whole-mount-retinal-organoid-visualization-with-cellular-resolution

Whole-mount Retinal Organoid Visualization with Cellular Resolution

Retinal organoids derived from human induced pluripotent stem cells are intricate 3D structures that mimic the human retina, offering a powerful platform ...

Hospital Universitari Vall d'Hebron, Vall d'Hebron Institut de Recerca (VHIR)

Chronic oxidative stress and immune dysregulation are key mechanisms involved in the pathogenesis of most retinal degenerative diseases, including age-related macular degeneration. The Ccl2/Cx3cr1/Crb1 mouse model develops a progressive degeneration phenotype, with photoreceptor atrophy, drusen-like lesions or pigment alterations at an early age; however, the role of oxidative stress and immune function in the pathogenesis of the model is poorly understood. We performed a comprehensive characterization of the Ccl2/Cx3cr1/Crb1 mouse to evaluate how these pathways influence pathogenesis. We generated a Ccl2/Cx3cr1 double-knockout (DKO) mouse on a C57BL/6N background (with the rd8 mutation of the Crb1 gene), assessed its retina status and function during 9 months in both in vivo and post-mortem analysis, and performed a comprehensive transcriptomic analysis. DKOrd8 mice presented focal retinal lesions with increased infiltration of microglia and involvement of Müller cells. Lesions progressed to thinning of the photoreceptor nuclear layer, causing a loss in retinal function. Transcriptomics analysis revealed major differential expression of genes involved in oxidative stress and neuronal function, in particular genes related to the mitochondrial electron transport chain and antioxidant cellular response. Our results suggest that alterations in chemokine signaling combined with the rd8 mutation in Ccl2/Cx3cr1/Crb1 mice involve early changes in several pathways associated with age-related macular degeneration, highlighting the relevance of these processes in the pathological retinal degeneration in the DKOrd8 model.

Transcriptomics analysis of Ccl2/Cx3cr1/Crb1 deficient mice provides new insights into the pathophysiology of progressive retinal degeneration.

The retinal extracellular matrix (ECM) provides architectural support, adhesion and signal guidance that controls retinal development. Decellularization of the ECM affords great potential to tissue engineering; however, how structural retinal ECM affects in vitro development, differentiation and maturation of ocular cells remains to be elucidated. Here, mouse and porcine retinas were decellularized and the protein profile analyzed. Acellular retinal ECM (arECM) scaffolds were then repopulated with human iPSC-derived retinal pigment epithelial (RPE) cells or ocular progenitor cells (OPC) to assess their integration, proliferation and organization. 3837 and 2612 unique proteins were identified in mouse and porcine arECM, respectively, of which 93 and 116 proteins belong to the matrisome. GO analysis shows that matrisome-related proteins were associated with the extracellular region and cell junction and KEGG pathways related to signalling transduction, nervous and endocrine systems and cell junctions were enriched. Interestingly, mouse and porcine arECMs were successfully repopulated with both RPE and OPC, the latter exhibiting cell lineage-specific clusters. Retinal cells organized into different layers containing well-defined areas with pigmented cells, photoreceptors, Müller glia, astrocytes, and ganglion cells, whereas in other areas, conjunctival/limbal, corneal and lens cells re-arranged in cell-specific self-organized areas. In conclusion, our results demonstrated that decellularization of both mouse and porcine retinas retains common native ECM components that upon cell repopulation could guide similar ocular cell adhesion, migration and organization.

Repopulation of decellularized retinas with hiPSC-derived retinal pigment epithelial and ocular progenitor cells shows cell engraftment, organization and differentiation.

Recently, great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes. Current protocols generally focus on isolating retinal cells, retinal pigment epithelium (RPE), or corneal cells and fail to recapitulate the complexity of the tissue during eye development. Here, the generation of more advanced in vitro multiocular organoids from human induced pluripotent stem cells (hiPSCs) is demonstrated.

Multiocular organoids from human induced pluripotent stem cells displayed retinal, corneal, and retinal pigment epithelium lineages.

All-trans retinoic acid (ATRA) plays an essential role during human eye development, being temporally and spatially adjusted to create gradient concentrations that guide embryonic anterior and posterior axis formation of the eye. Perturbations in ATRA signaling can result in severe ocular developmental diseases. Although it is known that ATRA is essential for correct eye formation, how ATRA influences the different ocular tissues during the embryonic development of the human eye is still not well studied. Here, we investigated the effects of ATRA on the differentiation and the maturation of human ocular tissues using an in vitro model of human-induced pluripotent stem cells-derived multiocular organoids.

All-trans retinoic acid modulates pigmentation, neuroretinal maturation, and corneal transparency in human multiocular organoids.

Retinitis Pigmentosa type 25 (RP25) is a form of inherited retinal dystrophy characterized by a progressive loss of rod photoreceptors, subsequent degeneration of cone photoreceptors, and eventually, the retinal pigment epithelium. Caused by mutations in the EYS gene, it is believed to be critical for the structural and functional integrity of the retina. Using a non-integrative RNA reprogramming method, we have generated human induced pluripotent stem cell (hiPSC) lines from RP25 patient and from carriers but asymptomatic daughters. These three hiPSC lines maintain a normal karyotype, exhibit pluripotency gene expression, and can differentiate into the three germ layers.

Generation of three human induced pluripotent stem cell lines from retinitis pigmentosa 25 patient and two carriers but asymptomatic daughters.

The Lund MetS rat (BBDR.cg-Lepr.cp/LundRj) is a novel animal model that has a congenic leptin receptor deficiency (LepR) and males exhibit a variety of metabolic abnormalities mimicking the human metabolic syndrome, including hyperglycemia, dyslipidemia, severe obesity, and a type 2 diabetes-like condition from 14 weeks of age. However, whether Lund MetS rats (LM rats) develop diabetic retinopathy is still unknown. The purpose is to investigate the features of diabetic retinopathy in this model. In this study, male LM rats aged 15 and 30 weeks were analyzed for pathological retinal changes, including vasculopathy, inflammation, reactive gliosis, oxidative stress, and neurodegeneration features on the retinas by histological, immunohistochemical, and gene and protein expression analysis. Compared with the non-diabetic LM rats, diabetic LM rats, mainly at 30 weeks of age, had a decrease in retinal thickness and loss of retinal ganglion cells and photoreceptors, indicating retinal neurodegeneration. They also presented an increase in VEGF-A expression, Endra, Icam-1, Vcam-1, and Endrb vascular genes, and albumin suggesting neurovascular unit dysfunction. Furthermore, retinas presented reactive gliosis and infiltration of microglia, TNF-α-positive vessels and expressed elevated levels of inflammatory genes Tnf-α, IL-18 and IL-6, and oxidative stress markers Sod2 and 8-hydroxy-2-deoxyguanosine (8-OHdG). Our results suggest that diabetic LM rats reproduce the early neurodegenerative and altered neuro-vascular features that also occur in the human diabetic eye.

Article	Year
Transcriptomics analysis of Ccl2/Cx3cr1/Crb1 deficient mice provides new insights into the pathophysiology of progressive retinal degeneration. Experimental eye research\| PubMed ID: 33373623	2021
Repopulation of decellularized retinas with hiPSC-derived retinal pigment epithelial and ocular progenitor cells shows cell engraftment, organization and differentiation. Biomaterials\| PubMed ID: 34332373	2021
Multiocular organoids from human induced pluripotent stem cells displayed retinal, corneal, and retinal pigment epithelium lineages. Stem cell research & therapy\| PubMed ID: 34809716	2021
All-trans retinoic acid modulates pigmentation, neuroretinal maturation, and corneal transparency in human multiocular organoids. Stem cell research & therapy\| PubMed ID: 35902874	2022
Generation of three human induced pluripotent stem cell lines from retinitis pigmentosa 25 patient and two carriers but asymptomatic daughters. Stem cell research\| PubMed ID: 39742628	2025
Diabetic retinopathy features in lund MetS rats. Experimental eye research\| PubMed ID: 39923911	2025

Anna Duarri

Biography

JoVE Journal Publications

Other Publications