June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Generation of choroid and RPE cells from iPSC with CFH knockout for modeling of AMD
Author Affiliations & Notes
  • Tea Soon Park
    National Institutes of Health, Bethesda, Maryland, United States
  • Russell Quinn
    National Institutes of Health, Bethesda, Maryland, United States
  • Rishabh Hirday
    National Institutes of Health, Bethesda, Maryland, United States
  • Amir Ali
    National Institutes of Health, Bethesda, Maryland, United States
  • Eric Nguyen
    National Institutes of Health, Bethesda, Maryland, United States
  • Devika Bose
    National Institutes of Health, Bethesda, Maryland, United States
  • Ruchi Sharma
    National Institutes of Health, Bethesda, Maryland, United States
  • Kapil Bharti
    National Institutes of Health, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Tea Soon Park None; Russell Quinn None; Rishabh Hirday None; Amir Ali None; Eric Nguyen None; Devika Bose None; Ruchi Sharma None; Kapil Bharti None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1474. doi:
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    • Get Citation

      Tea Soon Park, Russell Quinn, Rishabh Hirday, Amir Ali, Eric Nguyen, Devika Bose, Ruchi Sharma, Kapil Bharti; Generation of choroid and RPE cells from iPSC with CFH knockout for modeling of AMD. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1474.

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      © ARVO (1962-2015); The Authors (2016-present)

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  • Supplements
Abstract

Purpose : Complement factor H (CFH) inhibits the alternate pathway of the complement system. Compromised CFH expression causes immune system imbalance and complement-mediated damage to healthy cells. CFH expression and activity changes are associated with increased risk of age-related macular degeneration (AMD), but the relative contributions of CFH from the retinal pigmented epithelium (RPE) and the choroid are not known. Here we aim to study the effects of CFH in choroid versus RPE using CFH knockout (KO) iPSCs and the 3D bioprinting technique.

Methods : iPSC line from a healthy donor (wildtype, WT-iPSC) was genetically modified to generate a CFH-KO-iPSC using CRISPR/Cas9 technology. Both WT- and CFH-KO-iPSC lines were differentiated into RPE, endothelial cells (EC), pericytes, and fibroblasts and validated by immunophenotyping. Capillary formations by iPSC-derived cells were assessed by a hydrogel tubulogenesis assay combining ECs, pericytes, and fibroblasts matured for 3 weeks. 3D-bioprinting was performed on a biodegradable PLGA scaffold for choroid formation and the RPE monolayer seeded on the other side of the scaffolds. Live images of GFP expression from ECs were documented for capillary formation and maturation for 5 weeks after 3D-bioprinting.

Results : All differentiated cell components from WT- and CFH-KO-iPSC were cryopreserved and thawed successfully prior to functional validation and 3D bioprinting. CD31+ EC was verified by acetylated-Dil-LDL uptake, hydrogel tubulogenesis, and expression of CD146 and vWF, ETV2, PLVAP, CA4, and RGCC. Pericytes were differentiated from CD31- cells with TGF-b3 and PDGF-bb and confirmed the expression of PDGFR-b, NG2, CD44, and a-SMA. Fibroblasts were differentiated from pericytes and expressed vimentin, connexin43, and collagen-I. EC, pericytes, and fibroblasts were assembled into capillary-like structures in 3D hydrogel tubulogenesis assay up to 3 weeks. Bruch’s membrane formation in bioprinted tissues was confirmed by the expression of fibronectin, collagen-I, laminin, and elastin.

Conclusions : Our results show that all four cell components (RPE, EC, pericytes, and fibroblasts) were differentiated from WT- and CFH-KO-iPSC with compatible protein expressions. Using these cell components we generated 3D-bioprinted ex-vivo choroid-RPE system that provides as a tool to investigate the disease mechanism and the role of anaphylatoxins in RPE versus the choroid.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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