Epithelial Keratins and Stem Cells

Based on the unique keratin expression pattern of corneal epithelium, we proposed in 1986 that corneal epithelial stem cells reside in the limbus, a previously ignored, transitional zone between cornea and conjunctiva (J Cell Biol 1986). This work and a subsequent 1989 Cell paper done in collaboration with Robert Lavker of the Northwestern University led to the rejection of the previous concept of “conjunctival epithelial transdifferentiation” which proposes that conjunctival epithelial cells can migrate onto the cornea proper forming a bona fide corneal epithelium. The limbal stem cell concept explains why an earlier surgical procedure, in which conjunctival epithelium was used to repair damaged corneal epithelium, was ineffective. This concept led to the introduction of a new surgical procedure called “limbal transplantation” in which limbal stem cells are used to repair a damaged or denuded corneal epithelium. Without limbal stem cell transplantation, corneal transplants in patients who are deficient in limbal stem cells invariably fail due to blood vessel in-growth and corneal opacity. Limbal stem cell transplantation can solve this problem and restore the eyesight of many patients; this procedure is therefore being performed by ophthalmologists worldwide. In addition, the limbal stem cell concept has led to an improved understanding and classification of various anterior ocularepithelial disorders. The limbal stem cell concept is now widely accepted and is included in most ophthalmology textbooks.

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Sun and Green. Nature. 1977. In vitro growth of human skin epidermal and corneal epithelial cells cultured in the presence of lethally irradiated 3T3 feeder cells.
Sun, T.-T. and H. Green. Nature. 1977. Figure 2. (a) Similar keratins are synthesized by cultured human corneal epithelial cells (lane 2, insoluble keratins), conjunctival epithelial cells (3), and skin epidermal cells (4). Lanes 1 and 5 are total proteins of cultured corneal epithelial cells and insoluble proteins of cultured human corneal fibroblasts, respective. (b) Cultured rabbit corneal epithelial cells (RCE) expressed, however, an additional 64Kd keratin (later identified as K3 ‘corneal epithelial keratin’ and a 40K keratin (K19).
Sun and Green. PNAS. 1978. Keratins can be detected using rabbit antibodies to human epidermal keratins in virtually all epithelial cells in the body including those of the skin epidermis and hair follicles (b), cornea (c), tongue (d), anal skin (e), esophagus (f and g), sweat gland ducts and myoepithelium (h and i), Hassall's corpuscles of the thymus (j and k), bladder (l), trachea (m), small intestine (n), kidney collecting ducts (o and p), cervix (q), uterus (r), pancreas (s and t) and submaxillary glands (u and v).
Sun and Green. PNAS. 1978. Immunofluorescent staining of keratin fibers in cultured rabbit bladder epithelial cells (a), small intestinal epithelial cells (b), renal epithelial cells (c and d), showing that the fibrous staining of the intermediate filament network.
Doran et al. Cell. 1980. Cultured rabbit skin epidermal (a), corneal (b), and esophageal (c) epithelial colonies grown with the support of lethally irradiated 3T3 feeder cells, showing distinct morphology supporting their intrinsic divergence.
Doran et al. Cell. 1980. Intrinsic and extrinsic regulation of keratin patterns in rabbit skin, esophageal and corneal epithelia. For each tissue, its keratin pattern in vivo (first lane), in cultured cells (second) or in in vivo reconstituted epithelium (after cultured cells are injected subcutaneously into athymic mouse).
Lavker and Sun. J Invest Derma. 1983. Rabbit epidermis (left panel) is ‘keratinized’ consisting of basal (B), spinous (S), granular (G) and cornified (C) cell layers. However, cultured rabbit epidermal cells (right panel), although stratified, undergo a strikingly different pathway of differentiation. Note that cultured epidermial cells fail to form granular and cornified layers. This illustrates the striking effects of the extrinsic environment on epidermal differentiation.
Lavker and Sun. J Invest Derma. 1983. Cultured rabbit epidermal cells, that undergo ‘altered differentiation’, were trypsinized and a single cell suspension was injected subcutaneously into immunodeficient nude mice.
Lavker and Sun. Science. 1982. These results provide a striking example of epidermal basal cell heterogeneity. The data also suggest that basal cells located at the tip of the deep rete ridges are slow cycling stem cells that give rise to the rapidly proliferating, suprabasally located transit amplifying cells, and that the basal cells of the shallow ridges play the role of anchoring the palm epidermis to the dermis.
Lavker and Sun. J Invest Derma. 1983. Nonserrated basal cells (NS) at the tips of the deep rete ridges are believed to be slow cycling stem cells. These cells give rise to suprabasally located transit amplifying cells (TA) which actively incorporate tritiated thymidine. The TA cells give rise to the more superficially located, nonlabeled postmitotic (PM) cells. The serrated (S) cells located in the shallow rete ridges are believed to have an anchoring function. B=basal; S=spinous; G=granular; SC=stratum corneum.
Lavker and Sun. J Invest Derma. 1983. Significance of finding proliferating cells in the suprabasal compartment of the epidermis.
Eichner and Sun. J Cell Biol. 1984. SDS polyacrylamide gel analysis of the cytoskeletal proteins from human epidermis and cultured human epidermal cells.
Eichner and Sun. J Cell Biol. 1984. Two-dimensional polyacrylamide gel electrophoresis of keratins from normal human epidermis (left) and cultured human epidermal cells (right).
Tseng et al. J Cell Biol. 1984. Light microscopy of various rabbit epithelial tissues from normal and vitamin A-deficient animals.
Tseng et al. J Cell Biol. 1984. Electron microscopy of corneal epithelia from (a) control and (b) vitamin A-deficient rabbits. Note the presence of nuclei (N) in superficial cells of normal corneal epithelium, and the formation of keratohyalin granules (K) and anucleated stratum corneum during vitamin A-deficiency.
Tseng et al. J Cell Biol. 1984. Electron microscopy of conjunctival epithelia from (a) normal and (b) vitamin A-deficient rabbits. Note in normal conjunctival epithelium the round, nonsquamous superficial cells, and mucous-filled goblet cells (G), and in vitamin A-deficient epithelium the lack of goblet cells and the appearance of the squamous, anucleated superficial stratum corneum (SC) cells.
Tseng et al. J Cell Biol. 1984. Immunoblot analysis of epithelial keratins using AF1 and AE3 monoclonal antikeratin antibodies. A+ and A- denote specimens from control and vitamin A-deficient rabbits, respectively.
Tseng et al. J Cell Biol. 1984. Indirect immunofluorescent staining of frozen sections of rabbit corneal epithelia with AE2 monoclonal antikeratin antibody.
Weiss and Sun. Journal of cell biology. 1984. Lane 2, ichthyosis vulgaris; lane 3, atopic dermatitis (bar and arrowhead designate the 48- and 46-kd keratins, respectively, which are only partially resolved by 1-D gels); lanes 4 and 5, two different cases of psoriasis showing different amounts of 65-67 kd keratins; lane 6, keratoacanthoma; lane 7, actinic keratosis; lane 8, wart; lane 9, verrucous carcinoma; lanes 10 and 11, two different cases of squamous cell carcinoma showing slightly different keratin patterns; lane 12, basal cell carcinoma; lane 13, cultured normal human epidermal keratinocytes.
Weiss and Sun. Journal of cell biology. 1984. Analysis of the acidic human epidermal keratins by two-dimensional gel electrophoresis (equilibrium; IEF-SIDS). a and a', normal abdominal epidermis ; b and b', cultured human epidermal cells ; c and c', psoriasis.
Weiss and Sun. Journal of cell biology. 1984. Analysis of total human epidermal keratins by two-dimensional gel electrophoresis (nonequilibrium; NEpHG-SDS).
Weiss and Sun. Journal of cell biology. 1984. A spectrum of keratin expression in psoriasis.
Weiss and Sun. Journal of cell biology. 1984. A schematic representation of the spectrum of keratin expression in epidermal diseases. The keratin pattern of normal in vivo epidermis is shown to the left, that of cultured keratinocytes to the right, and those of various diseases studied so far in between.
Cooper and Sun. Laboratory investigation. 1985. One- and two- dimensional immunoblotting of human keratins using AE1 (recognizing many acidic keratins) and AE3 (basic keratins) monoclonal antibodies.
Cooper and Sun. Laboratory investigation. 1985. Separation of all the human keratins into the acidic (AE1-positive) and basic (AE3-positive) families.
Cooper and Sun. Laboratory investigation. 1985. Classification, immunoreactivity and rules of expression of human epithelial keratins.
Cooper and Sun. Laboratory investigation. 1985. Classification of human epithelia and carcinomas according to their keratin composition.
Sun et al. Annals of the NY Academy of Sci. 1985. Sequential and coordinate expression of the four major epidermal keratins accompanying morphological differentiation of normal human epidermis.
Sun et al. Annals of the NY Academy of Sci. 1985. A schematic drawing illustrating the morphological changes that occur when human epidermal cells are grown in tissue culture media containing various growth-stimulating substances such as vitamin A, hydrocortisone (HC), epidermal growth factor (EGF), cyclic AMP (CAMP), cholera toxin (CT), etc.
Sun et al. Annals of the NY Academy of Sci. 1985. Tissue distribution of some human keratins. Keratins are identified by their MWs. The 40K (K19), 45K (K18), 50K (K14), 51K (K13), 55K (K12) and 56.5K (K10) keratins belong to the acidic (Type 1) subfamily, whereas the 52K (K8), 58K (K5), 59K (K4), 64K (K3) and 65-67K (K1) keratins belong to the basic (Type II) subfamily. For simplicity, four other keratins (46K-Kx and 48K-K16 acidic and 54K-Kx and 56K-K6 basic keratins that show less clear-cut relationship with epithelial cell type are not included in this diagram. Note, however, that K6-K16 pair represent markers for hyperproliferation in all stratified squamous epithelial cells.
Sun et al. Annals of the NY Academy of Sci. 1985. The sequential expression of the 40K, 50K, and 56.5K keratins in rabbit epidermis and several other epithelia during embryonic development, as detected by immunoblotting using AE1 antibody. (a) 12-day whole embryo, (b) intestine, (c) corneal epithelium, (d) conjunctival epithelium, (e) esophageal epithelium, and (f) skin epidermis.
Sun et al. Annals of the NY Academy of Sci. 1985. A unifying model of keratin expression. A unifying model of keratin expression. Keratins of the acidic (type I) and basic (type II) subfamilies are arranged vertically according to their size (see the 5000-dalton scale). Keratins below the horizontal line are mainly expressed by simple epithelial cells and those above are mainly expressed in stratified epithelia.
Schermer et al. J Cell Bio. 1986. This table is based on an analysis of keratins from rabbit skin, cornea, esophagus, bladder, trachea, mesothelium, intestine, and cultured skin, corneal and esophageal keratinocytes.
Schermer et al. J Cell Bio. 1986. One-dimensional immunoblot analyses of rabbit epithelial keratins.
Schermer et al. J Cell Bio. 1986. Two-dimensional immunoblot analyses of rabbit corneal keratins.
Schermer et al. J Cell Bio. 1986. Immunofluorescent staining of cultured rabbit corneal epithelial cells with AE5.
Schermer et al. J Cell Bio. 1986. Immunofluorescent staining of frozen rabbit corneal sections, a to e are immunofluorescent micrographs; a'-c' are corresponding phase-contrast images of a-c, respectively. Note that the 63-kD K3 keratin can be detected in all cell layers of central corneal epithelium (basal cells included; panel a), but only in suprebasal cells in limbal epithelium (b). These results show that K3 is expressed differently in central vs peripheral corneal epithelium.
Schermer et al. J Cell Bio. 1986. Immunoblot analyses of keratins from cultured rabbit corneal epithelial cells during successive stages of growth. Lanes 1 to 6 are water-insoluble cytoskeletal proteins of cultured rabbit corneal epithelial cells for increasing number of days, while lane 7 shows the cytoskeletal proteins of in vivo normal rabbit corneal epithelium. Proteins were visualized using Fast Green (FG), anti-intermediate filaments (a IF), AE1 (acidic keratins), AE3 (basic) and AE5 (the corneal keratin K3).
Schermer et al. J Cell Bio. 1986. Two-dimensional immunoblot analyses of keratins from cultured rabbit corneal epithelial cells.
Schermer et al. J Cell Bio. 1986. Suprabasal staining of cultured rabbit corneal epithelial cells with AE5 antibody. (a) Phase-contrast, center of a large colony. (b) AE5 staining of the same field as in a showing preferential decoration of suprabasal cells. (c and d) Double staining of a vertical (7 pan) frozen section of a rabbit corneal epithelial colony (day 11) with a rabbit antikeratin antiserum, and AE5, respectively.
Schermer et al. J Cell Bio. 1986. Morphology of basal cells prepared from rabbit corneal epithelial cultures by the EGTA technique.
Schermer et al. J Cell Bio. 1986. One-dimensional immunoblot analyses of the keratins of of basal (lanes 1 and 3) and suprabasal cells (lanes 2 and 4). (a) Fast green. (b) Immunoblotting using a mixture of mouse anti-intermediate filament and AE1 antibodies (MaK). These results established that K3 keratin is associated exclusively with suprabasal cells thus representing a molecular marker for an advanced stage of corneal epithelial differentiation.
Schermer et al. J Cell Bio. 1986. A model of corneal epithelial maturation. (a) Expression of the 64K K3 keratin in corneal and limbal epithelia. (b) A model showing that corneal epithelial stem ceils are located in the basal layer of the limbal region. Arrows indicate the directions of cell migration during normal corneal epithelial maturation.
Schermer et al. J Cell Bio. 1986. A model of corneal epithelial maturation.
Cotsarelis et al. Cell. 1989. Histology of Anterior Segment of Mouse Eye.
Cotsarelis et al. Cell. 1989. Identification of Label-Retaining Cells in Limbal Epithelium. Autoradiograms demonstrate the labeling pattern of limbal (a, c, e) and central cornea1 (b, d, f) epithelia after long-term labeling under various conditions.
Cotsarelis et al. Cell. 1989. Preferential Stimulation of Limbal Epithelial Incorporation of %TdA by a Distant Corneal Epithelial Wound.
Cotsarelis et al. Cell. 1989. Localization of Epithelial Stem Cells in Cornea (a), Palm (b), Trunk Skin (c), Ear Skin (d), Hair Follicle (e), Dorsal Tongue Epithelium (f), and Small Intestinal Epithelium (g).
Kenyon, K.R., and Tseng, S.C. Ophthalmology. 1989. Limbal autograft transplantation for ocular surface disorders. (Photo courtesy of Sheffer Tseng, Ocular Surface Research and Education Foundation, Miami, FL)
Lavker et al. Experimental eye research. 2004. Clinical examples of limbal stem cell transplantation. The left panel represents the preoperative appearance of total LSCD caused by acid burn and the right panel represents their corresponding post-operative appearance. (Photo source: Sheffer Tseng, Ocular Surface Research and Education Foundation, Miami, FL)
Wu et al. J Biol Chem. 1994. The DNA sequence and potential transcription factor binding motifs of the 5’-upstream sequence of rabbit K3 keratin gene.
Chen et al. Molecular and Cellular Biology. 1997. The ratio of Sp1 to AP-2 DNA-binding activity increases in a differentiation- dependent manner in cultured rabbit corneal epithelial cells.
Chen et al. Molecular and Cellular Biology. 1997. 1997. Sp1 and AP-2 bind to overlapping binding sites in the E element of the 300-bp K3 promoter.
Chen et al. Molecular and Cellular Biology. 1997. This model indicates that (i) the activating Sp2 and inhibiting AP-2 bind to overlapping motifs thus their binding to the composite site is mutually exclusive; (ii) although Sp1 and AP-2 are both present in the undifferentiated limbal basal cells, Sp1 binding is suppressed by polyamine thus K3 keratin expression is suppressed; (iii) the differentiated (suprabasal) cells in limbal epithelium (as well as the ‘basal cells’ of central corneal epithelium) contains Sp1 but little inhibitory AP-2 thus K3 gene expression is activated.
Lavker and Sun. Eye. 2003. Schematic diagram showing the location of corneal epithelial stem cells (SC) in the basal layer of the limbus. These stem cells overlie a mesenchyme (niche) that is distinctively more cellular and blood vessel (bv) rich than the corneal stroma.
Miller and Sun. Biochimica et Biophysica Acta. 2005. The proliferative hierarchy in epithelia: the stem cell concept.
Miller and et al. Biochimica et Biophysica Acta. 2005. Mode of division and the role of the niche in determining stem cell pool size.
Miller et al. Biochimica et Biophysica Acta. 2005. Proliferative strategies during epithelial repair.
Miller and Sun. Biochimica et Biophysica Acta. 2005. Role of the niche in maintaining/preserving stem cell proliferative potential.
Miller and Sun. Biochimica et Biophysica Acta. 2005. The role of stem cells in the clonality of pretumor progression.
Sun et al. Nature. 2010. "Location of corneal epithelial stem cells."