June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Lineage tracing in retinal organoids as a platform for studying retinoblastoma
Author Affiliations & Notes
  • Michelle G Zhang
    Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Zenith Acosta Torres
    Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Dawn Owens
    Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Robert Suter
    Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Nagi Ayad
    Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, Florida, United States
  • J William Harbour
    Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Daniel Pelaez
    Bascom Palmer Eye Institute, Sylvester Comprehensive Cancer Center, and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, Florida, United States
  • Footnotes
    Commercial Relationships   Michelle Zhang, None; Zenith Acosta Torres, None; Dawn Owens, None; Robert Suter, None; Nagi Ayad, None; J William Harbour, None; Daniel Pelaez, None
  • Footnotes
    Support  This work was supported by Alex’s Lemonade Stand Foundation, Alcon Research Institute, the University of Miami Miller School of Medicine Medical Scientist Training Program, the University of Miami Sheila and David Fuente Graduate Program in Cancer Biology, and a generous gift from Dr. Mark J. Daily. The Bascom Palmer Eye Institute also received funding from NIH Core Grant P30 EY014801 and a Research to Prevent Blindness Unrestricted Grant. The Sylvester Comprehensive Cancer Center also received funding from the National Cancer Institute Core Support grant P30 CA240139. We are grateful to the patients who generously contributed samples for this research. We acknowledge the support of the Biostatistics & Bioinformatics and Oncogenomics Shared Resources at the Sylvester Comprehensive Cancer Center, and the University of Miami Center for Computational Science.
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3815. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Michelle G Zhang, Zenith Acosta Torres, Dawn Owens, Robert Suter, Nagi Ayad, J William Harbour, Daniel Pelaez; Lineage tracing in retinal organoids as a platform for studying retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3815.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Retinoblastoma is the most common primary intraocular malignancy in children. Approximately 25% of the cases fail first line chemotherapy, and the most significant barrier to the treatment strategy is the gap in knowledge regarding biological mechanisms driving retinoblastoma progression. The purpose of this project is to characterize the susceptibility to RB1 loss in the terminal differentiation of the various retinal phenotypes.

Methods : We employ an iPSC-derived 3-Dimensional retinal organoid platform, along with CRISPR gene editing, CellTag barcoding libraries for lineage tracing, and single cell RNA-sequencing (scRNA-seq) to analyze the development of retinal phenotypes from RB1 wild-type (RB1-WT) and RB1 knockout (RB1-KO) retinal organoids. This approach allows us to trace shared retinal cell lineages and identify developmental bifurcations affected by RB1 loss. WT and RB1-KO organoids were established from dental pulp stem cells and photoreceptor markers were probed for in Week 20 using immunofluorescence. At Week 14, both organoids were scRNA-sequenced and analyzed for changes in differentiation markers specific to each major retinal cell type. After differences were confirmed, organoid microinjections were performed with GFP-lentivirus to ensure the feasibility of capturing a diverse representation of retinal cells for downstream analyses. Meanwhile, CellTag libraries were amplified, sequenced for quality control, and packaged into lentivirus. WT and RB1-KO organoids were then marked at three different timepoints using these lentivirus in preparation for scRNA-seq.

Results : Retinal organoids form an ordered structure that can be maintained as far as Week 20, and photoreceptor markers CRX and RCVRN are present along the periphery. By Week 14, there is already an imbalance in the distribution of early-stage and late-stage differentiation markers caused by RB1 loss. GFP-lentivirus microinjections allow for a deeper penetration of lentivirus while keeping the organoids viable. CellTag barcoding libraries sequenced for quality control show a diverse number of unique identifiers.

Conclusions : These preliminary results demonstrate the feasibility of using this platform as a model for modeling retinoblastoma progression and identifying the developmental effects of RB1 loss in the distribution of nascent retinal phenotypes.

This is a 2020 ARVO Annual Meeting abstract.

×
×

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.

×