June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Modeling VHL-associated hemangioblastomas in vitro using patient-derived stem cells
Author Affiliations & Notes
  • Lola P Lozano
    Institute for Vision Research, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
  • Laura R Bohrer
    Institute for Vision Research, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
  • Nathaniel Kevin Mullin
    Institute for Vision Research, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
  • Edwin M Stone
    Institute for Vision Research, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
  • H Culver Boldt
    Institute for Vision Research, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
  • Robert Mullins
    Institute for Vision Research, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
  • Elaine Binkley
    Institute for Vision Research, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
  • Budd A. Tucker
    Institute for Vision Research, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
    Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Lola Lozano None; Laura Bohrer None; Nathaniel Mullin None; Edwin Stone None; H Boldt None; Robert Mullins None; Elaine Binkley None; Budd Tucker None
  • Footnotes
    Support  NIH Grants T32-GM139776, R21 EY032155
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3176. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Lola P Lozano, Laura R Bohrer, Nathaniel Kevin Mullin, Edwin M Stone, H Culver Boldt, Robert Mullins, Elaine Binkley, Budd A. Tucker; Modeling VHL-associated hemangioblastomas in vitro using patient-derived stem cells. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3176.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Mutations in the VHL gene can lead to von Hippel-Lindau (VHL) disease, which predisposes individuals to developing retinal hemangioblastomas. Mutations are inherited in an autosomal dominant manner with tumor development believed to result from dysregulation of the hypoxia response pathway. Clinical observations document significant phenotypic variability among related patients who carry the same mutation. This suggests that genetic modifiers may influence disease severity. The purpose of this study was to combine patient-derived induced pluripotent stem cells (iPSCs) and genome editing to generate an in vitro model system suitable for investigating VHL disease mechanisms and evaluating the role of novel genetic variants on phenotypic severity.

Methods : We obtained peripheral blood and dermal fibroblast cells from four family members diagnosed with VHL disease. DNA isolated from peripheral blood was Sanger sequenced. Dermal fibroblasts were characterized via a hypoxia gene expression array and reprogrammed into iPSCs via forced expression of the transcription factors OCT4, SOX2, KLF4 and C-MYC. CRISPR-Cas9 ribonucleoproteins designed to cut upstream of the desired VHL locus were designed, fabricated, and delivered to patient iPSCs via the CellFE Zephyr microfluidics platform. T7E1 assay was used to evaluate cutting efficiency and select guides for homology dependent repair.

Results : Sequencing the VHL locus revealed a heterozygous pathogenic Tyr98His mutation in all four individuals. Two of the patients (ages 44 and 60) were determined to have clinically severe disease and two of the patients (ages 61 and 82) were determined to have clinically mild disease. Patient iPSCs were demonstrated to be pluripotent via scorecard analysis and to have normal chromosomal structure and number via karyotyping. Following delivery of CRISPR-Cas9 ribonucleoproteins, a cutting efficiency of up to 30% at the targeted locus near the Tyr98His mutation was detected. Notably, delivery was achieved using the CellFE Zephyr microfluidics platform which avoids introduction of additional foreign reagents into cells, minimizing toxicity and potentially confounding variables.

Conclusions : These results show that iPSC cell cultures can be effectively established from dermal punch biopsies of patients affected with VHL disease. Furthermore, CRISPR technology will enable the ability to model disease-associated hemangioblastomas in vitro.

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

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×