Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture

Abstract

Background The BMP signaling pathway plays a key role in growth, differentiation and patterning during neural development. Recent work on the generation of a self-organization of three-dimensional retinal organoid (3D-retina) from human pluripotent stem cells (hPSCs) revealed that addition of recombinant human BMP4 (rhBMP4) promotes retinal differentiation in the early neural differentiation stage. For clinical application, efficient differentiation from hPSCs to retinal cells with minimal numbers of off-target non-retinal cells is desirable. We therefore aimed to further improve an efficient retinal differentiation method for future up-scaling of cell production. Methods hPSCs were differentiated into 3D-retina using a modified SFEBq method. The effect of rhBMP4 with or without Checkpoint kinase 1 (Chk1) inhibitor (PD407824), a modulator of BMP signaling pathway, at day 3 was compared by characterizing the differentiating 3D-retina by the use of the hPSCs and immunohistochemical analysis. Results The Chk1 inhibitor treatment promoted retinal differentiation from hPSCs, in combination with low-concentration rhBMP4. Addition of a Chk1 inhibitor generated a unique type of organoid with neural retina (NR) encapsulated in retinal pigment epithelium (RPE), possibly by promoting phosphorylation of SMAD1/5/9 in the cells inside the early aggregates. We confirmed that the Chk1-inhibitor-treated hPSC-3D-retina differentiated into rod and cone photoreceptor precursors and other types of retinal neurons, in long-term culture. Conclusions In this study, we found that combined use of rhBMP4 and a Chk1 inhibitor cooperatively promoted retinal differentiation from hPSCs. Our new retinal differentiation method is a promising option for the stable supply and up-scaling of production of 3D-retina for future cell therapy.

Reliability of human retina organoid generation from hiPSC-derived neuroepithelial cysts

“…Like the early 2D and retinal differentiation protocols, HRO protocols also involve several factors applied at defined time points throughout retinogenesis to potentially support differentiation at several stages (Llonch et al, 2018;Wagstaff et al, 2021). However, the optimal protocol has not yet been identified, some of the underlying developmental mechanisms are still incompletely understood, and further protocols optimized for specific retinal features are continuously being developed (Kaya et al, 2019;Mellough et al, 2019;Pan et al, 2020;Zerti et al, 2020;Wagstaff et al, 2021;Chen et al, 2022;Chew et al, 2022;Sanjurjo-Soriano et al, 2022;Yamasaki et al, 2022). Of note, many pluripotent stem cellbased differentiation protocols rely on the formation of embryoid bodies (EBs), 3D cell aggregates that mimic some features of a developing embryo and that have the ability to produce cells from all three germ layers (Hopfl et al, 2004).…”

One-stop assembly of adherent 3D retinal organoids from hiPSCs based on 3D-printed derived PDMS microwell platform

“…As a result, we speculated that GATA5, DLX5, BMPR1B, and other members of the BMP family except for BMP4 may be involved in hiPSCs differentiation into the ROs in the PDMS microwell platform. The low concentrations of BMP4 can promote early differentiation of aggregates toward the retina in the early stage [56,57]. However, the detailed mechanism of action is unclear.…”

NAxtra magnetic nanoparticles for low-cost, efficient isolation of mammalian DNA and RNA

“…Retinal, cardiac, and vascular organoids were generated following procedures described by Yamasaki et al . 21 , Sun et al 22 , and Hofbauer et al . 23 , respectively, with minor modifications, such as changes in medium components and/or differentiation mediators.…”