Reproducing diabetic retinopathy features using newly developed human induced‐pluripotent stem cell‐derived retinal Müller glial cells

Abstract

Muller glial cells (MGCs) are responsible for the homeostatic and metabolic support of the retina. Despite the importance of MGCs in retinal disorders, reliable and accessible human cell sources to be used to model MGC‐associated diseases are lacking. Although primary human MGCs (pMGCs) can be purified from post‐mortem retinal tissues, the donor scarcity limits their use. To overcome this problem, we developed a protocol to generate and bank human induced pluripotent stem cell‐derived MGCs (hiMGCs). Using a transcriptome analysis, we showed that the three genetically independent hiMGCs generated were homogeneous and showed phenotypic characteristics and transcriptomic profile of pMGCs. These cells expressed key MGC markers, including Vimentin, CLU, DKK3, SOX9, SOX2, S100A16, ITGB1, and CD44 and could be cultured up to passage 8. Under our culture conditions, hiMGCs and pMGCs expressed low transcript levels of RLPB1, AQP4, KCNJ1, KCJN10, and SLC1A3. Using a disease modeling approach, we showed that hiMGCs could be used to model the features of diabetic retinopathy (DR)‐associated dyslipidemia. Indeed, palmitate, a major free fatty acid with elevated plasma levels in diabetic patients, induced the expression of inflammatory cytokines found in the ocular fluid of DR patients such as CXCL8 (IL‐8) and ANGPTL4. Moreover, the analysis of palmitate‐treated hiMGC secretome showed an upregulation of proangiogenic factors strongly related to DR, including ANG2, Endoglin, IL‐1β, CXCL8, MMP‐9, PDGF‐AA, and VEGF. Thus, hiMGCs could be an alternative to pMGCs and an extremely valuable tool to help to understand and model glial cell involvement in retinal disorders, including DR.

Spatial and Temporal Development of Müller Glial Cells in hiPSC-Derived Retinal Organoids Facilitates the Cell Enrichment and Transcriptome Analysis

“…Both the P1 and P5 iMGCs abundantly expressed MGC markers SOX9 and GS, but rarely expressed markers VSX2, PAX6 or CRX for other retinal subtypes ( Supplementary Figure S6A ). Surprisingly, the MGC marker CRALBP became negative from passage 1 ( Supplementary Figure S6A ), which was consistent with a previous study on MGCs isolated from ROs (Couturier et al, 2021 ). RNA-seq analysis illustrated that the majority of genes including the classical MGC marker genes such as SOX9, VIM, GLUL, APOE , and SPP1 presented similar transcript level between the P1 and P5 iMGCs, demonstrating the phenotype stability of the iMGCs after several passages ( Supplementary Figures S6B–D ).…”

“…In addition, RPC-related genes such as OLIG2 and DLL3 were enriched in the early-stage ROs ( Figure 2F ). While MGC-related genes, such as CRYAB, RLBP1, CA2 , and SLC1A3 were progressively upregulated over the differentiation process ( Figure 2G ; Supplementary Figure S5C ), which were consistent with the previous studies (Hoshino et al, 2017 ; Kim et al, 2019 ; Yan et al, 2020 ; Couturier et al, 2021 ). The expression of RPC and MGC genes SOX9 and VIM significantly increased in the early-stage ROs when compared to hiPSCs and kept high expression level in late-stage ROs ( Figure 2G ).…”

“…In this study, we compared the cells isolated from ROs at early- (D60-90) and late-stages (D120 and afterwards) in detail. Although both were expandable, cells isolated from the late-stage ROs were the most optimal choice for the selection and enrichment of human MGCs, as they expanded to daughter MGCs with higher purity and expansion capability, and expressed typical MGC markers GS, which is consistent with the reported iMGCs expanded from ROs older than D160 (Couturier et al, 2021 ). In addition, Eastlake and colleagues reported that all of MGCs isolated from ROs aged at D34–D281 could express mRNA coding for the MGC markers CRALBP, GS, nestin and vimentin in two different passages (Eastlake et al, 2019 ).…”

“…Photoreceptor-related genes such as CRX and RCVRN , amacrine and horizontal cell-related genes such as PROX1 and TFAP2A , retinal ganglion cell-related genes such as ATOH7 and ISL1 , and bipolar cell-related genes such as GRM6 and CA10 , were downregulated in the iMGCs, but up-regulated in D150 ROs. While the expression of MGC-related genes such as VIM, CRYAB, APOE , and CCL2 (Hoshino et al, 2017 ; Kim et al, 2019 ; Yan et al, 2020 ; Couturier et al, 2021 ) were enriched in the passaged iMGCs, but down-regulated in D150 ROs ( Figures 5D–H ). Moreover, all of these passaged iMGCs expressed neither GFAP protein ( Supplementary Figure S7 ), nor GFAP, SOD3, S100B, PAX2, CSF1R , or CX3CR1 mRNA (FPKM <1, data not shown), indicating there were no contamination of non-retinal cells or tissue such as astrocyte, optic stalk and microglia in these passaged iMGCs (Westergard and Rothstein, 2020 ; Bosze et al, 2021 ).…”

“…A couple of groups have tried to expand iMGCs from ROs at different developmental stages ranged from D34 to D281 (Chung et al, 2019 ; Eastlake et al, 2019 ; Couturier et al, 2021 ). However, the optimal stage of ROs for enrichment of iMGCs has not been discussed.…”