June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Generation of human RGCs by small molecule-recruitment of developmental mechanisms.
Author Affiliations & Notes
  • Negar Firoozi
    Ophthalmology and Visual sciences, University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Helen Erickson
    Ophthalmology and Visual sciences, University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Pooja Teotia
    Ophthalmology and Visual sciences, University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Iqbal Ahmad
    Ophthalmology and Visual sciences, University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Footnotes
    Commercial Relationships   Negar Firoozi, None; Helen Erickson, None; Pooja Teotia, None; Iqbal Ahmad, None
  • Footnotes
    Support  National Institute of Health (2R01EY022051-05, R01EY029778-01)
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 2508. doi:
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      Negar Firoozi, Helen Erickson, Pooja Teotia, Iqbal Ahmad; Generation of human RGCs by small molecule-recruitment of developmental mechanisms.. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2508.

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

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Abstract

Purpose : Glaucoma is a complex group of diseases characterized by selective degeneration of retinal ganglion cells (RGCs). A comprehensive approach to glaucomatous RGC degeneration may include functional replacement of dead neurons through transplantation and understanding RGCs vulnerability using a disease in a dish model. Both approaches require the directed generation of stable, functional, and target-specific RGCs from pluripotent stem cells (PSCs). We recently demonstrated a rapid and safe, stage-specific, chemically defined protocol that selectively generates functional human RGCs by recapitulating developmental mechanisms. However, this method used factors of animal extraction that precluded its use for clinical purposes. Here, we present a small molecule-based chemically defined protocol where RGCs are generated from (PSCs) by the recruitment of developmental mechanisms.

Methods : We subjected human embryonic stem (ES) cells, in which tdTomato is expressed under the Brn3b promoter for real time analysis of RGC differentiation through our stage-specific, chemically defined protocol for RGC generation. In parallel, ES cells were carried through a protocol in which growth factors were replaced with small molecules that engaged specific pathways. The following substitutions (in parenthesis) were made: hDKK-1 (IWR1-endo), TGF-β (SB431542), hNoggin (LDN 193189), hShh (SAG), hFollistatin (LDN and SB431542), and hBDNF (7,8-DHF).

Results : The protocol consists of two broad stages: (1) derivation of neural rosettes (NRs) consisting of retinal progenitor cells (RPCs) and (2) differentiation of RPCs into RGCs, in three distinct sub-stages of initiation, differentiation, and maturation. In both, growth factor- and small molecule-based protocols, the appearance of NRs and generation of RGCs followed similar temporal sequences. FACS analysis of NR cells revealed the majority to be PAX6-positive. The temporal sequence of RGC differentiation was similar. At the end of differentiation (15DIV), the hRGC generated looked similar in morphology and number.

Conclusions : Our results demonstrate a facile and efficient protocol, amenable for clinical applications and disease modeling, in which hRGCs are generated from pluripotent stem cells by a stage specific small molecule recruitment of developmental mechanisms.

This is a 2020 ARVO Annual Meeting abstract.

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