June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Improving iPSC-RGC 2D differentiation yield and application to evaluating AAV transduction
Author Affiliations & Notes
  • Jarel Gandhi
    Ophthalmology Research, AbbVie Inc, Irvine, California, United States
  • Jacob Painter
    Ophthalmology Research, AbbVie Inc, Irvine, California, United States
  • Eswar Saraswathi-Mohan
    Ophthalmology Research, AbbVie Inc, Irvine, California, United States
  • Yuan-Xing Guo
    Ophthalmology Research, AbbVie Inc, Irvine, California, United States
  • Yong-Xin Li
    Ophthalmology Research, AbbVie Inc, Irvine, California, United States
  • Matthew Mason
    Ophthalmology Research, AbbVie Inc, Irvine, California, United States
  • Mohammed Dibas
    Ophthalmology Research, AbbVie Inc, Irvine, California, United States
  • Keith A Luhrs
    Ophthalmology Research, AbbVie Inc, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Jarel Gandhi Abbvie Inc, Code E (Employment); Jacob Painter Abbvie Inc, Code E (Employment); Eswar Saraswathi-Mohan Abbvie Inc, Code E (Employment); Yuan-Xing Guo Abbvie Inc, Code E (Employment); Yong-Xin Li Abbvie Inc, Code E (Employment); Matthew Mason Abbvie Inc, Code E (Employment); Mohammed Dibas Abbvie Inc, Code E (Employment); Keith Luhrs Abbvie Inc, Code E (Employment)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 764. doi:
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      Jarel Gandhi, Jacob Painter, Eswar Saraswathi-Mohan, Yuan-Xing Guo, Yong-Xin Li, Matthew Mason, Mohammed Dibas, Keith A Luhrs; Improving iPSC-RGC 2D differentiation yield and application to evaluating AAV transduction. Invest. Ophthalmol. Vis. Sci. 2023;64(8):764.

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

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Abstract

Purpose : Higher throughput screening assays require a large number of purified cells, an obstacle that has typically limited adoption of induced pluripotent stem cell (iPSC) derived cell models. In order to perform gene therapy construct screens targeting retinal ganglions cells (RGCs), we set out to improve RGC differentiation yield by investigating directed neuronal lineage differentiation.

Methods : A reporter iPSC line was generated tagging BRN3B expression to TdTomato and Thy1.2, as previously published. The reporter iPSC line was differentiated to RGCs using previously published 2D culture methods as control. Directed neuronal differentiation was performed towards various ectodermal lineages and compared for RGC differentiation by flow cytometry, immunofluorescence, qPCR, western blot, and functional calcium signaling. MACS purified RGCs were treated with MOIs ranging 103 to 106 with AAV2-CAG-eGFP and visualized over time.

Results : At day 28 of differentiation, expression of TdTomato was detected in 16% of live cells in the rostral floorplate neuroepithelium (RFN) condition compared to 4% in control condition. TdTomato signal was not detected in other groups. MACS purification of Thy1.2+ cells resulted in 4x more cell yield in the RFN group compared to the control. Purified cells in both conditions exhibiting colocalized expression of TdTomato and RBPMS. Western blot analysis resulted in clear bands for BRN3A, BRN3B, and RBPMS in both groups. qPCR showed high levels of RGC markers (ISL1, SNCG, ATOH7, ELAVL4), RGC maturation markers (FSTL4, OPN4, SPP1), and low levels of non-RGC markers (LIN28A, MiTF) in both groups. Stimulation of purified cells with glutamate and higher concentration potassium resulted in calcium signaling response in 80.0 ± 7.8% of cells in RFN condition and 98.0 ± 1.7% in control condition (p=0.02). Infection of RGCs with AAV2-CAG-eGFP with MOI up to 106 did not alter viability up to 1 week. GFP gene expression was detected at low levels, but GFP signal was not detected by microscopy.

Conclusions : Directed differentiation towards the rostral floorplate neuroepithelium improved overall yield efficiency of RGCs compared to previously established 2D culture protocols. An initial MOI ranging study with AAV2 capsid showed no effect on iPSC-RGC viability with relatively low levels of transgene expression after 1 week.

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

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