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M.A. Shatos, D.R. Talbot, T.F. Ng, T. Hirose, K. Lashari; Isolation And Characterization Of Cells From Human Infant Eyes With Persistent Fetal Vasculature . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5747.
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© ARVO (1962-2015); The Authors (2016-present)
Persistent fetal vasculature (PFV) is a congenital condition caused by failure of the primary vitreous to regress during development and can lead to vision loss. We have shown that excised retrolental tissues are potential sources of progenitor cells. Our goal here was to isolate, characterize, and propagate cells in PFV membranes for use in ocular transplantation.
Cells were isolated by digesting PFV membranes with Type I collagenase and seeding them in medium supplemented with FBS and growth factors. For immunocytochemistry (ICC) and RT–PCR, cells were grown in medium with or without FBS on glass coverslips or in culture dishes respectively. For ICC, cells were fixed in 4% paraformaldehyde and incubated with antibodies; for RT–PCR studies, total RNA was extracted using Trizol and used as template for cDNA synthesis. Neuroglial expression markers (vimentin, NF200, MAP 2+5, GFAP); endothelial (PECAM); vascular (VEGF, HGFR); neuronal (MAP5); progenitor (nestin) cell expression markers were studied with selected antibodies or by RT– PCR. For high sensitivity RNA measurements real time PCR with SYBR green was performed.
PFV–derived cells have grown in culture for 4 months with no decrease in proliferation rate. To date, two types of cells are evident: one slender with long intertwining processes and the other a rounder cell with small granular inclusions in its cytoplasm. ICC indicated positive expression of the neural markers NF200 and MAP2+5. Vimentin was also positive but no expression of GFAP or PECAM was observed. PCR indicated that cultures are mixtures of cell types. Cell populations contain relatively (versus actin controls) abundant MAP5 and nestin RNAs, lower amounts of GFAP and NF200 RNAs, and no CD–133 or Tal–1 RNAs.
Our cell cultures from PFV membranes appear to consist primarily of two populations of undifferentiated cells. One subpopulation appears to be of neuronal origin. These cells can proliferate in vitro and may be suitable for ocular transplantation studies.
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