June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Analysis of Retinal Organoids Long-term Development by Functional Imaging and Realtime Polymerase Chain Reaction
Yuntian Xue; Bryce McLelland; Gabriel Nistor; Johnny Garcia; Mojan Rouhizadeh; Vanna Giang; Hans Keirstead; William C Tang; Magdalene J Seiler; Andrew W Browne
Author Affiliations & Notes
  • Yuntian Xue
    Biomedical Engineering, University of California Irvine, Irvine, California, United States
  • Bryce McLelland
    AIVITA Biomedical, California, United States
  • Gabriel Nistor
    AIVITA Biomedical, California, United States
  • Johnny Garcia
    Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
  • Mojan Rouhizadeh
    Biological Sciences, University of California Irvine, Irvine, California, United States
  • Vanna Giang
    Biological Sciences, University of California Irvine, Irvine, California, United States
  • Hans Keirstead
    AIVITA Biomedical, California, United States
  • William C Tang
    Biomedical Engineering, University of California Irvine, Irvine, California, United States
  • Magdalene J Seiler
    Physical Medicine & Rehabilitation, University of California Irvine, Irvine, California, United States
    Stem Cell Research Center, University of California Irvine, Irvine, California, United States
  • Andrew W Browne
    Ophthalmology, University of California Irvine, Irvine, California, United States
    Biomedical Engineering, University of California Irvine, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Yuntian Xue, None; Bryce McLelland, AIVITA Biomedical (E); Gabriel Nistor, AIVITA Biomedical (E); Johnny Garcia, None; Mojan Rouhizadeh, None; Vanna Giang, None; Hans Keirstead, AIVITA Biomedical (E), AIVITA Biomedical (S); William Tang, None; Magdalene Seiler, None; Andrew Browne, None
  • Footnotes
    Support  CIRM TR1-10995; KL2 TR001416; Beth L. Koehler Endowed Fund, Fund #6630; The authors acknowledge departmental support from an RPB unrestricted grant.
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1672. doi:
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      Yuntian Xue, Bryce McLelland, Gabriel Nistor, Johnny Garcia, Mojan Rouhizadeh, Vanna Giang, Hans Keirstead, William C Tang, Magdalene J Seiler, Andrew W Browne; Analysis of Retinal Organoids Long-term Development by Functional Imaging and Realtime Polymerase Chain Reaction. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1672.

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Abstract

Purpose : Stem cell-derived retinal organoids (RtOgs) can be used for transplantation in retinal degeneration (RD) models. The purpose of this study was to understand RtOgs' long-term metabolic and genetic changes in vitro to improve quality control for biomanufactured transplantable tissues.

Methods : Ten GMP-compatible lots of RtOgs manufactured by AIVITA Biomedical Inc (Irvine, CA, USA) and were used for long-term functional imaging and qPCR analysis. We used two-photon microscopy (Zeiss LSM 780, 740 nm pulsed laser) coupled with fluorescence lifetime imaging (FLIM) and hyperspectral imaging (HSpec) to characterize the RtOgs’ metabolic activity and structure. RtOg samples ranged from days 51 to 159 of differentiation during longitudinal imaging. In qPCR analysis, we used 12 retinal progenitor and photoreceptor genes and 1 housekeeping gene to identify and quantify the gene expression profile. Human fetal (n=1) and adult retinal tissue (n=3) were used as positive controls. Stem cell line CSC14 was used as a negative control.

Results : The free/bound NADH (f/b NADH) ratio demonstrated that a metabolic shift from glycolysis to oxidative phosphorylation occurred between day 54 and 87. The total metabolic activity shifted slightly back toward glycolysis between day 90 and 100. Consistency in organoid development among production lots was shown. Organoids demonstrated similar metabolic signatures over 5-6 months. Retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor genes expression emerged around the 2nd month of differentiation, which corresponded to the shift in f/b NADH ratio. RtOgs between 3-6 months of differentiation had photoreceptor gene expression levels between the human fetal retina and human adult retina gene expression levels. The occurrence of OPN1 SW and OPN1 LW indicated the maturation of photoreceptors in 4 months of differentiation, which was concurrent to the stabilized level of f/b NADH ratio starting from 4 months.

Conclusions : Long-term imaging data showed that RtOgs in different lots exhibited a reproducible metabolic development from more proliferative to more differentiated at an early stage. After 4 months, the metabolic signature stabilized consistent with gene expression profile stabilization.

This is a 2021 ARVO Annual Meeting abstract.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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Copyright © 2024 Association for Research in Vision and Ophthalmology.
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