Generation of Human iPSC-Derived Retinal Organoids for Assessment of AAV-Mediated Gene Delivery

Tso, Amy; Costa, Bruna Lopes da; Fehnel, Alexandra; Levi, Sarah R.; Jenny, Laura A; Ragi, Sara D; Li, Yao; Quinn, Peter M J

doi:10.1007/978-1-0716-2651-1_27

Cited by 6 publications

(5 citation statements)

References 32 publications

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“…Previously validated and published WT hiPSC line ( 101 ) was maintained on Matrigel (BD)-coated plates in mTeSR Plus medium (STEMCELL Technologies) and passaged with ReleSR (STEMCELL Technologies). The retinal organoid differentiation was carried out using the agarose microwell array seeding and scraping (AMASS) method with previously described minor modifications ( 101 , 102 ). In brief, iPSC at 90% confluence were detached with ReleSR (STEMCELL Technologies).…”

Section: Methodsmentioning

confidence: 99%

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina

Stehle,

Imventarza,

Woerz

et al. 2024

Life Sci. Alliance

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Crumbs homolog 1(CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1–CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1–CRB2 interactions. We observed no or a mild loss of CRB1–CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.

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Section: Methodsmentioning

confidence: 99%

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina

Stehle,

Imventarza,

Woerz

et al. 2024

Life Sci. Alliance

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“…Previously validated and published wild-type hiPSC lines [21,22] and a commercially obtained Tom20-mEGFP hiPSC line (AICS-0011 cl.27) were maintained on Matrigel (BD)coated plates in mTeSR plus medium (STEMCELL Technologies, Vancouver, Canada) and passaged with ReleSR (STEMCELL Technologies, Vancouver, Canada). Retinal organoid differentiation was carried out using the agarose microwell array seeding and scraping method with minor modifications [27,28]. In brief, 90% confluent iPSCs were detached with ReleSR (STEMCELL Technologies, Vancouver, Canada).…”

Section: Cell Culture Of Human Adult Donor Rpe Ipsc-derived Rpe (Irpe...mentioning

confidence: 99%

“…TRAP1 silencing in ARPE-19 cells had no major effect on the mitochondrial ultrastructure, in which clear cristae and membranes were observed (Figure 5A,B). To complement the ultrastructural analysis, mitochondria were stained with Image-iT™ TMRM Reagent (TMRM), and morphological parameters such as mitochondrial interconnectivity and elongation were quantitated [27]. No fragmentation of mitochondria was observed in siTRAP1 cells compared with the control (Figure 5C,D).…”

Section: Trap1 Silencing Impairs Mitochondrial Structurementioning

confidence: 99%

TRAP1 Is Expressed in Human Retinal Pigment Epithelial Cells and Is Required to Maintain their Energetic Status

et al. 2023

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Age-related macular degeneration (AMD) is the leading cause of severe vision loss and blindness in elderly people worldwide. The damage to the retinal pigment epithelium (RPE) triggered by oxidative stress plays a central role in the onset and progression of AMD and results from the excessive accumulation of reactive oxygen species (ROS) produced mainly by mitochondria. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone that contributes to the maintenance of mitochondrial integrity by decreasing the production and accumulation of ROS. The present study aimed to evaluate the presence and the role of TRAP1 in the RPE. Here, we report that TRAP1 is expressed in human adult retinal pigment epithelial cells and is located mainly in the mitochondria. Exposure of RPE cells to hydrogen peroxide decreases the levels of TRAP1. Furthermore, TRAP1 silencing increases intracellular ROS production and decreases mitochondrial respiratory capacity without affecting cell proliferation. Together, these findings offer novel insights into TRAP1 functions in RPE cells, opening possibilities to develop new treatment options for AMD.

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“…Therefore, human stem cell-derived RO models have longer culture times, mainly due to natural interspecies differences in gestation periods between humans and mice. hiPSCs are grown close to confluence and made into small floating aggregates via chemical or mechanical means in suspension, forming EBs, undergoing neural induction, then ROs are [ 25 – 26 ] selected after which long term suspension culture allows formation of laminated and mature ROs. Researchers then developed an alternative approach, allowing hiPSCs to grow to confluence rather than generating small floating aggregates, maintaining adherent culture.…”

Section: Retinal Organoid Protocolsmentioning

confidence: 99%

“…Dispase treatment facilitated the detachment of these colonies to form laminated, mature RO in suspension culture [ 29 ]. All three methods have successfully employed both hESCs and hiPSCs and continue to undergo refinements and optimizations [ 24 – 26 , 30 – 33 ]. hiPSC RO recapitulate human fetal retinal structure (Fig.…”

Section: Retinal Organoid Protocolsmentioning

confidence: 99%

Retinal Organoids: A Next-Generation Platform for High-Throughput Drug Discovery

Zhao,

Yan

2023

Stem Cell Rev and Rep

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Retinal diseases are leading causes of blindness globally. Developing new drugs is of great significance for preventing vision loss. Current drug discovery relies mainly on two-dimensional in vitro models and animal models, but translation to human efficacy and safety is biased. In recent years, the emergence of retinal organoid technology platforms, utilizing three-dimensional microenvironments to better mimic retinal structure and function, has provided new platforms for exploring pathogenic mechanisms and drug screening. This review summarizes the latest advances in retinal organoid technology, emphasizing its application advantages in high-throughput drug screening, efficacy and toxicity evaluation, and translational medicine research. The review also prospects the combination of emerging technologies such as organ-on-a-chip, 3D bioprinting, single cell sequencing, gene editing with retinal organoid technology, which is expected to further optimize retinal organoid models and advance the diagnosis and treatment of retinal diseases.

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Generation of Human iPSC-Derived Retinal Organoids for Assessment of AAV-Mediated Gene Delivery

Cited by 6 publications

References 32 publications

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina

TRAP1 Is Expressed in Human Retinal Pigment Epithelial Cells and Is Required to Maintain their Energetic Status

Retinal Organoids: A Next-Generation Platform for High-Throughput Drug Discovery

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