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Isolation and Clonal Analysis of Human Epidermal Keratinocyte Stem Cells in Long-Term Culture

^a Immunobiology and Cell Differentiation Unit, Institute of Biomedical Technologies, Consiglio Nazionale delle Ricerche, Pisa, Italy;
^b Department of Oncology, University of Pisa, Pisa, Italy;
^c NASA/NIH Center for Three-Dimensional Tissue Culture, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development (NICHD), NIH, Bethesda, Maryland, USA

Key Words. Keratinocyte • Stem cells • Differentiation • Three-dimensional cultures • Cell cloning

Correspondence: Roberto P. Revoltella, M.D., Ph.D., Consiglio Nazionale delle Ricerche, Institute of Biomedical Technologies, Unit of Immunobiology and Cell Differentiation, Via Moruzzi 1, 56100 Pisa, Italy. Telephone: 39-050-315-2772; Fax: 39-050-315-3367; e-mail: r.revoltella{at}imd.pi.cnr.it

We developed a procedure for growing normal epidermal keratinocyte stem cells isolated from a single punch biopsy of adult human skin in long-term culture. Primary skin epithelial cells were maintained in collagen-coated plates with irradiated human neonatal foreskin fibroblasts (line HPI.1) as a feeder for more than 120 days, approximately 115 population doublings, without signs of replicative senescence. Clonal analysis revealed the presence of holoclones, meroclones, and paraclones. Only emerging colonies with high proliferative potentials and extensive capacities for division (holoclones and meroclones) were subcultured, favoring the expansion of stem cells and progenitors capable of prolonged self-maintenance when subcloned, thus accounting for the prevailing long-term proliferation of the original culture. We found that meroclones included bipotent progenitors capable of generating both keratinocytes and mucin-producing cells. The numbers of these cells were greater after confluence, suggesting that commitment for their differentiation occurred late in the life of a single clone. On a three-dimensional gelatin matrix and on a collagen layer containing the fibroblast feeder, cells isolated from the expansion of holoclones and meroclones formed stratified cohesive layers of keratinocytes that were able to further differentiate, as in normal skin. These results indicate that our procedure will serve as a valuable tool to study expansion of epidermal stem cells as well as the growth mechanisms and cell products associated with their growth and differentiation.

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