June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
In Vitro Dry-AMD Modeling using iPSC-derived Outer Blood Retinal Barrier Formed by RPE and 3D Bioprinted Choroid
Author Affiliations & Notes
  • Tea Soon Park
    NEI, NIH, Maryland, United States
  • Russell Quinn
    NEI, NIH, Maryland, United States
  • Rishabh Hirday
    NEI, NIH, Maryland, United States
  • Amir Ali
    NEI, NIH, Maryland, United States
  • Eric Nguyen
    NEI, NIH, Maryland, United States
  • Devika Abhijit Bose
    NEI, NIH, Maryland, United States
  • Ruchi Sharma
    NEI, NIH, Maryland, United States
  • Kapil Bharti
    NEI, NIH, 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 2023, Vol.64, 5069. doi:
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      Tea Soon Park, Russell Quinn, Rishabh Hirday, Amir Ali, Eric Nguyen, Devika Abhijit Bose, Ruchi Sharma, Kapil Bharti; In Vitro Dry-AMD Modeling using iPSC-derived Outer Blood Retinal Barrier Formed by RPE and 3D Bioprinted Choroid. Invest. Ophthalmol. Vis. Sci. 2023;64(8):5069.

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

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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 an increased risk of age-related macular degeneration (AMD), but the relative contributions of CFH from the retinal pigmented epithelium (RPE) and the choroid are unclear. Here we aim to study the effects of CFH in choroid versus the 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 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 vasculogenesis assay combining ECs, pericytes, and fibroblasts matured for 4 weeks. 3D-bioprinted choroid was combined with RPE monolayer that matured to form the outer-blood retinal barrier (oBRB) and Bruch’s membrane prior to activation of alternate complement pathway using human serum.

Results : All differentiated cell components from WT- and CFH-KO-iPSC were cryopreserved and thawed successfully prior to functional validation and 3D bioprinting. EC, pericytes and fibroblasts expressed differential proteins related to the alternate complement pathway and lipid accumulation upon human serum treatment. EC, pericytes, and fibroblasts were assembled into capillary-like structures in 3D hydrogel vasculogenesis assay up to 4 weeks. Bruch’s membrane formation in bioprinted tissues was confirmed by the expression of fibronectin, collagen-I, laminin, and elastin. When exposed to human serum, lipo-protein-rich deposits, cellular apoptosis, and capillary loss were observed. WT- and CFH-KO-choroid are matured with either WT- or CFH-KO-RPE to investigate the relative contribution to dry-AMD phenotypes.

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-oBRB system that provides a tool to investigate the disease mechanism and the role of alternate complement in the RPE versus the choroid.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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