of production (e.g., starting materials, facilities, and equipment), personnel (e.g., training, hygiene of staff), and documentation (e.g., standard operating procedures and registers to prove that they are consistently followed at each step in the manufacturing process) [6]. In fact, protocols for the development of human-induced pluripotent stem cell (iPSC)-derived retinal organoids have been successfully adapted to GMP standards required for the generation of transplantable photoreceptor cells for future clinical applications [11]. Similarly, Takebe and collaborators have further developed their initial liver buds protocol for massive production and reproducibility making it ready for further GMP adaptation in order to scale up production in GMP facilities for transplantation purposes [12].…”