March 1997
Volume 38, Issue 3
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Articles  |   March 1997
Clonal analysis of the in vivo differentiation potential of keratinocytes.
Author Affiliations
  • Z G Wei
    Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
  • T Lin
    Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
  • T T Sun
    Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
  • R M Lavker
    Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Investigative Ophthalmology & Visual Science March 1997, Vol.38, 753-761. doi:
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    • Get Citation

      Z G Wei, T Lin, T T Sun, R M Lavker; Clonal analysis of the in vivo differentiation potential of keratinocytes.. Invest. Ophthalmol. Vis. Sci. 1997;38(3):753-761.

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Abstract

PURPOSE: This study investigated the in vivo differentiation of conjunctival keratinocytes. METHODS: Keratinocytes from the fornical region of the conjunctival epithelium were isolated and plated at low density (5 x 102 per 100-mm dish) in Dulbecco's minimum essential medium containing 20% fetal bovine serum in the presence of mitomycin C-treated 3T3 feeder cells. At this density, only single, isolated cells were attached after overnight culture. Eight days later, small, well-isolated colonies separated from one another by the feeder cells were detached as a sheet from the dish and were injected subcutaneously into the flanks of BALB/c athymic mice through an 18-gauge needle. Within a day, a small firm nodule appeared at the site of injection. At different time points, the animals were killed, and the nodules were excised for morphologic, histogeometric, and cell kinetic analyses. RESULTS: Each implanted colony derived from a single cell gave rise to a single epithelial cyst lined with a reconstituted stratified epithelium. Goblet-like cells loaded with periodic acid-Schiff-positive cytoplasmic granules began to appear singularly in some of the cysts by day 8 postimplantation and were observed in approximately 85% of the cysts by day 14. CONCLUSIONS: Because the cysts formed were derived from clonal populations of epithelial cells and the majority of cysts had a mixed keratinocyte-goblet cell phenotype, these results suggest strongly the existence of a bipotent precursor cell in conjunctival epithelium that can give rise to both goblet and nongoblet cells. This system can be used to study factors that can influence the commitment of pluripotent epithelial stem cells to divergent pathways of differentiation.

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