Link:  Pharm/Biotech Resources

Title:  Method and composition for skin grafts

United States Patent:  6,964,869

Issued:  November 15, 2005

Inventors:  Allen-Hoffmann; B. Lynn (Madison, WI)

Assignee:  Wisconsin Alumni Research Foundation (Madison, WI)

Appl. No.:  131977

Filed:  April 24, 2002

A chimeric skin comprising immortalized human keratinocyte cells cocultured with donor keratinocytes is disclosed.

BACKGROUND OF THE INVENTION

The skin is the largest organ in the human body and functions as a protective barrier against the agents, such as infectious agents, in the external environment, and as a waterproof barrier that maintains fluid homeostasis and prevents evaporation of tissue moisture. Intact skin prevents local infection of the dermis or other underlying tissue with microorganisms. If unchecked, such local infections can become invasive and can result in sepsis or systemic infection.

The temporary loss of skin often results in mortality or morbidity. Widespread loss of skin integrity is most often associated with major burns. The importance of the barrier function of skin is demonstrated by the fact that for a given age, mortality is directly related to burn size. Current standards of care recommend resurfacing of the patient as soon as possible to restore fluid homeostasis and barrier function.

Burn patients are most often resurfaced with autologous skin grafts. Autologous skin grafts techniques are widely used, but frequently fail. For example, a patient with large burns may not have sufficient donor skin available to cover the recipient site.

Two major alternative patient salvage techniques are currently available. One is to completely excise all the burned tissue and cover the patient with a temporary epidermis. INTEGRA (Johnson and Johnson, New Brunswick, N.J.) is a neo-dermis bilayer having an inner layer of bovine collagen and chondroitin-6-sulfate and an outer layer of silicone. After neo-dermal angiogenesis 14 to 21 days later, the silicone outer layer is surgically removed and the patient is resurfaced with thin, widely meshed autografts. The thin autografts offer the distinct advantage of allowing limited donor sites to heal and be serially harvested. Although the burn community has embraced INTEGRA, the susceptibility to infection has limited its use in patients with dirty or infected burn wounds (1). The other major limiting factor has been donor site availability. Patients with catastrophic burn injury may not have sufficient donor site available despite the obvious advantage of requiring much thinner autografts.

The second commonly employed technique is to excise the burn wound and to provide a temporary dermal/epidermal cover of cadaver skin to restore the skin's barrier function. Autologous keratinocytes are harvested and placed into culture. EPICEL (Genzyme Corporation, Cambridge, Mass.), the only commercially available permanent skin replacement, forms keratinocytes 2 to 6 cell layers thick attached to fine mesh gauze. These autografts are available in 3 to 4 weeks from the time of biopsy. In the case of large burns, this timetable often coincides with development of burn wound sepsis. Bacterial contamination and other factors cause high failure rates of EPICEL that are not seen with traditional split-thickness skin grafts (2).

Further advances in burn wound care may come from the arena of tissue engineering. One proposal has been to restore the barrier function with chimeric cultures of autologous keratinocytes mixed with established allogeneic cell cultures (3, 4). This could permit earlier wound coverage by significantly decreasing the time required for definitive culture formation. Slower permanent resurfacing of the skin would occur with autologous keratinocytes as the allogeneic cells are rejected. One major obstacle is the need for an established, cultured, and tested allogeneic keratinocyte line. This has heretofore been impractical as keratinocytes senesce in culture and new keratinocyte lines would have to be continually established and tested.

Skin is composed of two layers, the dermis and the epidermis. The dermis is a connective tissue containing fibroblasts embedded in a matrix of collagen and elastic fibers. The epidermis, in contrast, consists primarily of cells with little connective tissue.

Keratinocytes are the major cellular component of the epidermis and comprise about 80% of the cells in adult human skin. They are the epidermal component responsible for providing the skin's barrier, reparative, and regenerative properties. Their name derives from their predominant cytoskeletal component, keratins. Keratins are the subunits of keratin filaments and are divided into two types: type I (acidic) keratins and type II (basic or neutral) keratins. All epithelia express type I and type II keratins, which range in molecular weight from 40 kDa to 70 kDa. Different epithelial tissues express specific pairs of keratins. Heterodimers of type I and II keratins form intermediate filaments that confer tensile strength and structural support to the cells and resulting epithelial tissue.

The epidermis consists of four morphologically and biochemically distinct layers. The basal layer of keratinocytes is in contact with the basement membrane, which separates the epidermis from the dermis. Basal cells are the only keratinocytes in intact skin capable of mitosis and, as such, are the source of all other keratinocytes in the epidermis. They attach to the substratum through hemidesmosomes and to adjacent cells through desmosomes and adherens junctions (reviewed in Jensen and Wheelock, 1996). Basal layer cells are columnar in shape and produce keratins K5 and K14.

The first suprabasal keratinocyte layer is the stratum spinosum, so named for the "spiny" appearance of the many desmosomal contacts between adjacent cells. Keratinocytes in this layer no longer produce K5 and K14 but, instead, synthesize differentiation-specific keratins K1 and K10. Cells begin to produce involucrin and epidermis-specific transglutaminases in the upper stratum spinosum. Morphologically, spinous cells are larger and more flattened than basal cells (reviewed in Holbrook, 1994).

Involucrin expression is localized to the cytoplasm of suprabasal and granular cells (Mansbridge and Knapp, 1987; Murphy et al., 1984) of normal skin. Involucrin has also been shown to localize pericellularly in these layers, but tissues used for these immunohistochemistry experiments were not fixed, and it was hypothesized that the protein diffused to the cell borders during or after the sectioning process (Smola et al., 1993; Watt, 1983).

Transglutaminases are a family of calcium-dependent enzymes that catalyze the covalent cross-linking of proteins to each other or to polyamines (reviewed in Rice et al., 1994). The keratinocyte transglutaminase isozyme, TG_K, is membrane-bound in suprabasal epidermis and catalyzes the cross-linking of involucrin and at least six other membranous proteins to form the cornified envelope (CE) (reviewed in Rice et al., 1994). The CE is a highly stable insoluble protein structure formed beneath the plasma membrane that is resistant to detergents and reducing agents and confers strength and rigidity to the terminally differentiated cells of the uppermost epidermal layer. Many CE components, including TG_K, are synthesized in the stratum spinosum although the envelope is not formed until the cells transition from the stratum granulosum to the stratum corneum. TG_K is also found in all subsequent layers of the epidermis (Michel et al., 1997; Mansbridge et al., 1987; Asselineau et al., 1989). The enzyme is inactive until, in the final stage of differentiation, a loss of membrane integrity results in an influx of calcium into the cell (Aeschlimann and Paulsson, 1994).

As keratinocytes differentiate further, they form the stratum granulosum. Cells of this layer are characterized by distinct electron-dense keratohyalin granules containing profilaggrin, the protein precursor of filaggrin (reviewed in Dale et al., 1994). Granular cells also contain lipid-filled granules that, during the transition zone between the stratum granulosum and stratum corneum, fuse with the plasma membrane and release their contents into the extracellular space, conferring hydrophobicity to the epidermal surface.

As the differentiating keratinocytes transition from the granular to the cornified layer, the profilaggrin is cleaved to yield filaggrin, which is involved in the alignment and aggregation via disulfide bonds of keratin bundles called macrofibrils (reviewed in Holbrook, 1994). Macrofibrils are the basic structural unit of the cornified envelope. In normal skin sections, filaggrin is localized in the granular layer, with some faint, continued staining in the cornified sheets (Michel et al., 1997; Asselineau et al., 1989; Mansbridge et al., 1987). It should be noted that antibodies against filaggrin detect profilaggrin as well as its cleavage products, which accounts for the strong, punctate staining pattern of the keratohyalin granules of the stratum granulosum.

The uppermost epidermal layer is the stratum corneum. Cells of this layer, having completed the differentiation process, have lost their nucleus and all metabolic function. They consist primarily of keratin filaments encased by the now-complete CE and overlying plasma membrane. Corneal cells are joined together by modified desmosomes and are ultimately sloughed off in sheets from the skin's surface.

Keratinocytes also produce cadherin adhesion molecules. The classical cadherins, N-, E-, and P-cadherin, are a subfamily of cadherins that localize to the adherens junctions and mediate cell-cell adhesion through homotypic interactions. These calcium-dependent, transmembrane glycoproteins play major regulatory roles in tissue formation and facilitate intercellular interactions. Cadherin complexes are also thought to participate in the transduction of intracellular signals through their association with the actin cytoskeleton (Knudsen et al., 1998). Keratinocytes produce both E- and P-cadherins. E-cadherin is found in all living layers of the epidermis (reviewed in Jensen and Wheelock, 1996) while P-cadherin is found in the stratum basale and immediately suprabasal cells.

The skin regenerates every 28 days (reviewed in Sams, 1996). As basal keratinocytes lose contact with the basement membrane they produce daughter cells that terminally differentiate as they move upward through the suprabasal layers to the skin surface. Terminal differentiation involves a series of biochemical and morphologic changes that result in a layer of dead, flattened squames that carries out the barrier and protective functions of the skin. These cornified cells are routinely sloughed off and replaced with newly differentiated cells, maintaining the controlled balance between proliferation and differentiation involved in tissue homeostasis.

Keratinocyte Culture

Cultivated cells isolated from disaggregated human skin have been used for over two decades to study keratinocyte growth and differentiation (reviewed in Leigh et al., 1994). Human foreskin keratinocytes cultured in the presence of a 3T3 mouse embryo fibroblast feeder layer or in serum-free medium formulations exhibit sustained growth for approximately 80 population doublings prior to senescence (reviewed in Leigh and Watt, 1994). Human keratinocytes cultured under these conditions can express differentiation-specific proteins, such as involucrin and keratins K1 and K10, in a position-specific manner (reviewed in Fuchs and Weber, 1994).

Although features of squamous differentiation and limited stratification are consistently observed in cultured keratinocyte monolayers, normal tissue architecture is not evident. The discovery that epidermal cells in traditional submerged culture grow optimally when plated on top of non-proliferating fibroblasts was a significant contribution to the study of keratinocyte cell biology (Rheinwald, 1980; reviewed in Fuchs, 1993). The use of this culturing system allowed investigators to serially cultivate keratinocytes for a wide range of purposes. Unfortunately, the submerged culture system allows for limited, aberrant stratification consisting of only a few layers of keratinocytes, which lack the specific morphological and biochemical characteristics of a true stratified epithelium. For example, several markers of normal epidermal differentiation are not produced in submerged culture, such as keratins 1 and 10 and the late stage marker, filaggrin (reviewed in Fuchs, 1993). Several factors limit this in vitro system. First, in vivo epidermis sits atop the dermis and receives its nutrients and growth signals via diffusion from the dermis through the basement membrane to the overlying basal cells. This imparts a polarity to the tissue that cannot be achieved in traditional submerged cell cultures that are fed through all upper surfaces to the bathing medium. Second, epithelial cells grown in this manner do not produce a basement membrane and lack exposure to its mesenchymal cues for normal differentiation and growth (reviewed in Fusenig, 1994). Third, while the fibroblast "feeder" layer promotes keratinocyte proliferation, the cultivation of cells in this manner results in the same aberrant or absent differentiation characteristics as is seen in cultures grown on a plastic substratum. This indicates that keratinocytes require a more complex mesenchyme of fibroblasts and extracellular matrix proteins to produce a functional epidermis. Accordingly, the traditional submerged culture system is appropriate only for relatively simplistic studies.

Several systems developed and tested over the past 20 years are designed to develop more in vivo-like keratinocyte culturing conditions. In 1979, collagen gels were first used as physiologic "rafts" upon which to grow keratinocytes at the air-medium interface (Bell et al., 1979; reviewed in Fusenig, 1994; Parenteau et al., 1992). This allowed the nutrients and growth factors from the medium to diffuse through the collagen to the basal layer of keratinocytes and exposed the uppermost keratinocyte layers to the air. These added in vivo-like conditions improved the histological architecture of the cultured epidermis. Full stratification and histological differentiation can be achieved using these three-dimensional "organotypic" culture methods, which have been continually modified to more closely recapitulate the in vivo growth environment.

In later organotypic systems, live fibroblasts were incorporated into collagen gels, keratinocytes were placed atop contracted collagen "rafts", and the entire raft was lifted to the air-medium interface (reviewed in Fusenig, 1994) to emulate intact skin, where resident dermal cells such as dermal fibroblasts are involved in signaling keratinocytes to produce a basement membrane and an epithelium with a significantly improved differentiation program (reviewed in Watt and Hertle, 1994). The viable fibroblasts provide valuable signals and promoted production by cultured keratinocytes of basement membrane proteins.

Keratinocyte Differentiation

In both intact skin and in organotypic culture, differentiating keratinocytes produce proteins unique to particular differentiation stages. The presence and localization of these protein markers can be detected using biochemical and immunohistochemical methods and used to determine whether an epithelial tissue is differentiating (stratifying) normally. The expression profiles of several such proteins are presented below.

The most well-studied proteins for determining epidermal differentiation are the keratins, most notably K5/14 and K1/10, which make up the intermediate filament network in epidermal keratinocytes. This network provides a cellular framework that extends from the nucleus to specific adhesion junctions called desmosomes and hemidesmosomes (reviewed in Fuchs and Cleveland, 1998). These cell-cell and cell-substratum interactions, when connected to keratin filaments, are responsible for anchoring keratinocytes to each other and to the underlying basement membrane. The K5/K14 pair of keratin filaments is expressed in the basal cells of stratified squamous epithelia (reviewed in Fuchs, 1993). As expected, K14 mRNA is present only in the basal layer of normal human epidermis (Stoler et al., 1988). As basal cells of the epidermis begin to differentiate, they downregulate their expression of K5/14 and begin to produce differentiation-specific keratins. The K1/K10 pair of keratin filaments is expressed in the suprabasal layers of the epidermis (reviewed in Fuchs, 1993). K1 and K10 are early markers of terminal differentiation as they are produced by keratinocytes upon leaving the basal layer. In samples of intact human skin, K1 is from the first suprabasal layer throughout the surface of the tissue (Stoler et al., 1988; Stark et al., 1999; Asselineau et al., 1989; Boukamp et al., 1990).

In primary human keratinocyte organotypic cultures, initiation of K1 protein synthesis is delayed relative to intact skin. Protein localization begins 5-8 cell layers above the basement membrane as opposed to 1-2 layers in normal skin samples. Induction of K1 production normalizes following transplantation of the organotypic cultures onto nude mice (Smola et al., 1993; Stark et al., 1999). These studies did not examine K1 mRNA expression.

Involucrin is localized to the suprabasal cell membranes in primary keratinocyte organotypic cultures, even in fixed tissue (Boukamp et al., 1990; Smola et al., 1993; Stark et al., 1999; Watt et al., 1987), and several groups also found that the membranous pattern remained after transplantation onto nude mice (Breitkreutz et al., 1997; Watt et al., 1987).

TG_K expression seems to vary in organotypic cultures of normal human keratinocytes, first appearing either in the stratum spinosum or the stratum granulosum and continuing up through the stratum corneum (Michel et al., 1997; Stark et al., 1999).

Organotypic cultures of normal human keratinocytes also display localization of filaggrin in the granules of the uppermost strata of the epidermal tissue (Boukamp et al., 1990; Michel et al., 1997; Stark et al., 1999).

In addition to differentiation markers, one can also assess the structural and functional integrity of an epithelial tissue by monitoring for the presence and localization of adhesion proteins.

To date, neither of E- nor P-cadherin has been examined in organotypic culture of primary keratinocytes, although E-cadherin has been detected immunohistochemically in normal skin (Haftek et al., 1996).

Smola and coworkers (Smola et al., 1998) have clearly shown that organotypic cultures composed of normal dermal fibroblasts and keratinocytes develop a basement membrane zone capable of supporting cell type specific adhesion structures such as hemidesmosomes.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized in that a coculture of a human immortalized keratinocyte cells and human donor cells is advantageously employed as a chimeric skin for use in skin grafting and other plastic surgery methods.

DESCRIPTION OF THE INVENTION

The present invention involves using a combination of in vitro cultured conditionally immortal carrier human keratinocytes and donor patient-derived keratinocytes to form skin grafts. We disclose herein that such carrier keratinocytes derived from in vitro cell culture sources can be cocultured with donor patient keratinocytes using monolayer or organotypic culture methods to produce an engineered chimeric skin suitable for grafting. A conditionally immortal keratinocytes are keratinocytes that are immortal under defined growth conditions. A keratinocyte is considered immortal if it can be cultured under the defined growth conditions for more than 20 passages, preferably more than 30 passages, still more preferably more than 40 passages, and yet more preferably for more than 50 passages. In this application, the terms "conditionally immortal" and "immortal" are used interchangeably. The in vitro cultured conditionally immortal carrier human keratinocytes are allogeneic to the graft recipient. The donor or patient-derived keratinocytes are preferably autologous to the graft recipient.

In some preferred embodiments, the spontaneously immortalized NIKS (Near-Diploid Immortalized KeratinocyteS) cell line is utilized as the source of carrier keratinocytes. NIKS cells (ATCC CRL-12191) have not been exposed to mutagenic agents, possess wild-type p53 and Rb genes, retain cell type-specific growth requirements and differentiation properties, are non-tumorigenic in nude and SCID mice, are virus free, and recapitulate full skin architecture in monolayer and organotypic culture and respond to growth factors that regulate keratinocyte growth, such as EGF and TGF-β1. This is in contrast to HaCaT cells (Boelsma et al., 1999; Schoop et al., 1999) which at later passages exhibit anchorage independent growth, have high colony forming efficiency, reach high saturation densities, and do not require cell type-specific culture conditions for serial cultivation (Fusenig and Boukamp, 1998).

Unlike other spontaneously immortalized keratinocyte cell lines such as HaCaT, NIKS keratinocytes differentiate to the same extent and at the same rate as the parental BC-1-Ep keratinocytes in organotypic culture with dermal fibroblasts to form a stratified epithelium that is histologically identical to the parental BC-1-Ep cells and to other normal keratinocyte strains. The multilayered keratinizing epithelium is highly organized and exhibits features typical of intact skin such as hemidesmosomes, desmosomes, keratin tonofilaments, and keratohyalin granules. Both the parental and NIKS keratinocytes produce hemidesmosomes in organotypic culture suggesting that the synthesis, deposition, and assembly of extracellular matrix glycoproteins has occurred.

In standard monolayer- or organotypic coculture, NIKS cells do not overgrow the human keratinocytes, do not exhibit aberrant differentiation protein expression patterns (among those tested), and obey tissue compartment boundaries, neither dropping below the basement membrane nor acting like a tumor cell. NIKS cells do not cause aberrant replication of dermal fibroblasts in the collagen gel.

NIKS were originally cultured from neonatal foreskin and subsequently developed a spontaneous stable genetic addition of the long arm of chromosome 8 that allows these cells to have a significant survival advantage in culture. U.S. Pat. Nos. 5,989,837 and 6,214,567, incorporated herein by reference, disclose the creation and use of NIKS cells. See also, Allen-Hoffmann, B. L., et al., "Normal Growth and Differentiation in a Spontaneously Immortalized Near-Diploid Human Keratinocyte Cell Line, NIKS," J. Invest. Dermatol. 114:444-455, 2000, incorporated herein by reference, which describes suitable monolayer and organotypic culture conditions for conditionally immortal maintenance of the NIKS cells.

Such engineered chimeric monolayer and organotypic cell cultures and/or engineered chimeric skin equivalent tissue grafts would have the unique characteristics of providing immediate wound coverage and also providing autologous cells for late formal wound closure. The new skin equivalent tissue can be used, for example, as a skin replacement using an autologous (NIKS+patient cells), allogeneic (NIKS+unrelated cells on a patient), or xenogeneic graft (NIKS+pig cells or primate cells) for, e.g., wound closure (diabetic ulcers, skin burns, necrotizing skin disease, etc.) or cosmetic purposes (face lifts, other plastic surgery procedures). The chimeric skin equivalent tissue of the invention can be provided in sizes and thicknesses suitable for use in grafting methods and other methods in the manner in which the artisan skilled in such methods would use existing grafts and tissues.

The present invention is not limited, however, to the use of NIKS cells in chimeric coculture. Indeed, the present invention contemplates the use of a variety of other conditionally immortalized, nontumorigenic carrier cells and cell lines that form cornified envelopes when induced to differentiate and that undergo normal squamous differentiation and maintain cell type-specific growth requirements. Other sources of such cells can include keratinocytes and dermal fibroblasts biopsied from humans and cavaderic donors (Auger et al., In Vitro Cell. Dev. Biol.—Animal 36:96-103; U.S. Pat. Nos. 5,968,546 and 5,693,332, each incorporated herein by reference), neonatal foreskins (Asbill et al., Pharm. Research 17(9): 1092-97 (2000); Meana et al., Burns 24:621-30 (1998); U.S. Pat. Nos. 4,485,096; 6,039,760; and 5,536,656, each of which is incorporated herein by reference), and immortalized keratinocytes cell lines such as NM1 cells (Baden, In Vitro Cell. Dev. Biol. 23(3):205-213 (1987)) and HaCaT cells (Boucamp et al., J. cell. Biol. 106:761-771 (1988)). Each of these cell lines can be cultured or genetically modified as described in more detail below. The scope of the invention also extends to use of cells and cell lines derived, directly or indirectly, from the aforementioned suitable cell types, including derivatives of NIKS cells, where such cells and cell lines retain the ability to function in the method of the invention.

In general, the method of the present invention is characterized by the following steps: One would obtain an in vitro carrier keratinocyte culture, preferably an organotypic culture of immortalized keratinocytes such as NIKS cells. One may wish to genetically manipulate the cultured keratinocytes. For example, the cells can be engineered to express or enhance expression of a protein or other gene product, or to suppress a protein or gene product. Other manipulations can include the knockin or knockout (ablation) of a gene or mutation of an existing gene or gene product. For example, in some preferred embodiments, either the carrier keratinocyte cells or patient derived cells are transfected or transformed with a gene of interest (for example, the gene encoding human Kruppel-like factor (GKLF) 4). In further preferred embodiments, the gene of interest is operably linked to promoter in an appropriate vector. In some preferred embodiments, tissue specific promoters such as the involucrin or transglutaminase 3 promoters are utilized. In other preferred embodiments, the expression of GKLF is driven by the inducible promoter system of the pTetOn plasmid (Clontech, Palo Alto, Calif.). In still other embodiments, a constitutive promoter can be used. It is contemplated that other mammalian expression vectors are suitable for use in the present invention, including, but not limited to, pWLNEO, pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Any other plasmid or vector can be used as long as it can replicate and remain viable in the host. In some preferred embodiments of the present invention, mammalian expression vectors comprise an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking non-transcribed sequences. In other embodiments, DNA sequences derived from the SV40 splice, and polyadenylation sites can be used to provide the required non-transcribed genetic elements. Additionally, the KLF 4 gene can be inserted via a retroviral vector. Transfection can be accomplished by any method known in the art, including but not limited to calcium-phosphate coprecipitation, electroporation, microparticle bombardment, liposome mediated transfection, or retroviral infection.

In still further embodiments, the graft is engineered to provide a therapeutic agent to a subject. The present invention is not limited to the delivery of any particular therapeutic agent. Indeed, it is contemplated that a variety of therapeutic agents can be delivered to the subject, including, but not limited to, enzymes, peptides, peptide hormones, other proteins, ribosomal RNA, ribozymes, and antisense RNA. These therapeutic agents can be delivered for a variety of purposes, including but not limited to the purpose of correcting genetic defects. In some particular preferred embodiments, the therapeutic agent is delivered for the purpose of detoxifying a patient with an inherited inborn error of metabolism (e.g., aminoacidopathesis) in which the graft serves as wild-type tissue. It is contemplated that delivery of the therapeutic agent corrects the defect. In some embodiments, the keratinocytes used to form the skin equivalent include a polynucleotide that encodes encode a therapeutic agent (e.g., insulin, clotting factor IX, erythropoietin, etc.) and the skin equivalent is grafted onto the subject. The therapeutic agent is then delivered to the patient's bloodstream or other tissues from the graft. In preferred embodiments, the polynucleotide that encodes the therapeutic agent is operably linked to a suitable promoter. The present invention is not limited to the use of any particular promoter. Indeed, a variety of promoters is contemplated, including, but not limited to, inducible, constitutive, tissue specific, and keratinocyte-specific promoters. In some embodiments, the nucleic acid encoding the therapeutic agent is introduced directly into the keratinocytes (i.e., by calcium phosphate co-precipitation or via liposome transfection). In other preferred embodiments, the nucleic acid encoding the therapeutic agent is provided as a vector and the vector is introduced into the keratinocytes by methods known in the art. In some embodiments, the vector is an episomal vector such as a plasmid. In other embodiments, the vector integrates into the genome of the keratinocytes. Examples of integrating vectors include, but are not limited to, retroviral vectors, adeno-associated virus vectors, and transposon vectors.

In still other embodiments, techniques such as homologous recombination can be used to knock in or knock-out genes. In particularly preferred embodiments, the genes for α-2 macroglobulin, or the major histocompatibility complex (MHC) genes are deleted or inactivated. Techniques and reagents for homologous recombination are described in U.S. Pat. Nos. 5,416,260; 5,965,977; and 5,981,214; each of which is incorporated herein by reference.

Next, one would identify a patient and isolate cells for coculture. For cultivating human keratinocytes in monolayer culture, a tissue sample is obtained. Keratinocytes are isolated from human skin or other stratified squamous epithelia. Keratinocyte cultures are established by plating aliquots of a single cell suspension in the presence of mitomycin C-treated Swiss mouse 3T3 fibroblasts as previously described (Allen-Hoffmann and Rheinwald, 1984). The standard keratinocyte culture medium is composed of a mixture of Ham's F-12 medium:Dulbecco's modified Eagle's medium (DME), (3:1, final calcium concentration 0.66 mM) supplemented with 2.5% fetal calf serum (FCS), 0.4 μg/ml hydrocortisone (HC), 8.4 ng/ml cholera toxin (CT), 5 μg/ml insulin (Ins), 24 μg/ml adenine (Ade), 10 ng/ml epidermal growth factor (EGF), 100 units penicillin and 100 μg/ml streptomycin (P/S). The cells are routinely subcultured at weekly intervals at 3×10⁵ cells per 100-mm tissue culture dish (approximately a 1:25 split) with a mitomycin C-treated feeder layer. Recombinant human EGF and transforming growth factors-β1 (TGF-β1) are obtained from R & D Systems (Minneapolis, Minn.).

To produce a chimeric culture of donor keratinocytes and carrier keratinocytes, preferably NIKS keratinocytes, the desired ratio of donor keratinocytes and carrier keratinocytes is used at the time of subculture or at any other time during the culture process. For example, NIKS cells can be added to an adherent donor keratinocyte culture, donor keratinocytes can be added to an adherent NIKS monolayer culture, or the carrier cells and donor keratinocytes can be mixed together at time of subculture.

In some preferred embodiments for cultivation of the carrier keratinocyte cells (e.g., NIKS cells) and patient keratinocytes in organotypic culture, a collagen base is formed by mixing normal human fibroblasts, with Type I collagen in Ham's F-12 medium containing 10% FCS and P/S. The collagen base is allowed to contract for 5 days to form contracted collagen rafts. The patient keratinocytes and the NIKS keratinocytes are plated on the contracted collagen rafts at 3.5×10⁵ cells in 50 μl of a mixture of Ham's F-12:DME, (3:1, final calcium concentration 1.88 mM) supplemented with 0.2% FCS, 0.4 μg/ml HC, 8.4 ng/ml CT, 5 μg/ml Ins, 24 μg/ml Ade, and P/S. Cells are allowed to attach for 2 hours before flooding culture chamber with media (day 0). On days 1 and 2 cells are re-fed. On day 4, cells are lifted to the air interface with cotton pads and switched to cornification medium containing Ham's F-12:DME, (3:1, final calcium concentration 1.88 mM) supplemented with 2% FCS, 0.4 μg/ml HC, 8.4 ng/ml CT, 5 μg/ml Ins, 24 μg/ml Ade, and P/S. Cells are fed cornification medium every 3 days until complete stratafication is achieved (approximately 15 days). One can identify successful monolayer culture by visual presence of keratinocytes in the culture dish. Successful organotypic culture is identified by the observing the ratios of patient-derived keratinocytes to carrier keratinocytes.

The ratios of patient-derived keratinocytes to carrier keratinocytes exemplified herein are not intended to be limiting. Indeed, based on the guidance given herein, it is clear that a variety of ratios can be used in the present invention. Accordingly, in some embodiments, a suitable ratio of patient-derived keratinocytes to carrier keratinocytes ranges from about 0.5%:99.5% to about 80%:20%, preferably from about 10%:90% to about 60%:40%; and most preferably a ratio of about 20%:80%.

A skin graft of the present invention will have the tissue architecture and differentiation and adhesion markers of normal skin Advantageously, a skin graft of the present invention can be grown faster than skin grafts made from patient-derived skin.

Organotypic chimeric cocultures of donor and carrier keratinocytes, and resulting skin grafts of the present invention, display a tissue architecture that closely resembles the architecture of normal skin and organotypic human keratinocyte cultures substantially as described in the Background of the Invention, supra and as shown in FIG. 5, taking into account the typical variations observed in organotypic cultures. For example, organotypic chimeric cocultures of primary human keratinocytes and NIKS cells were paraffin-embedded, sectioned, and stained with hematoxylin and eosin for histological examination. The cocultures exhibit a discrete stratum basale consisting of columnar basal cells. The overlying stratum spinosum is composed of several larger, progressively flattened cell layers. The upper layers display a granular layer having hematoxylin-stained keratohyalin granules.

The cultures exhibit normal distribution of cell-type specific proteins associated with various stages of squamous differentiation. The localization of differentiation markers was visualized using indirect immunofluorescence. The early stage differentiation marker keratin 1 appears as diffuse cytoplasmic staining in all cultures, with expression being initiated in the first or second suprabasal cell layer and continuing upward through the stratum.

Immunofluorescent staining shows a diffuse cytoplasmic protein localization of involucrin beginning in the first several suprabasal cell layers. NIKS cultures exhibit identical spatial localization of involucrin at 8, 11, and 13 days as at 21 days (Loertscher et al., 2000). Prior analyses of intact, normal human skin samples have demonstrated the expected cytoplasmic localization of involucrin (Mansbridge and Knapp, 1987; Murphy et al., 1984). Our data shows involucrin protein localized in the cytoplasmic compartment, as expected. This finding supports our claim of normal, in vivo-like differentiation of chimeric, organotypic cultures of human keratinocytes and NIKS keratinocytes.

The intermediate differentiation marker TG_K exhibits identical localization patterns and intensity in chimeric, organotypic NIKS and human keratinocyte cultures. Its distinct, honeycomb-like appearance reflects the localization of the membrane-bound TG_K enzyme.

The late stage differentiation marker filaggrin is localized to the keratohyalin granules in the cells of the stratum granulosum of NIKS and primary keratinocyte rafts, as indicated by its punctate staining pattern. Chimeric organotypic cultures of human keratinocytes and NIKS keratinocytes displayed normal spatial localization of filaggrin at 15 days.

Immunohistochemistry was also used to determine the presence and localization of E- and P-cadherin. In the cocultures of the invention, E-cadherin appears in the regions of cell-cell contact in all cultures, with expression beginning in the immediate suprabasal layer and continuing up through the living strata. The P-cadherin expression pattern is also similar in all cultures, but is initiated in the stratum basale and continues only through the first several suprabasal layers. These findings mirror those observed in intact skin and demonstrate that chimeric, organotypic human keratinocyte and NIKS keratinocyte cultures produce an appropriate pattern of cadherin molecules as compared to organotypic cultures of human keratinocytes alone and/or intact human skin.
 

Claim 1 of 20 Claims

1. A chimeric skin comprising an immortalized human keratinocyte cell in coculture with donor keratinocytes, wherein the skin comprises normal tissue architecture and differentiation markers of stratified squamous epithelia and wherein expression and location of late-stage differentiation markers are typical of intact human skin.

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