Retinal organoids and microfluidic chip-based approaches to explore the retinitis pigmentosa with USH2A mutations

Abstract

Retinitis pigmentosa (RP) is a leading cause of vision impairment and blindness worldwide, with limited medical treatment options. USH2A mutations are one of the most common causes of non-syndromic RP. In this study, we developed retinal organoids (ROs) and retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (iPSCs) of RP patient to establish a sustainable in vitro RP disease model. RT-qPCR, western blot, and immunofluorescent staining assessments showed that USH2A mutations induced apoptosis of iPSCs and ROs, and deficiency of the extracellular matrix (ECM) components. Transcriptomics and proteomics findings suggested that abnormal ECM-receptor interactions could result in apoptosis of ROs with USH2A mutations via the PI3K-Akt pathway. To optimize the culture conditions of ROs, we fabricated a microfluidic chip to co-culture the ROs with RPE cells. Our results showed that this perfusion system could efficiently improve the survival rate of ROs. Further, ECM components such as laminin and collagen IV of ROs in the RP group were upregulated compared with those maintained in static culture. These findings illustrate the potential of microfluidic chip combined with ROs technology in disease modelling for RP.

Keywords: USH2A; induced pluripoten stem cells; microfludic chip; retinal organoids; retinitis pigmentosa.

FIGURE 1

Generation of ROs derived from iPSCs with USH2A mutations. (A) Schematic representation of iPSCs differentiation along the ROs lineage. (B) Bright images of ROs at D8-D180 derived from RPiPSCs and NiPSCs with induction of BMP4, taurine and retinoic acid at different time points (×5). (C) Immunostaining images of cytosections of ROs were positive for markers of rod (rhodopsin), cone (opsin and S-arrestin), müller (GS), astrocyte (GFAP) and retinal progenitor cells (VSX2 and Nestin).

FIGURE 2

Directed differentiation protocol of RPE cells. (A) Schematic representation of the protocol. (B) Representative images of RPE cells at days 20, 63 and 84. (C) SEM images of RPE cells at D84. (D) Immunofluorescent staining of RPE specific markers. (Scale bar, 50 μm) (E–F) Positive cells or areas analysis. (G) Confocal micrographs of RPE cells stained with BEST1 (green) and PMEL (red). Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance (n = 3).

FIGURE 3

Transcriptomics and proteomics analysis for early ROs with USH2A mutations. (A) Volcano plot showing 693 DEGs were upregulated and 309 DEGs were downregulated in the RP group. (B) The 13 significantly changed signaling pathways based on DEGs in the RP group. (C)The 15 GO biological process terms based on DEGs. (D) Volcano plot showing 422 differentially expressed proteins, 151 proteins were upregulated and 170 proteins were downregulated. (E) The 11 significantly changed signaling pathways based on differentially expressed proteins. (F) The 15 GO biological process terms based on up-regulated and down-regulated proteins were selected.

FIGURE 4

Increased apoptosis in iPSCs and ROs with USH2A variants (A,B) Cell numbers of iPSCs at different days and passages. (C) Cell cycle distribution analysis of two iPSC lines. (D) The percentage of EdU-positive cells in both iPSC lines. (E) Compared to control group, apoptosis rate of iPSCs significantly increased in the RP group. (F) Apoptotic cells were labeled with TUNEL (FITC, green). Nuclei are labeled with DAPI (blue) and the ratio of TUNEL-positive cells in retina organoids in both groups at D80. (G) Western blotting results of bcl2 and bax at D18 and D80. The grayscale ratio of RP group to the control group quantified in bar graphs. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3). (H) Heatmap of gene expression changes and proteins related to regulation of apoptotic process. Upregulated and downregulation expression are in red or blue respectively. Bar is representing Z-score. Con: control.

FIGURE 5

RPiPSCs derived ROs showing abnormal ECM organization functions. (A) Immunostaining of laminin (red) and collagen IV (red) of ROs in both groups. (B) Quantification of positive areas with laminin and collagen IV. (C) RT-qPCR results showing the expression of ECM-related molecules. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance (n = 3). (D) Heatmap of gene and protein expression changes related to ECM organization of ROs at D18. Upregulated and downregulation expression are in red or blue respectively. Bar is representing Z-score. Con: control.

FIGURE 6

Microfluidic chip of perfusable ROs in co-culture with RPE. (A) Schematic of the microfluidic system. (B) Immunostaining images of laminin (red) and collagen IV (red) of ROs in response to perfusion with 200 μL/min after 30 days compared to static control condition. (C,D) Measurements of positive areas with laminin and collagen IV. (E) The survival rate of ROs. (F) After 30 days perfusion, RT-qPCR results show COL4A6, LAMB2 and NID1 mRNA expression levels of ROs. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3). Scale bar:100 μm.

FIGURE 7

Quantitative comparison of RPE cells in two culture methods. (A) Bright images of RPE cells under two culture conditions and immunostaining images of PMEL (red). (B) Positive areas analysis. Data are shown as mean ± SD. **p < 0.01 (n = 3) (C) Perfused culture of RPE cells leads to an upregulation of BEST1 and PMEL. Scale bar:50 μm.

FIGURE 8

Schematic overview of ECM-receptor interactions of molecules with special emphasis on signaling pathways based on transcriptional and proteomic profiles.