COCO enhances the efficiency of photoreceptor precursor differentiation in early human embryonic stem cell-derived retinal organoids

Abstract

Background: Significant progress has been made in cell replacement therapy for neural retinal diseases using retinal cells differentiated from human pluripotent stem cells. Low tumorigenicity and the ability to mature to form synaptic junctions make precursor cells a promising donor source. Here, we attempted to improve the yield of photoreceptor precursor cells in three-dimensional retinal organoids from human embryonic stem cells (hESCs). Methods: A CRX-tdTomato-tagged hESC line was generated to track retinal precursors in 3D retinal organoids. COCO, a multifunctional antagonist of the Wnt, TGF-β, and BMP pathways, was employed to 3D organoid differentiation schemes for enhanced photoreceptor precursor cells. Organoid fluorescence intensity measurement was used to monitor retinalization tendency with the number of precursors further checked by flow cytometry. Signature gene expression during organoid differentiation were assessed by qPCR and immunocytochemistry after COCO supplementation. Results: CRX-positive cells can be spatiotemporally tracked by tdTomato without affecting retinalization during retinal organoid differentiation. Fluorescence intensity of organoids, which turned out highly consistent with flow cytometry measurement, allowed us to determine the differentiation efficiency of precursors during organoid culturing directly. Using COCO as an auxiliary supplement, rather than alone, can yield an increased number of photoreceptor precursors in the early stage of organoid differentiation. Over a longer time-frame, photoreceptor precursors enhanced their fate of cones and decreased fate of rods after treatment with COCO. Conclusions: Tracing with the CRX-reporter system showed that in retinal organoids derived from human pluripotent stem cells, COCO increased the differentiation efficiency of photoreceptor precursors and cones.

Human embryonic stem cell-derived organoid retinoblastoma reveals a cancerous origin

“…Human pluripotent stem cells (hPSCs) are capable of generating self-organized 3D retinal organoids (hROs), which could recapitulate retinogenesis in vitro ( 16 – 18 ). This model has provided an extraordinary research path for disease modeling and drug screening of human retinal diseases, especially monogenic disorders ( 19 – 21 ). As a genetic malignancy, Rb is attributed mainly to biallelic inactivation of RB1 , a tumor-suppressor gene ( 2 , 4 , 22 ).…”

Modeling human retinoblastoma using embryonic stem cell-derived retinal organoids

“…RB1 Mut/Mut hESC lines can further differentiate into human Rb organoids in a stepwise manner. Like normal retinal organoid derived from WT hESCs ( Kuwahara et al., 2015 ; Jin et al., 2019 ; Pan et al., 2020 ), the morphogenetic and molecular properties of RB1 Mut/Mut hESC-derived Rb organoids recapitulate the developing human retina normally before the differentiation day 60, i.e., multilayered neural retina (NR) containing all retinal cell types ( Liu et al., 2020 ). Remarkably, tumor-like “primary foci” were clearly visible in Rb organoids at day 60–75( Figure 5 B).…”

“…A low number of hESCs per well leads to small embryoid bodies with insufficient differentiation capacity, while an excessive number of cells per well results in large embryoid bodies with apoptotic cells. The recommended planting density (12,000 live cells/well) is suitable for most of the stem cell lines ( Deng et al., 2018 ; Pan et al., 2020 ). For different cell lines, optimization may be needed to obtain optimal plating density if the efficiency of EB formation is still low after several attempts.…”

Modeling retinitis pigmentosa through patient-derived retinal organoids

“…Moreover, the experimental procedures and the culture environment can also affect the differentiation efficiency. To improve the differentiation efficiency, we suggest that readers should refer to our recent publication about enhancing the efficiency of the photoreceptor precursor during retinal organoids differentiation ( Pan et al., 2020 ).…”