June 2020
Volume 61, Issue 7
ARVO Annual Meeting Abstract  |   June 2020
Human ONLR-Neural progenitor cell characterization using AAV technology
Author Affiliations & Notes
  • Zara Mehrabyan
    Ophthalmology, Univ of Maryland Sch of Medicine, Baltimore, Maryland, United States
  • Yan Guo
    Ophthalmology, Univ of Maryland Sch of Medicine, Baltimore, Maryland, United States
  • Steven L. Bernstein
    Ophthalmology, Univ of Maryland Sch of Medicine, Baltimore, Maryland, United States
    Neuroanatomy, University of Maryland, Baltimore, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Zara Mehrabyan, None; Yan Guo, None; Steven Bernstein, None
  • Footnotes
    Support  NEI R01-Ey05304 to SLB and supported by Donner Foundation and the Holt Fund
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3792. doi:
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      Zara Mehrabyan, Yan Guo, Steven L. Bernstein; Human ONLR-Neural progenitor cell characterization using AAV technology. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3792.

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

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Purpose : Neural progenitor/stem cells were recently discovered in the optic nerve lamina region (ONLR) of rodents and humans in our lab (ONLR-NPCs). We previously evaluated mouse ONLR-NPCs and traced their lineage using transgenic technology. Here we report human ONLR progenitor cell characteristics and trace their lineage using adeno-associated virus (AAV) reporters. We wanted to identify: 1). If we can use AAV transduction containing nestin-GFP promoter for NPC identification to help in tracing their lineage and 2). If human ONLR-NPCs in culture can give rise to all macroglial forms.

Methods : Human ONLR and optic nerve (ON) cultures were prepared from human tissue obtained immediately post mortem from 4 donors, 21-42y/o without ocular disease, via the Living Legacy Foundation. 1.5mm of the ONLR and a similar sized distal ON portion were dissected, minced, and triturated; cells were isolated following digestion with collagenase, and re-suspended in NT2 medium and plated on 15% matrigel coated dishes. Cells were placed in a humidity controlled incubator at 370C supplemented with 5% CO2 and 5% O2. We generated an AAV 2/1 vector containing the minimal nestin promotor and GFP reporter, via VectorBuilder (Santa Clara, CA ). Nestin, while not exclusively found in stem cells, is usually strongly expressed in NPCs. We transduced the AAV 2/1- nestin-GFP vector into ONLR- and ON cultures, and analyzed them for GFP co-localization with other NPC markers, including Sox2 and GFAP. We also evaluated cells for astrocyte and oligodendrocyte precursor- and mature oligodendrocyte markers, using immunocytochemistry.

Results : Vigorous culture growth was seen in primary ONLR isolates, much less vigorously from distal ON isolates. Human ONLR-NPCs formed smooth sided neurospheres when grown on low adhesion surfaces. Nestin-driven-GFP expression was detectable at approximately 3 days post-infection and increased up to day 7DIV (n= results of 4 cultures). GFP expression co-localized with antibodies to Nestin, Sox and GFAP, and to a minimal extent with NG2.

Conclusions : NPCs can be isolated and cultured from human ONLR. The current ex vivo approach is suitable for investigating NPC/NSC properties and their ultimate fates. ONLR-NPCs can be transfected using custom-generated AAV 2/1 viral particles, and they express GFP using the Nestin promotor, enabling identification and isolation of these cells, and further investigation of their properties.

This is a 2020 ARVO Annual Meeting abstract.


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