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

Suguru Yamasaki; Atsushi Kuwahara; Akiyoshi Kishino; Toru Kimura; Masayo Takahashi; Michiko Mandai

doi:10.1016/j.reth.2021.12.003

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

Regen Ther. 2022 Jan 1:19:24-34. doi: 10.1016/j.reth.2021.12.003. eCollection 2022 Mar.

Authors

Suguru Yamasaki^{1

2}, Atsushi Kuwahara², Akiyoshi Kishino², Toru Kimura², Masayo Takahashi¹, Michiko Mandai^{1

3}

Affiliations

¹ Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo, 650-0047, Japan.
² Regenerative & Cellular Medicine Kobe Center, Sumitomo Dainippon Pharma Co., Ltd., Kobe, Hyogo, 650-0047, Japan.
³ RIKEN Program for Drug Discovery and Medical Technology Platforms (DMP), RIKEN Cluster for Science, Technology and Innovation Hub, Saitama, 351-0198, Japan.

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.

Keywords: 3D-retina, three-dimensional retinal organoid; BMP, Bone morphogenetic protein; Chk1 inhibitor; Chk1i, Checkpoint kinase 1 inhibitor; Organoid; PD, PD407824; PSCs, Pluripotent stem cells; Photoreceptor; Pluripotent stem cell; RP, Retinitis pigmentosa; Retina; Self-organization; iPSC.