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      Location and Clonal Analysis of Stem Cells and Their Differentiated Progeny in the Human Ocular Surface

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          Abstract

          We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45–50 cell doublings and at ∼15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic “cell doubling clock.” These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.

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          Most cited references48

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          The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.

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            Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.

            Inconsistent with the view that hair follicle stem cells reside in the matrix area of the hair bulb, we found that label-retaining cells exist exclusively in the bulge area of the mouse hair follicle. The bulge consists of a subpopulation of outer root sheath cells located in the midportion of the follicle at the arrector pili muscle attachment site. Keratinocytes in the bulge area are relatively undifferentiated ultrastructurally. They are normally slow cycling, but can be stimulated to proliferate transiently by TPA. Located in a well-protected and nourished environment, these cells mark the lower end of the "permanent" portion of the follicle. Our findings, plus a reevaluation of the literature, suggest that follicular stem cells reside in the bulge region, instead of the lower bulb. This new view provides insights into hair cycle control and the possible involvement of hair follicle stem cells in skin carcinogenesis.
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              Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.

              Human diploid epidermis epidermal cells have been successfully grown in serial culture. To initiate colony formation, they require the presence of fibroblasts, but proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largely suppressed, but epidermal cells grow from single cells into colonies. Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the colony morphology more oderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isolate keratinocyte clones free of viable fibroblasts. Like human diploid fibroblasts, human diploid keratinocytes appear to have a finite culture lifetime. For 7 strains studied, the culture lifetime ranged from 20-50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1-1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1-5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons.
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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                17 May 1999
                : 145
                : 4
                : 769-782
                Affiliations
                [* ]Laboratory of Tissue Engineering, I.D.I.-IRCCS, Istituto Dermopatico dell'Immacolata, Rome, Italy; []Division of Ophthalmology, Ospedale “SS. Giovanni e Paolo,” Venice, Italy; and [§ ]Department of Ophthalmology, University of “Tor Vergata,” Rome, Italy
                Author notes

                Address correspondence to Dr. Michele De Luca, Laboratory of Tissue Engineering, IDI, Istituto Dermopatico dell'Immacolata, Via dei Castelli Romani, 83/85, 00040 Pomezia (Roma) Italy. Tel.: 39-6-9112192. Fax: 39-6-9106765. E-mail: m.deluca@ 123456idi.it

                Article
                10.1083/jcb.145.4.769
                2133195
                10330405
                14317a6f-431b-4388-ae28-5f8ca6abc6fc
                Copyright @ 1999
                History
                : 15 October 1998
                : 19 March 1999
                Categories
                Regular Articles

                Cell biology
                keratinocyte,stem cells,differentiation,eye,goblet cells
                Cell biology
                keratinocyte, stem cells, differentiation, eye, goblet cells

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