Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. Jul 1, 1986; 103(1): 49–62.
PMCID: PMC2113783

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells

Abstract

In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."

Full Text

The Full Text of this article is available as a PDF (1.7M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Breitkreutz D, Bohnert A, Herzmann E, Bowden PE, Boukamp P, Fusenig NE. Differentiation specific functions in cultured and transplanted mouse keratinocytes: environmental influences on ultrastructure and keratin expression. Differentiation. 1984;26(2):154–169. [PubMed]
  • Buck RC. Measurement of centripetal migration of normal corneal epithelial cells in the mouse. Invest Ophthalmol Vis Sci. 1985 Sep;26(9):1296–1299. [PubMed]
  • Cavanagh HD, Colley A, Pihlaja DJ. Persistent corneal epithelial defects. Int Ophthalmol Clin. 1979 Summer;19(2):197–206. [PubMed]
  • Cooper D, Schermer A, Pruss R, Sun TT. The use of aIF, AE1, and AE3 monoclonal antibodies for the identification and classification of mammalian epithelial keratins. Differentiation. 1984;28(1):30–35. [PubMed]
  • Cooper D, Schermer A, Sun TT. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications, and limitations. Lab Invest. 1985 Mar;52(3):243–256. [PubMed]
  • Cooper D, Sun TT. Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression. J Biol Chem. 1986 Apr 5;261(10):4646–4654. [PubMed]
  • Davanger M, Evensen A. Role of the pericorneal papillary structure in renewal of corneal epithelium. Nature. 1971 Feb 19;229(5286):560–561. [PubMed]
  • Doran TI, Vidrich A, Sun TT. Intrinsic and extrinsic regulation of the differentiation of skin, corneal and esophageal epithelial cells. Cell. 1980 Nov;22(1 Pt 1):17–25. [PubMed]
  • Eichner R, Bonitz P, Sun TT. Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression. J Cell Biol. 1984 Apr;98(4):1388–1396. [PMC free article] [PubMed]
  • Eichner R, Sun TT, Aebi U. The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments. J Cell Biol. 1986 May;102(5):1767–1777. [PMC free article] [PubMed]
  • Franke WW, Appelhans B, Schmid E, Freudenstein C, Osborn M, Weber K. Identification and characterization of epithelial cells in mammalian tissues by immunofluorescence microscopy using antibodies to prekeratin. Differentiation. 1979;15(1):7–25. [PubMed]
  • Franke WW, Schiller DL, Hatzfeld M, Winter S. Protein complexes of intermediate-sized filaments: melting of cytokeratin complexes in urea reveals different polypeptide separation characteristics. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7113–7117. [PMC free article] [PubMed]
  • Franke WW, Schiller DL, Moll R, Winter S, Schmid E, Engelbrecht I, Denk H, Krepler R, Platzer B. Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues. J Mol Biol. 1981 Dec 25;153(4):933–959. [PubMed]
  • Franke WW, Schmid E, Osborn M, Weber K. Different intermediate-sized filaments distinguished by immunofluorescence microscopy. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5034–5038. [PMC free article] [PubMed]
  • Franke WW, Schmid E, Wellsteed J, Grund C, Gigi O, Geiger B. Change of cytokeratin filament organization during the cell cycle: selective masking of an immunologic determinant in interphase PtK2 cells. J Cell Biol. 1983 Oct;97(4):1255–1260. [PMC free article] [PubMed]
  • Friedenwald JS, Buschke W. Some Factors Concerned in the Mitotic and Wound-Healing Activities of the Corneal Epithelium. Trans Am Ophthalmol Soc. 1944;42:371–383. [PMC free article] [PubMed]
  • Fuchs E. Evolution and complexity of the genes encoding the keratins of human epidermal cells. J Invest Dermatol. 1983 Jul;81(1 Suppl):141s–144s. [PubMed]
  • Fuchs E, Green H. The expression of keratin genes in epidermis and cultured epidermal cells. Cell. 1978 Nov;15(3):887–897. [PubMed]
  • Fuchs E, Hanukoglu I, Marchuk D, Grace MP, Kim KH. The nature and significance of differential keratin gene expression. Ann N Y Acad Sci. 1985;455:436–450. [PubMed]
  • Gipson IK, Anderson RA. Comparison of 10 nm filaments from three bovine tissues. Exp Cell Res. 1980 Aug;128(2):395–406. [PubMed]
  • Goldberg MF, Bron AJ. Limbal palisades of Vogt. Trans Am Ophthalmol Soc. 1982;80:155–171. [PMC free article] [PubMed]
  • Hatzfeld M, Franke WW. Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol. 1985 Nov;101(5 Pt 1):1826–1841. [PMC free article] [PubMed]
  • Kim KH, Marchuk D, Fuchs E. Expression of unusually large keratins during terminal differentiation: balance of type I and type II keratins is not disrupted. J Cell Biol. 1984 Nov;99(5):1872–1877. [PMC free article] [PubMed]
  • Kinoshita S, Friend J, Kiorpes TC, Thoft RA. Keratin-like proteins in corneal and conjunctival epithelium are different. Invest Ophthalmol Vis Sci. 1983 May;24(5):577–581. [PubMed]
  • Kinoshita S, Friend J, Thoft RA. Sex chromatin of donor corneal epithelium in rabbits. Invest Ophthalmol Vis Sci. 1981 Sep;21(3):434–441. [PubMed]
  • Knapp LW, O'Guin WM, Sawyer RH. Rearrangement of the keratin cytoskeleton after combined treatment with microtubule and microfilament inhibitors. J Cell Biol. 1983 Dec;97(6):1788–1794. [PMC free article] [PubMed]
  • Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. [PubMed]
  • Lavker RM, Sun TT. Heterogeneity in epidermal basal keratinocytes: morphological and functional correlations. Science. 1982 Mar 5;215(4537):1239–1241. [PubMed]
  • Lavker RM, Sun TT. Epidermal stem cells. J Invest Dermatol. 1983 Jul;81(1 Suppl):121s–127s. [PubMed]
  • Moll R, Moll I, Franke WW. Differences of expression of cytokeratin polypeptides in various epithelial skin tumors. Arch Dermatol Res. 1984;276(6):349–363. [PubMed]
  • Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. [PubMed]
  • Nelson WG, Battifora H, Santana H, Sun TT. Specific keratins as molecular markers for neoplasms with a stratified epithelial origin. Cancer Res. 1984 Apr;44(4):1600–1603. [PubMed]
  • Nelson WG, Sun TT. The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies. J Cell Biol. 1983 Jul;97(1):244–251. [PMC free article] [PubMed]
  • O'Farrell PZ, Goodman HM, O'Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. [PubMed]
  • Potten CS, Schofield R, Lajtha LG. A comparison of cell replacement in bone marrow, testis and three regions of surface epithelium. Biochim Biophys Acta. 1979 Aug 10;560(2):281–299. [PubMed]
  • Pruss RM, Mirsky R, Raff MC, Thorpe R, Dowding AJ, Anderton BH. All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell. 1981 Dec;27(3 Pt 2):419–428. [PubMed]
  • Quinlan RA, Schiller DL, Hatzfeld M, Achtstätter T, Moll R, Jorcano JL, Magin TM, Franke WW. Patterns of expression and organization of cytokeratin intermediate filaments. Ann N Y Acad Sci. 1985;455:282–306. [PubMed]
  • Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975 Nov;6(3):331–343. [PubMed]
  • Roberson MC. Corneal epithelial dysplasia. Ann Ophthalmol. 1984 Dec;16(12):1147–1150. [PubMed]
  • Scheving LE, Burns ER, Pauly JE, Tsai TH. Circadian variation in cell division of the mouse alimentary tract, bone marrow and corneal epithelium. Anat Rec. 1978 Aug;191(4):479–486. [PubMed]
  • Schweizer J, Kinjo M, Fürstenberger G, Winter H. Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: basal cells with properties of terminally differentiating cells. Cell. 1984 May;37(1):159–170. [PubMed]
  • Skerrow D, Skerrow CJ. Tonofilament differentiation in human epidermis, isolation and polypeptide chain composition of keratinocyte subpopulations. Exp Cell Res. 1983 Jan;143(1):27–35. [PubMed]
  • Srinivasan BD, Worgul BV, Iwamoto T, Eakins KE. The reepithelialization of rabbit cornea following partial and complete epithelial denudation. Exp Eye Res. 1977 Oct;25(4):343–351. [PubMed]
  • Sun TT, Green H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope. Cell. 1976 Dec;9(4 Pt 1):511–521. [PubMed]
  • Sun TT, Green H. Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states. Nature. 1977 Oct 6;269(5628):489–493. [PubMed]
  • Sun TT, Green H. Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation. J Biol Chem. 1978 Mar 25;253(6):2053–2060. [PubMed]
  • Sun TT, Green H. Immunofluorescent staining of keratin fibers in cultured cells. Cell. 1978 Jul;14(3):469–476. [PubMed]
  • Sun TT, Shih C, Green H. Keratin cytoskeletons in epithelial cells of internal organs. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2813–2817. [PMC free article] [PubMed]
  • Sun TT, Tseng SC, Huang AJ, Cooper D, Schermer A, Lynch MH, Weiss R, Eichner R. Monoclonal antibody studies of mammalian epithelial keratins: a review. Ann N Y Acad Sci. 1985;455:307–329. [PubMed]
  • Thoft RA, Friend J. The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci. 1983 Oct;24(10):1442–1443. [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Tseng SC, Hatchell D, Tierney N, Huang AJ, Sun TT. Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. J Cell Biol. 1984 Dec;99(6):2279–2286. [PMC free article] [PubMed]
  • Tseng SC, Jarvinen MJ, Nelson WG, Huang JW, Woodcock-Mitchell J, Sun TT. Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies. Cell. 1982 Sep;30(2):361–372. [PubMed]
  • Vidrich A, Gilmartin ME, Mitchell J, Freedberg IM. Postsynthetic modifications of epithelial keratins. Ann N Y Acad Sci. 1985;455:354–370. [PubMed]
  • Waring GO, 3rd, Roth AM, Ekins MB. Clinical and pathologic description of 17 cases of corneal intraepithelial neoplasia. Am J Ophthalmol. 1984 May;97(5):547–559. [PubMed]
  • Watt FM, Green H. Stratification and terminal differentiation of cultured epidermal cells. Nature. 1982 Feb 4;295(5848):434–436. [PubMed]
  • Weiss RA, Eichner R, Sun TT. Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes. J Cell Biol. 1984 Apr;98(4):1397–1406. [PMC free article] [PubMed]
  • Weiss RA, Guillet GY, Freedberg IM, Farmer ER, Small EA, Weiss MM, Sun TT. The use of monoclonal antibody to keratin in human epidermal disease: alterations in immunohistochemical staining pattern. J Invest Dermatol. 1983 Sep;81(3):224–230. [PubMed]
  • Woodcock-Mitchell J, Eichner R, Nelson WG, Sun TT. Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell Biol. 1982 Nov;95(2 Pt 1):580–588. [PMC free article] [PubMed]
  • Wu YJ, Parker LM, Binder NE, Beckett MA, Sinard JH, Griffiths CT, Rheinwald JG. The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell. 1982 Dec;31(3 Pt 2):693–703. [PubMed]
  • Wu YJ, Rheinwald JG. A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas. Cell. 1981 Sep;25(3):627–635. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • MedGen
    MedGen
    Related information in MedGen
  • OMIM
    OMIM
    OMIM record citing PubMed
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...