May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Isolation, Characterization and Expansion of Porcine Retinal Progenitor Cells
Author Affiliations & Notes
  • M.A. Shatos
    Schepens Eye Research Institute, Boston, MA, United States
  • H. Klassen
    Stem Cell Research, Children's Hosp of Orange Co, Orange, CA, United States
  • E. Scherfig
    Dept of Ophthalmology, Rigshospitalet and Eye Pathology Inst, Univ of Copenhagen, Copenhagen, Denmark
  • J.F. Kiilgaard
    Dept of Ophthalmology, Rigshospitalet and Eye Pathology Inst, Univ of Copenhagen, Copenhagen, Denmark
  • K. Warfvinge
    Dept of Ophthalmology, Lund, Sweden
  • J.U. Prause
    Dept of Ophthalmology, Lund, Sweden
  • M.J. Young
    Dept of Ophthalmology, Lund, Sweden
  • Footnotes
    Commercial Relationships  M.A. Shatos, Schepens Eye Research Institute P; H. Klassen, Schepens Eye Research Institute P; E. Scherfig, None; J.F. Kiilgaard, None; K. Warfvinge, None; J.U. Prause, None; M.J. Young, Schepens Eye Research Institute P.
  • Footnotes
    Support  NIH Grant EY09595, MDRCRT, and Massachusetts Lions Foundation
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1694. doi:
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      M.A. Shatos, H. Klassen, E. Scherfig, J.F. Kiilgaard, K. Warfvinge, J.U. Prause, M.J. Young; Isolation, Characterization and Expansion of Porcine Retinal Progenitor Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1694.

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

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Abstract

Abstract: : Purpose: To isolate, characterize and expand porcine retinal progenitor cells for use in transplantation experiments. Methods: Fetal Dorak pigs were sacrificed at 60 days gestation. Neurosensory retinae (excluding the optic nerve head and ciliary marginal zone) were surgically removed and minced. Retinal tissue was subjected to repeated cycles of collagenase (type 1, 0.1%) digestion and then seeded into either uncoated or laminin-treated culture plates in Neurobasal media with B-27 and 20 ng/ml EGF. For immunocytochemistry, cells were grown on glass coverslips in either EGF-supplemented media to maintain a proliferative, undifferentiated state or in media with 10% serum to promote differentiation. Cells were fixed at Day 0, 3, 5 and 7 after serum exposure and evaluated for expression of neural progenitor cell markers, nestin and Ki-67 and for mature CNS lineage markers, MAP 5, NF-200 and GFAP. In separate experiments, cells were seeded at low density and examined for their ability to expand and self-renew. Results: Cells fulfilling several criteria associated with stem cells in other species were isolated from retinae of fetal pigs. Cells grown on plastic primarily formed non-adherent spheres while those grown on laminin were largely adherent. When transferred to glass coverslips both groups of cells expressed identical markers: At day 0 in EGF-containing medium, both highly expressed nestin and Ki-67. Exposure to serum initiated differentiation measured by a gradual decrease in expression of nestin and Ki-67, and increased expression of mature CNS lineage markers MAP5, NF-200 and GFAP. Upon seeding at low densities, the cells repopulated their vessels, although the rate of expansion was less than that seen for murine retinal progenitor cells. These porcine retinal progenitor cells have been maintained, and expanded in culture for 6 months with no apparent change in phenotype, growth rate, or expression of cellular markers. Conclusions: Retinal progenitor cells can be isolated from fetal porcine neuroretina, and can self-renew in vitro for at least 6 months. The cells are multipotent, as they can differentiate into both neuronal and glial lineages. Self-renewal and multipotency are the hallmarks of stem cells, but more studies are needed to determine whether the progenitor cells isolated here fall into this category.

Keywords: retinal culture • retinal development • transplantation 
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