June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Improved generation and long-term engraftment of retinal organoids from Tankyrase/PARP-Inhibitor-Regulated Naïve human pluripotent stem cells (TIRN-hPSC)
Author Affiliations & Notes
  • Ludovic Zimmerlin
    Oncology, Johns Hopkins University, Baltimore, Maryland, United States
    Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, United States
  • Imran Ahmed Bhutto
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Riya Kanherkar
    Oncology, Johns Hopkins University, Baltimore, Maryland, United States
    Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, United States
  • Tea Soon Park
    Oncology, Johns Hopkins University, Baltimore, Maryland, United States
    Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, United States
  • Michael Barbato
    Oncology, Johns Hopkins University, Baltimore, Maryland, United States
  • Michael Koldobskiy
    Oncology, Johns Hopkins University, Baltimore, Maryland, United States
  • Ying Liu
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Mandeep Singh
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Gerard A Lutty
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, United States
  • Elias T Zambidis
    Oncology, Johns Hopkins University, Baltimore, Maryland, United States
    Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Ludovic Zimmerlin None; Imran Bhutto None; Riya Kanherkar None; Tea Soon Park None; Michael Barbato None; Michael Koldobskiy None; Ying Liu None; Mandeep Singh None; Gerard Lutty None; Elias Zambidis None
  • Footnotes
    Support  This work was supported by grants from the NIH/NEI (R01EY032113; R01EY023962), NIH/NICHD (R01HD082098), Research to Prevent Blindness (Stein Innovation Award), The Maryland Stem Cell Research Fund (2020-MSCRFD-5374), and The Lisa Dean Moseley Foundation.
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3725 – F0331. doi:
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      Ludovic Zimmerlin, Imran Ahmed Bhutto, Riya Kanherkar, Tea Soon Park, Michael Barbato, Michael Koldobskiy, Ying Liu, Mandeep Singh, Gerard A Lutty, Elias T Zambidis; Improved generation and long-term engraftment of retinal organoids from Tankyrase/PARP-Inhibitor-Regulated Naïve human pluripotent stem cells (TIRN-hPSC). Invest. Ophthalmol. Vis. Sci. 2022;63(7):3725 – F0331.

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

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Abstract

Purpose : Generation of retinal organoids (RO) from conventional, primed hPSCs is inefficient and optimized in only a few ‘permissive’ lines. We recently established naïve epiblast-like TIRN-hPSC with reduced interline variability of differentiation for improved vascular progenitor engraftment. Here, we tested whether TIRN-hPSC-derived ROs also possess improved in vivo development to mature photoreceptors.

Methods : Isogenic primed vs TIRN-hPSC were differentiated with an established 3D RO protocol. Horse-shoe (HS)-shaped domains matured to retinal cups (RC) with laminated layers. Neuroepithelial specification and epigenomic integrity were validated at 4-20 weeks by RT-PCR, immunofluorescence, RNA-Seq, and CpG methylation sequencing. Interline variability of RC differentiation was quantitated to 16 weeks. RC neural sheets were transplanted into the subretinal space of NOG-SCID mice, and human photoreceptor specification and engraftment was evaluated at 6-9 months post-transplant.

Results : Although several conventional hPSC lines failed to differentiate into RCs, all TIRN-reverted hPSC efficiently generated HS domains and well-differentiated RC organoids with efficiencies comparable to “permissive” hPSC lines. Eye field-specific transcripts and photoreceptor progenitor markers (2-20 weeks) were detected in greater quantities in TIRN RCs (e.g., CRX+RCV+, HuC/D+ ganglion/amacrine cells, PROX1+ horizontal cells, MITF+ pigmented epithelium). TIRN-derived RO displayed improved maturation of rhodopsin+ photoreceptors with proper histo-architecture. Confocal microscopic evaluation of NOG eyes transplanted with TIRN-derived RC sheets demonstrated long-term engraftment (10 months) of mature human cells in tabulation-like structures with development of a full repertoire of mature rod/cone photoreceptors (e.g., rhodopsin+, L/M opsin+, recoverin+), astrocytes (GFAP+vimentin-), and Mueller cells (GFAP+vimentin+). Transcriptomic and epigenetic studies revealed that chemical PARP inhibition of the TIRN method mediated the improved epigenetic plasticity to retinal lineages.

Conclusions : TIRN reversion of conventional hPSCs abolished interline variability of RO development across genetic background, augmented retinal fate specification, and potentiated long-term survival of transplanted photoreceptors for at least 10 months.

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

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