When organs of the body are formed, they develop in neatly organized arrays. Often, cell types are separated by connective tissue called basement membranes. In skin, for instance, the superficial layer of epidermal cells adheres to the underlying basement membrane. This skin basement membrane acts as a barrier between the epidermal cells on the outside, and the dermal cells underneath. A similar arrangement of cells occurs in the lining of the gut and in the oral cavity.
Basement membranes have been implicated in the growth, attachment, migration, repair and differentiation of their overlying cell populations. Three layers have been defined in basement membranes: a) the lamina lucida, an electronmicroscopically clear region in close approximation to the overlying cells; b) the lamina densa, an electron dense region of 20-300 nm in width; and c) the sublamina densa which contains anchoring fibrils, microfibrillar bundles and collagen fibers.
Many epithelial cells interact with the underlying extracellular matrix, a network of proteins to which cells attach by a junction called the hemidesmosome (Staehelin, (1974) Structure and Function of Intercellular Junctions, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo., 191-283). The hemidesmosome, with its associated structures, including intermediate filaments and anchoring fibrils, forms an adhesion complex. Disruptions of the epithelial-connective tissue interaction are often accompanied by disruption of the hemidesmosome complex. For example, in certain blistering skin diseases, such as junctional epidermolysis bullosa, where epithelial cell-connective tissue interaction is abnormal, it has been proposed that there is biochemical modification in, or loss of, a basement membrane zone-associated component of the hemidesmosome.
Bullous pemphigoid (BP) is an autoimmune disease which results in a disruption of the interactions between epithelial cells and connective tissue simultaneously with loss of hemidesmosome integrity (Chapman et al., Br. J. Dermatol., 123:137-144, 1990). In particular, it has been determined that BP patients produce auto-antibodies against hemidesmosome components, and two high molecular weight components of the hemidesmosome have been identified and characterized using these BP antibodies (Klatte et al., J. Cell Biol., 109:3377-3390, 1989). One component is a 230 kDa polypeptide (BP230) that may act as an anchor for cytoskeletal elements in the hemidesmosomal plaque (Jones and Green, Curr. Opin. Cell Biol., 3:127-132, 1991). The second component is a 180 kDa type II membrane protein (BP180) which has a collagen-like extracellular domain (Giudice et al., J. Clin. Invest., 87:734-738, 1991); Hopkinson et al., J. Invest. Dermatol, 3:264-267, 1992).
In addition, it has been demonstrated that the interaction of the hemidesmosome with the underlying connective tissue involves the .alpha..sub.6 .beta..sub.4 integrin cell adhesion receptor (Stepp et al., Proc. Natl. Acad. Sci. USA, 87:8970-8974, 1990; Jones et al., Curr. Opin. Cell Biol., 3:127-132, 1991; Sonnenberg et al., J. Cell Biol., 113:907-917, 1991; Kurpakus et al., J. Cell Biol., 115:1737-1750, 1991). The .alpha..sub.6 .beta..sub.4 heterodimer has been localized to hemidesmosomes along the basal surfaces of the 804G rat bladder carcinoma cell line (Jones et al., Cell Regulation, 2:427-438, 1991). These results suggest that integrins (e.g., .alpha..sub.6 .beta..sub.4) may play an important role in the assembly and adhesive functions of hemidesmosomes.
For a recent review describing the molecular structure and assembly of hemidesmosomes, see Jones et al., Experimental Cell Research, 213, 1-11 (1994).
It has generally been reported that most epithelial cells do not assemble bona fide hemidesmosomes when cultured in vitro, despite the fact that they appear to express all of the necessary plaque and hemidesmosomal components. Indeed, it is only recently that cell lines, such as the 804G and NBT-II rat bladder carcinoma cell lines, have been found to be capable of assembling hemidesmosomes in vitro under standard culture conditions (Riddelle et al., (1991), J. Cell Biol., 112:159-168; allowed U.S. application Ser. No. 08/152,460, the entire contents of which are hereby incorporated by reference; Jones et al., Experimental Cell Research, 213, 1-11 (1994)). Even more recently, Bergstraesser et al. found that primary normal human breast epithelial cells produced hemidesmosomes after prolonged culture in vitro (Bergstraesser et al., American Journal Of Pathology, 147, 1823-1839 (December 1995)).
When epithelial cells unable to form hemidesmosomes are plated on the cell matrix deposited by 804G rat bladder carcinoma cells, hemidesmosome formation is induced (Langhofer et al., (1993) J. Cell Sci., 105:753-764; allowed U.S. application Ser. No. 08/324,367, the entire contents of which are hereby incorporated by reference). In addition, U.S. Pat. No. 5,422,264, the entire contents of which are hereby incorporated by reference, discloses that a soluble matrix equivalent produced by 804G cells also induces attachment and hemidesmosome formation in cells contacted with the soluble matrix.
The 804G matrix comprises four glycosylated proteins of approximately 135 kDa, 140 kDa, 150 kDa and 400 kDa and one non-glycosylated protein of about 85 kDa (Langhofer et al., J. Cell Sci., 105, 753-764 (1993); allowed U.S. application Ser. No. 08/324,367). The 140 kDa and 85 kDa proteins are immunologically related to laminin B2t (Langhofer et al., J. Cell Sci., 105, 753-764 (1993)). The 804G matrix and soluble matrix equivalent comprise similar major protein components.
The molecular weights of the 804G matrix elements are similar to those of components of high-molecular-weight complexes of human proteins secreted by keratinocytes. These human complexes are BM600 (described in Verrando et al., Biochim. Biophys. Acta., 942:45-56 (1988) and Hsi et al., Placenta 8:209-217 (1987)), kalinin (described in Rouselle et al., J. Cell Biol., 114:567-576 (1991) and Burgeson et al., PCT WO 92/17498 and PCT WO 94/05316), and epiligrin (described in Carter et al., Cell, 65:599-610 (1991) and Carter et al., PCT WO 95/06660). In addition, kalinin has been shown to be immunologically related to 804G matrix (Jones et al., Experimental Cell Research, 213, 1-11 (1994)). However, kalinin, BM600 and epiligrin, unlike 804G matrix, do not induce hemidesmosome formation.
Because the organization of cells growing on the hemidesmosome-inducing 804G matrix is significantly more advanced and more tissue-like than cells grown without the matrix, it would be desirable to grow epithelial cells for various applications on such a matrix. However, 804G matrix is derived from a rat cell line. For uses related to humans, it would be preferable to use a human matrix. Specifically it would be particularly desirable to use a human cell-derived hemidesmosome-inducing cell matrix for the growth of epithelial cells thereon. The present invention provides such a matrix.
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