Abstract
BackgroundSignificant 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).MethodsA 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.ResultsCRX-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.ConclusionsTracing 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.
Highlights
The majority of retinal cell types can be formed by in vitro differentiation of pluripotent stem cells (PSCs)
Compared with the single cell type obtained from most 2D differentiation, the multiple types of retinal cells obtained in a 3D differentiation system form well-laminated and properly stratified structures similar to the real retina, which are obviously closer to the physiological state [10, 11]
Correlation between fluorescence intensity and number of fluorescent cells in retinal organoids To intuitively and quickly judge whether the differentiation scheme effectively improved the production of photoreceptor precursor cells, we first constructed a cell line based on Embryonic stem cells (ESCs) H9 with Cone-Rod homeobox (CRX) labeled by tdTomato
Summary
The majority of retinal cell types can be formed by in vitro differentiation of pluripotent stem cells (PSCs). Inhibiting Notch signaling during the early period of differentiation accelerates the transformation of progenitor cells into precursor cells, while eliminating the inhibition of GSK3 FGFR promotes the transition of RPE cells to the neural retina [12, 13]. It is unknown whether an earlier stage of intervention during stem cell differentiation would achieve more effective production of photoreceptor precursor cells. We attempted to improve the yield of photoreceptor precursor cells in three-dimensional retinal organoids from human embryonic stem cells (hESCs)
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