GB2483622A

GB2483622A - Fibroblast feeder cells

Info

Publication number: GB2483622A
Application number: GB0913469.3A
Authority: GB
Inventors: Colin Albert Buchanan Jahoda
Original assignee: University of Durham
Current assignee: University of Durham
Priority date: 2009-08-03
Filing date: 2009-08-03
Publication date: 2012-03-21
Also published as: JP2013500738A; EP2461817A1; GB0913469D0; WO2011015862A1

Abstract

A cell culture support comprises a fibroblast cell feeder layer, characterized in that said fibroblast cells are isolated from hair bearing skin tissue. The dermal fibroblasts from hair-bearing skin express greater levels of markers including periostin and SPARC (osteonectin) than fibroblasts from non-hair-bearing skin. The cell culture support is used to culture epidermal cells, in particular keratinocytes or epidermal progenitor cells. Fibroblast cells isolated from hair bearing skin tissue are used in wound-healing compositions, and in the production of skin-derived precursors (SKPs).

Description

Cell Support The present invention relates to an improved cell support. More particularly the invention relates to a cell support comprising fibroblast cells isolated from hair bearing skin tissue.

Background

Tissue engineering has implications with respect to many areas of clinical and cosmetic surgery and relates to the replacement, restoration or repair of damaged or diseased tissues Tissue engineering has particular application in wound healing in the provision of skin grafts for skin Skin is a highly complex organ covering the external surface of the body. Skin is composed of two layers, the dermis and the epidermis. The dermis is primarily formed of connective tissue containing fibroblasts embedded in a matrix of collagen. The epidermis is the outer layer, which is several cells thick. The epidermis is composed primarily of keratinocytes, which make up over 95% of the cell population. The remainder of the cell population is comprised of dendritic cells, such as Langerhans cells and pigmented cells called melanocytes. The epidermis is essentially cellular and non vascular, there being relatively little extra cellular matrix except for the layer of collagen and other proteins beneath the basal layer of keratinocytes.

The keratinocytes involved in for providing the epidermal barrier, reparative, and regenerative properties.

Skin functions, amongst other things, to prevent water loss from the body and to act as a protective barrier against the action of physical, chemical or infectious agents. Loss of skin may result in mortality or morbidity and in the treatment of large wounds, for example burns, it is recommended to restore the barrier function of the skin, for example by resurfacing with autologous skin grafts. One obstacle in tissue engineering, and in particular in skin grafting, is the time required for keratinocyte culture formation. Accordingly, there remains a need for improved methods of establishing keratinocyte culture formation.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Brief Summary of the Disclosure

In a first aspect the invention provides a cell support comprising a fibroblast cell feeder layer, characterized in that said fibroblast cells are isolated from hair bearing skin tissue.

Preferably said support further comprises an epidermal cell culture, such as a keratinocyte cell culture or an epidermal progenitor.

In one embodiment said fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least one of marker selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITGA1 1, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, RAPLN1, THBS4, CYP26B1, WISP1 and 1L7.

Preferably said fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least 5, 10, 15, 20 or 25 markers selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITGA1 1, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7.

In one embodiment said fibroblast cells isolated from hair bearing skin tissue are characterized as expressing increased levels compared to a fibroblast cell isolated from non-hair bearing tissue of a protein marker selected from the group consisting of: SPARC and periostin.

Preferably said hair bearing skin bears actively growing hair follicles. More preferably said hear bearing tissue is selected from the group consisting of: scalp tissue, face tissue, pubic tissue.

In one embodiment said culture support further comprises a feeder layer support, such as a matrix, dish, a well, a flask or a plate.Preferably said matrix is a collagen matrix.

In a further aspect the invention provides a method of epidermal cell culture comprising co-culturing at least one epidermal cell type together with one or more fibroblast cells, characterized in that said one or more fibroblast cells are isolated from hair bearing skin tissue.

Preferably said one or more fibroblast cells are provided as a fibroblast feeder layer.

In one embodiment said fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least one of marker selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGIR1, ITOA1 1, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH2SH, KCNJ1S, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7.

Preferably said fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least 5, 10, 15, 20 or 25 markers selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, ACTR1, ITGA11, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH2SH, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7.

In a further embodiment said fibroblast cells isolated from hair bearing skin tissue are characterized as expressing increased levels compared to a fibroblast cell isolated from non-hair bearing tissue of a protein marker selected from the group consisting of: SPARC and periostin.

In one embodiment said co-culture is in a serum free medium.

In one embodiment said epidermal cell is a keratinocyte, an epidermal progenitor cell or a keratinocyte progenitor. Preferably said epidermal cells are human epidermal cells.

Preferably said fibroblast cells are human fibroblasts.

In a further aspect the invention provides a co-culture vessel comprising a fibroblast feeder layer according to the present invention and one or more epidermal cells.

Preferably said one or more epidermal cells is a keratinocyte, an epidermal progenitor cell, or a keratinocyte progenitor cell.

In one embodiment said vessel further comprises a serum free medium.

In a further aspect the invention provides a fibroblast cell isolated from hair bearing skin tissue for use as a medicament.

In a further aspect the invention provides use of a fibroblast cell isolated from hair bearing skin tissue in the manufacture of a medicament for skin wound healing.

In one embodiment said medicament is prepared for administration with one or more epidermal cells. Preferably said one or more epidermal cells are keratinocytes.

In one embodiment said fibroblast cell is associated with a matrix, such as a collagen matrix.

In one embodiment said medicament is prepared for topical administration.

In a further aspect the invention provides a method of healing a skin wound comprising applying one or more fibroblast cells isolated from hair bearing skin tissue to the skin wound.

In one embodiment said one or more fibroblast cells is applied to a wound bed of the skin In one embodiment the method further comprises applying one or more epidermal cells to the skin wound or to the wound bed of the skin wound.

In a further aspect the invention provides a wound healing composition comprising one or more fibroblast cells isolated from hair bearing skin tissue and a pharmaceutically acceptable carrier.

In one embodiment the composition further comprising one or more epithelial cells for simultaneous, separate or sequential administration. Preferably said one or more epidermal cells are keratinocytes.

Preferably said composition is prepared for topical administration.

In a further aspect the invention provides a wound dressing comprising a medicament in accordance with the present invention or a composition in accordance with the present invention.

In a further aspect the invention provides use of a culture of fibroblasts isolated from hair bearing skin to obtain a skin derived precursor (SKP), wherein said culture of fibroblasts has been subject to at least one passage.

Brief Description of the Drawings

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 Illustrates a summary diagram of the method for the extraction and processing of RNA for microarray analysis.

Figure 2 Illustrates the results of Immunohistochemistry and Western analysis of samples from different human dermal fibroblast cultures show that the fibroblasts from hairy skin (DF) express levels of alpha smooth muscle actin less than hair follicle dermal papilla (DP) and dermal sheath (DS) cells, but greater than fibroblasts from non-hairy foreskin, breast and face skin.

Figure 3 Shows the growth of human epidermal cells (keratinocytes) in association with different supporting human dermal fibroblasts in 2 dimensional culture. The relative growth of the epidermal cells is measured by the strength of the red Rhodamine dye which stains epidermal keratinocytes. Greater epidermal cell growth is seen with supporting hairy dermal fibroblasts compared with dermal fibroblasts from two non-hairy sites, foreskin and breast, and with mouse 3T3 cells.

Figure 4 is a graphical representation showing the growth of epidermal cells (keratinocytes) in association with different supporting dermal fibroblasts. The relative growth of the epidermal cells is measured by absorbance of the red Rhodamine dye (by spectrophometer) which stains keratinocytes. Three replicate samples of dermal cells from a region of hairy skin (pubic) support epidermal cells significantly better than dermal cells from three non-hairy skin regions, breast, foreskin and scalp (from a bald individual). Mouse 3T3 cells are also significantly less good at supporting epidermal growth than the hair skin dermal fibroblasts.

Figure 5 is a graphical representation showing the growth of epidermal cells (keratinocytes) in association with different supporting dermal cells. The relative growth of the epidermal cells is measured by absorbance of the red Rhodamine dye (by spectrophometer) which stains keratinocytes. Three replicate samples of dermal cells from a region of hairy skin (hairy scalp) support epidermal cells significantly better than dermal cells from three non-hairy skin regions, breast, foreskin and scalp (from a bald individual). Mouse 3T3 cells are also significantly less good at supporting epidermal growth than the hair skin dermal fibroblasts.

Figure 6 is a graphical representation showing the growth of epidermal cells (keratinocytes) in association with different supporting dermal fibroblasts. The relative growth of the epidermal cells is measured by absorbance of the red Rhodamine dye (by spectrophometer) which stains keratinocytes. Three replicate samples of dermal cells from a region of hairy skin (beard) support epidermal cells significantly better than dermal cells from three non-hairy skin regions, breast, foreskin and scalp (from a bald individual). Mouse 3T3 cells are also significantly less good at supporting epidermal growth than the hair skin dermal fibroblasts.

Detailed Description

The inventors have surprisingly identified that a sub-population of dermal fibroblasts isolated from hair bearing skin improved cell support properties, particularly epidermal support properties, when compared to the cell support properties of fibroblasts from non-hair bearing skin.

Using this sub-population of dermal fibroblasts the present invention provides an improved method of in vitro epidermal culture and propagation. The invention also provides a method of wound healing and re-epithelialisation using the sub-population of dermal fibroblasts.

Fibroblasts are involved in the maintenance of the structural integrity of connective tissue. They secrete precursors of the extracellular matrix and collagen. The sub-population of fibroblasts of the present invention are isolated from hair bearing skin tissue. Preferably said hair bearing skin is a tissue containing hair follicles, preferably active hair follicles, e.g. follicles active in the hair cycle. More preferably said tissue is isolated from the scalp, the beard area, the neck, the arms or the pubic (axilla) region.

Biological markers allow the identification and characterization of fibroblasts isolated from hair bearing skin tissue. In particular these biological markers can be used to distinguish fibroblasts isolated from hair bearing skin from fibroblasts isolated from non-hair bearing skin, e.g. foreskin, palm skin, plantar skin and breast.

Fibroblasts isolated from hair bearing skin show an increase, when compared to a fibroblast isolated from non-hair bearing skin, e.g. foreskin, palm skin, plantar skin, in the level of expression of at least one polypeptide selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITGA11, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7 or an increase of expression of a nucleic acid molecule e.g. an mRNA encoding a polypeptide selected from ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITOA1 1, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7.

Preferably, a fibroblast isolated from hair bearing skin shows an increased expression of at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or 25 of the aforementioned polypeptides and or nucleic acid markers when compared to a fibroblast isolated from non-hair bearing skin.

In addition, fibroblasts isolated from hair bearing skin also show an increase, when compared to a fibroblast isolated from non-hair bearing skin, e.g. foreskin, palm skin, plantar skin, in the level of at least one secreted protein marker selected from the group consisting of: SPARC (Secreted Protein Acidic and Rich in Cysteine) and periostin, or a nucleic acid molecule, e.g. an mRNA encoding SPARC or periostin. Preferably a fibroblast isolated from hair bearing skin shows an increased level of extracellular SPARC and I or periostin protein expression, secretion or activity when compared to a fibroblast isolated from non-hair bearing skin.

SPARC (also known as osteonectin or BM4O) was first discovered in bone (Termine et al., 1981 Cell. 26, 99-105) but has been found in other tissue, including skin where its expression is most intense directly below the basement membrane and in the papillary dermis, around vascular and glandular structures (Hunzelmann Invest dermatol (1998) 110:122-126). The role of SPARC in tissue repair and remodelling has been expertly reviewed by Phan et al 2006 who describes the main activities of SPARC include: modulating cell-ECM interactions, delaying cell-cycle progression, inhibiting proliferation and angiogenesis, and regulating the expression of a number of growth factor and ECM proteins.

Preferably, a fibroblast isolated from hair bearing skin shows increased expression of a least one polypeptide selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITGA1 1, POSTN, ISLR, DSP, ITGA8, MYO1 D, KCNK2, CH25H, KCNJ1 5, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, HAPLN1, THBS4, CYP26B1 WISP1 and 1L7, or a nucleic acid molecule, e.g. an mRNA encoding said polypeptide and an increased expression of a least one polypeptide selected from the group consisting of SPARC and periostin or a nucleic acid molecule, e.g. an mRNA encoding said polypeptide, when compared to a fibroblast isolated from non-hair bearing skin.

Still more preferably, a fibroblast isolated from hair bearing skin shows increased expression of each polypeptide of the group consisting of ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITOA11, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH2SH, KCNJ15, CLIC2, DPT, TME PAl, FOXD1, LGR5, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7, or increased expression of a nucleic acid molecule, e.g. an mRNA encoding said polypeptide and an increased expression of each polypeptide selected from the group consisting of SPARC and periostin or a nucleic acid molecule, e.g. an mRNA encoding said polypeptide, when compared to a fibroblast isolated from non-hair bearing skin.

The inventors have surprisingly identified that fibroblast cells isolated from hair bearing skin tissue are capable of stem cell potential after serial passaging in culture as demonstrated by their capacity to form multipotent skin-derived precursors (SKPs). Fibroblast cells isolated from hair bearing skin tissue are further characterized as having an increased stem cell potential compared to a fibroblast cell isolated from non-hair bearing tissue after serial passaging in culture, preferably said stem cell potential is demonstrated by the capacity to said fibroblasts to form multipotent skin-derived precursors (SKPs).

As used herein, a "passage" refers to a round of subculturing. Accordingly, when cells are subcultured, they are referred to as having been passaged.

Preferably, the fibroblasts are human fibroblasts.

Fibroblasts may be isolated from hair bearing skin using methods known in the art. For example, fibroblasts may be isolated from hair bearing skin by explant culture, enzymatic dissociation, cell sorting or a combination thereof.

Fibroblasts isolated from hair bearing skin in accordance with the present invention can be used to advantageously promote the growth of cells, preferably epidermal cells, both in vitro, e.g. in tissue engineering and cell culture, and in vivo, in epidermal wound repair and dermal regeneration.

The cells supported by the fibroblast sub-population in accordance with the present invention are preferably epidermal cells. Said epidermal cells may be differentiated epidermal cells or epidermal progenitor cells. By epidermal progenitor cell is meant a multipotent cell having epidermal potential, e.g. a cell capable of differentiating into an epidermal cell.

Preferably an epidermal cell in accordance with the invention is selected from a keratinocyte, a melanocyte, a Langerhans cell or a Merkels cell. Alternatively said epidermal cell is an epidermal progenitor, more preferably a cell having keratinocyte, melanocyte, Langerhans cell or Merkel cell potential. Preferable the epidermal cell is a keratinocyte, more preferably an epidermal keratinocyte or a corneal keratinocyte.

Preferably, the epidermal cells are mammalian cells, more preferably human epidermal cells.

Alternatively the cell supported by the fibroblast sub population is an embryonic stem cell, a neural progenitor cell or a blood progenitor cell.

As used herein, the terms "culture" and "cell culture" are used interchangeably refer to the process whereby cells, taken from a living organism, are grown under controlled conditions, preferably in vitro.

In one aspect fibroblasts isolated from hair bearing skin in accordance with the invention are used as a feeder layer to support a cell or cell culture, preferably an epidermal cell culture, as described above. The fibroblast feeder layer is preferably provided on culture support, such as a matrix, dish, a well, a flask or a plate.

In a further aspect the invention provides an improved method of epidermal cell culture, which comprises co-culturing at least one epidermal cell type together with one or more fibroblast cells which is isolated from hair bearing skin tissue. The inventors have demonstrated that fibroblasts isolated from hairy skin provide keratinocytes with factors for growth and differentiation which are distinct from the factors provided by fibroblasts from non-hair bearing skin and advantageously promote improved keratinocyte culture.

The cell culture of the invention may be conducted in the presence of an appropriate cell culture medium. Appropriate epidermal and keratinocyte culture medium are known in the art and include for fibroblasts Minimal Essential Medium (Eagles or Dulbecco"s); for keratinocytes, Dulbecco's MEM, Keratinocyte Basal Medium 2 from PromoCell's Cryo-SFM, a serum-free cryo-medium, Keratinocyte-SFM,Keratinocyte nutrient MCDB 153 medium or EpiLife® Medium from Invitrogen. In one embodiment the culture medium is preferably a serum free culture medium.

The culture support and method can be used regenerate tissue, for example in the preparation of skin-grafts. Tissues cultured in accordance with the methods and products described herein can be transplanted into patients to initiate wound healing and repair.

In a further aspect the invention provides a fibroblast from hair bearing skin tissue for use as a medicament. In a still further aspect the invention provides the use of a fibroblast cell isolated from hair bearing skin tissue in the manufacture of a medicament for skin wound healing.

Preferably the fibroblast cells are associated with a matrix. Preferably the matrix is a biocompatible matrix. More preferably, the matrix is formed from polyhydroxy acids, polyorthoesters, polyanhydrides, proteins, polysaccharides, polyphosphazenes or combinations thereof. Still more preferably, the support is formed from or comprises collagen, e.g. a collagen-glucosaminoglycan support.

In a further aspect the invention provides a wound healing composition comprising one or more fibroblast cells isolated from hair bearing skin tissue. Preferably the fibroblast cells are provided with any standard physiologically and pharmaceutically acceptable carrier. The compositions are sterile and contain a therapeutically effective amount of fibroblasts in an amount suitable for administration to a patient. Fibroblasts may be associated with a matrix as described above. In one embodiment the composition further comprises one or more epithelial cells for simultaneous, separate or sequential administration. Preferably, said one or more epidermal cells are keratinocytes. Preferably said epithelial cells are provided in a matrix as described above.

The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human. When administered, the pharmaceutical compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, cytokines and optionally other therapeutic agents, preferably agents for use in wound healing such as growth factors, peptides, proteolytic inhibitors, extracellular matrix components, steroids and cytokines.

The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. As used herein, a pharmaceutically acceptable carrier includes any conventional carrier, such as those described in Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co, Easton, PA, 15th Edition (1975).

The compositions of the invention can be administered by any conventional route, including injection. The administration may, for example, be topical, intracavity, subcutaneous, or transdermal. Preferably the composition is prepared for topical administration.

The compositions of the invention are administered in effective amounts. An "effective amount" is the amount of a composition that alone, or together with further doses, produces the desired response. The compositions used in the foregoing methods preferably are sterile and contain an effective amount of the active ingredient for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by measuring the physiological effects of the composition upon the rate of or extent of wound healing.

In a further aspect the invention provides a method for the treatment of skin or a skin wound, comprising applying to the skin, skin wound or skin wound bed a fibroblast isolated from hair bearing skin or composition described herein. The method is of use in skin re-epithelialisation.

The term "re-epithelialisation" relates to the repair, replacement, functional recovery and ultimate regeneration of damaged epithelium inside the body (including skin), or outside the body.

Fibroblasts isolated from hair bearing skin according to the invention can thus be used in the manufacture of a medicament for application to a living body, preferably a human. Preferably, fibroblasts isolated from hair bearing skin according to the invention are used in the manufacture of a medicament for the treatment of a wound, for wound healing or re-epithelialisation.

As used herein the term wound relates to damaged tissues, preferably damaged skin, where the integrity of the skin or tissue is disrupted as a result from i.e. external force, bad health status, aging, exposure to sunlight, heat or chemical reaction or as a result from damage by internal physiological processes. Wounds where the epidermis is damaged are considered an open wound. Wound healing is the process of regenerating the covering cell layers of a tissue, preferably by re-epithelialisation or reconstruction.

In one embodiment fibroblasts isolated from hair bearing skin are administered to wounds with one or more epithelial cells as described herein. The fibroblasts and optionally one or more epithelial cells are administered with one or more other wound healing agents such as growth factors, peptides, proteolytic inhibitors, extracellular matrix components, steroids or cytokines, oxygen donators or vitamins. Such additional wound healing agent(s) may be administered separately, simultaneously or sequentially. Such combinations may also be used in the manufacture of the medicament.

In one embodiment a patient may be administered the fibroblasts isolated from hair bearing skin and the said one or more epithelial cells as a single medicament. Alternatively the fibroblasts isolated from hair bearing skin and the said one or more epithelial cells may be administered separately.

Preferably, said fibroblasts isolated from hair bearing skin and or said one or more epithelial cells is autologous, i.e. said cells are derived from the individual to be treated or that biological material added to tissue cultures comes from the donor of the cells for tissue culture.

Alternatively the cells may be non-autologous.

In a further aspect the invention provides a wound dressing comprising a fibroblast isolated from hair bearing tissue, a composition or a medicament as described herein. As used herein wound dressing refers to a dressing for topical application to a wound. For example, the at least fibroblast isolated from hair bearing tissue, a composition or a medicament may be dispersed in or on a solid sheet of wound contacting material such as a woven or nonwoven textile material, or may be dispersed in a layer of foam such as polyurethane foam.

Examples

Biomarker Identification In order to find "unique" markers for skin fibroblasts isolated from hair bearing skin a comparison was conducted between dermal sheath v "hairy" skin fibroblasts v newborn skin fibroblasts. Comparisons were performed in 3D cultures in "integra".

The following cell types were compared: Dermal sheath -male -3 strains, Dermal fibroblasts from hair bearing skin -male -3 strains, Newborn dermal fibroblasts: -male -3 stains.

Approximately 300,000 cells for each cell type were seeded onto integra patch in MEM and 10% FBS. 3 patches were seeded for each cell type. Cells allowed to attach and grow for 3 days.

After 3 days RNA from cells was recovered by conventional techniques and hybridized on Affymetrix full human genome arrays. Total RNA was isolated from cells in integra initially using liquid nitrogen to denature the samples. RNA was isolated using the (RNeasy mini kit, Qiagen). RNA samples were prepared for hybridisation using the one cycle cDNA synthesis kit and applied to an Affymetrix human expression array following the manufacturer's instructions.

Data were analysed using GeneSpring® * (Silicon Genetics, USA).

443 genes show differences (increase or decrease) at> 2 fold in Hairy Dermal Fibroblasts v Dermal Sheath. 1130 genes show differences at >2 fold in Hairy Dermal Fibroblasts v Newborn Fibroblasts.

Those genes identified as biomarkers of particular interest for fibroblasts isolated from hair bearing skin ("hairy fibroblasts") are listed in table 1 below.

Table 1

Affymetrix FOLD Gene identifier and name Probe No. INCREASE!

DECREASE

HAIRY

Fl BROBLASTS

V FORESKIN

206373_at 104.8734 up ZIC1 Zic family member 1 (odd-paired homolog 22361 isat 8.084196 up LNX1 ligand of numb-protein X 1 235228_at 19.81083 up CCDC85A coiled-coil domain containing 85A 204779 sat 18.49246 up HOXB7 homeoboxB7 220559_at 16.01584 up EN1 engrailed homeobox 1 v-maf musculoaponeurotic fibrosarcoma 218559 sat 15.84178 up MAFB oncogene homolog B (avian) 228904_at 14.37703 up HOXB3 homeobox B3 235944 at 13.8787 up HMCN1 hemicentin 1 228564_at 12.18865 up L0C375295 hypothetical gene supported by BC01 3438 leucine-rich repeat-containing 0 protein- 218326sat 2.357926 up L0R4 coupled receptor4 leucine-rich repeat-containing 0 protein- 213880_at 12.46195 up LGR5 coupled receptors 203180 at 11.39927 up ALDH1A3 aldehyde dehydrogenase 1 family 205357_s_at 11.29129 up AGTR1 angiotensin II receptor 204776 at 9.982841 up THBS4 thrombospondin 4 205523_at 9.838161 up HAPLN1 hyaluronan and proteoglycan link protein 1 222899 at 9.498495 up ITGA11 integrin 1555778_a_at 6.432005 up POSTN periostin immunoglobulin superfamily containing 207191_s_at 9.369685 up ISLR leucine-rich repeat 1556209_at 8.825037 up CLEC2B C-type lectin domain family 2 203868_s_at 8.70770 1 up VCAM1 vascular cell adhesion molecule 1 235301_at 8.480307 up KIAA1324L KIAA1 324-like 205433_at 8.290045 up BCHE butyrylcholinesterase 206030_at 7.805743 up ASPA aspartoacylase (Canavan disease) 232523_at 7.688089 up MEOF1O multiple EGF-like-domains 10 200606_at 7.657114 up DSP desmoplakin 205568_at 7.560288 up AQP9 aquaporin 9 235666_at 7.395729 up ITGA8 integrin 212338_at 7.362677 up MYO1D myosin ID 228486_at 7.328847 up SLC44A1 solute carrier family 44 1552398_a_at 7.298154 up CLEC12A C-type lectin domain family 12 209160_at 7.283046 up AKR1C3 aldo-keto reductase family 1 1554062_at 7.152701 up X0 Xg blood group 219825_at 7.09656 up CYP26B1 cytochrome P450 209732_at 7.094428 up CLEC2B C-type lectin domain family 2 1556579_s_at 7.070232 up lGSFlO immunoglobulin superfamily 206201sat 6.882109 up MEOX2 mesenchyme homeobox 2 227461_at 6.826688 up STON2 stonin 2 231 906 at 6.752029 up HOXD8 Homeobox D8 210261_at 6.317289 up KCNK2 potassium channel 213994sat 6.247891 up SPON1 spondin 1 206932_at 6.107118 up CH25H cholesterol 25-hydroxylase 21011 9at 6.09470 1 up KCNJ1 5 potassium inwardly-rectifying channel 229127_at 6.026437 up CDNAFLJ31S17fis 206796 at 6.013537 up WISP1 WNT1 inducible signaling pathway protein 1 222860_s_at 5.941892 up PDGFD platelet derived growth factor D fibroblast growth factor receptor 2 (bacteria- 203638sat 5.915175 up FGFR2 expressed kinase 213415_at 5.862984 up CLIC2 chloride intracellular channel 2 207977sat 5.760128 up DPT dermatopontin 213993_at 5.745455 up SPON1 spondin 1 244317 at 5.720125 up KIAA1324L K1AA1324-like 212187_x_at 5.025631 up PTGDS prostaglandin D2 synthase 2lkDa (brain) 2071 55at 4.894668 down TBX5 T-box 5 219213_at 4.87695 up JAM2 junctional adhesion molecule 2 214587_at 4.872727 up COL8A1 collagen 222835_at 4.854392 down THSD4 thrombospondin 205932_s_at 4.849974 up MSX1 msh homeobox 1 205975_s_at 4.827469 up HOXD1 homeobox Dl 204456_s_at 4.793625 up GAS1 growth arrest-specific 1 228262_at 4.761 652 up MAP7D2 MAP7 domain containing 2 206693_at 4.67829 up 1L7 interleukin 7 222450_at 4.248641 up TMEPAI transmembrane 206307_s_at 3.889674 up FOXD1 forkhead box Dl 206377_at 44.02156 down FOXF2 forkhead box F2 206163_at 67.04833 down MAB21L1 mab-21 -like 1(0. elegans) 212224_at 63.5042 down ALDH1A1 aldehyde dehydrogenase 1 family 206377_at 44.02156 down FOXF2 forkhead box F2 204584_at 24.7118 down Li CAM Li cell adhesion molecule 221933_at 18.59671 down NLGN4X neuroligin 4 Moreover, in dermal cell culture microarray, expression of Periostin was shown to be 6.4 fold increased in Hairy fibroblasts vs Newborn fibroblasts.

Further biomarkers were identified by immunolabelling. Cells were cultured on glass coverslips for 4 days and fixed using 95% (v/v) MeOH: 5% (v/v) acetone for 15 mm at -20°C. Slides were then washed with PBS (3 x 5 minutes) and blocked with 3% (wt/v) BSA., then incubated with primary antibody e.g. mouse monoclonal anti-aiphaSMA 1:200 (v/v) for 1 hour at room temperature. Slides were then washed with PBS (3 x 5 minutes) and incubated with secondary antibody and DAPIe.g. alexoflour goat anti-mouse 1:500 (v/v) for 1 hour at room temperature.

Antigen binding (alpha-SMA) was revealed under FITC incident blue light (Xex=490nm and Xem=523nm as illustrated in figure 2. The results indicate that dermal fibroblasts isolated from hair bearing skin (DF hairy) show higher ASMA in comparison to dermal fibroblasts from non hair bearing sites.

Keratinocyte support A coculture approach was used to investigate dermal fibroblasts isolated from hair bearing skin (hairy DF cells) for an ability to support keratinocyte proliferation. Cocultures of dermal and epidermal cells were stained with rhodamine B (rhodamine B specifically staining keratinocytes) and eluted stains were quantified at 550 nm.

When dermal fibroblasts isolated from hair bearing skin were employed as a feeder layer for human keratinocytes their performance was equivalent to the conventional gold standard' method of keratinocyte culture (using murine 3T3 cells as a feeder layer plus R&G media).

isolation and establishment of fibroblast cultures from hair bearing and hair free skin tissue Human skin tissue was obtained according to ethically approved guidelines rom Durham University Hospital (Durham, UK), The Royal Victoria Infirmary (Newcastle upon Tyne, UK) and The James Cook Hospital (South Tees, UK). Dermal fibroblast cultures were established from papillary dermis explants, approximately 3mm x 3mm in size. Explants of hair-free (breast, face and abdomen) and hair-bearing (beard, scalp, arm and abdomen) skin tissue were dissected under a stereo-dissecting microscope to ensure exclusion of any contaminating hair follicle dermal cells. Explants were cultured for 14 days in MEM supplemented with 20% FBS (Sigma), 2 mM L-glutamine (Invitrogen), 100 units/mI penicillin and 100 pg/mI streptomycin (Sigma) and 250 pg/mI amphotericin-B (Invitrogen). Established dermal cell cultures were cultured in identical media as above, but with a reduction in FBS content from 20% to 10% (v/v).

Culture of human keratinocytes Human keratinocytes were cultured in EpilifeTM (Invitrogen) supplemented with Human Keratinocyte Growth Supplement (HKGS) at 1:100 v/v (Invitrogen) and used at passage 3 for experimentation.

Rhodamine B staining of human keratinocytes To compare the ability of DS, DR and DF to support epithelial cell growth, each dermal cell type was co-cultured with human keratinocytes (at passage 3). For comparative purposes keratinocytes were cultured using growth arrested murine 3T3 cells (3T3 cells were a kind gift from Dr SE James (University of Brighton) in MEM or Green's media. (DMEM and Ham's F12 medium in a 3:1 (v/v) ratio supplemented with 10% (v/v) FBS, 10 ng/mL epidermal growth factor (EGF; R&D Systems, City, UK), 0.4 pg/mL hydrocortisone (Sigma), 10b0 mol/L cholera toxin (Source), 1.8x104mo1/L adenine (Source), 5 pg/mL insulin (Sigma), 5 pg/mL transferrin (Source), 2x10 mol/L glutamine (Sigma), 2x1Y7 mol/L triiodothyrionine, 0.625 pg/mL amphotericin B (Sigma), 100 IU/mL penicillin and 100 pg/mL streptomycin (Sigma).

Epidermal keratinocytes specifically stain with rhodamine B and the extent of staining can be quantified as previously described by Castro-Munozledo (1997). Briefly, dermal cells (20,000 per 35 mm dish) were incubated for 3.5 hours with 8 pg/mI mitomycin C (Sigma) and washed with PBS (x3) before addition of human keratinocytes (120,000 per 35 mm dish). After eight days, the co-cultures were fixed for 2 hours with 3.7% (v/v) formalin (BDH, city, UK), rinsed with distilled water and incubated with 1% (w/v) rhodamine B solution (Sigma) for 30 minutes at room temperature. Stained samples were washed with 0.2N HCI then dried at 32 00 overnight.

Rhodamine B was eluted from keratinocytes by incubation with 0.2N NaOH for 30 minutes at room temperature and the extracted dye was analysed at 550 nm using a S2100 Diode array spectrophotometer (Scientific Laboratory Supplies, city, UK).

Keratinocyte attachment and proliferation was observed using DF, feeder cell layers, demonstrated by positive rhodamine B staining of keratinocytes (Figure 3). Keratinocytes cultured for eight days in the presence of growth arrested DS and DR cells were observed to form larger colonies than those cultured in the presence of growth arrested fibroblasts or murine 3T3 cells in MEM.

The results illustrated in figures 4, 5 and 6 illustrate that Dermal cells from hairy sites (DF, DS and DP) provide better support to keratinocytes than non hairy DF and 3T3 cells. Collectively figures 4, 5 and 6 illustrate consistent differences between human dermal fibroblasts from hairy and non-hairy skin regions and their support of epidermal keratinocyte growth.

SKP formation Human DF SKRS were generated from DF cells at early passage numbers (2 and 3) and late passage numbers (11 and 12) using methods previously described for SKP formation from cells derived from whole fresh dermal tissue (Biernaskie 2006). Briefly, 25,000 cells per ml were seeded in SKP proliferation media (DMEM (Sigma) and F12 (Invitrogen) in a 3:1 ratio and supplemented with 40 ng/ml FGF2 (R&D Systems); 20 ng/ml EGF (Sigma); 2% B27 (Invitrogen)) and cultured for 21 days in a T25 vented flask (Nunc, Scientific Laboratory Supplies). Addition of 1.5% methycellu lose (Sigma) to the SKP proliferation media showed no difference in SKP forming capacity.

Routine SKP formation from passaqed dermal fibroblast cells Cultured human dermal fibroblasts at low passage numbers and high passage numbers incubated in SKPs proliferation media formed spheres of 123.7 pm to 147.6 pm in diameter.

For both early and late passage SKPs, we did not observe any difference in the time taken to form SKPs, (first fibroblast SKPs were identifiable between 7 to 11 days in proliferation media).

Variation was only observed between lines, with some fibroblast SKPs taking up to 18 days to become visible.

Claims

CLAIMS1. A cell support comprising a fibroblast cell feeder layer, characterized in that said fibroblast cells are isolated from hair bearing skin tissue.
2. The support according to claim 1, further comprising an epidermal cell culture.
3. The support according to claim 2, wherein said epidermal cell culture is a keratinocyte cellculture.
4. The support according to claim 2, wherein said epidermal cell culture is an epidermal progenitor.
5. The support according to any one of the preceding claims, wherein said fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least one of marker selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITOA1 1, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LORS, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7.
6. The support according to claim 5, wherein said fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least 5, 10, 15, 20 or 25 markers selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITOA1 1, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LORS, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7.
7. The support according to any one of claims 1 to 6, wherein said fibroblast cells isolated from hair bearing skin tissue are characterized as expressing increased levels compared to a fibroblast cell isolated from non-hair bearing tissue of a protein marker selected from the group consisting of: SPARC and periostin.
8. The support according to any one of the preceding claims, wherein said hair bearing skin bears actively growing hair follicles.
9. The support according to any one of the preceding claims, wherein said hear bearing tissue is selected from the group consisting of: scalp tissue, face tissue, pubic tissue.
10. The support according to any one of claims ito 9, wherein said culture support further comprises a feeder layer support.
ii. The support according to claim 5, wherein said feeder layer support is a matrix, dish, a well, a flask or a plate.
12. The support according to claim 11, wherein said matrix is a collagen matrix.
13. A method of epidermal cell culture comprising co-culturing at least one epidermal cell type together with one or more fibroblast cells, characterized in that said one or more fibroblast cells are isolated from hair bearing skin tissue.
14. The method according to claim 13, wherein said one or more fibroblast cells are provided as a fibroblast feeder layer.
15. The method according to claim 13 or claim 14, wherein said fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least one of marker selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITOA1 1, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, HAPLN1, THBS4, CYP26B1, WISP1 and 1L7.
16. The method according to claim 15, wherein said fibroblast cells isolated from hair bearing skin tissue are characterized as having an increased expression compared to a fibroblast cell isolated from non-hair bearing tissue of at least 5, 10, 15, 20 or 25 markers selected from the group consisting of: ZIC1, LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITGA1 1, POSTN, ISLR, DSP, ITGA8, MYO1D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1, RAPLN1, THBS4, CYP26B1, WISP1 and 1L7.
17. The method according to any one of claims 13 to 16, wherein said fibroblast cells isolated from hair bearing skin tissue are characterized as expressing increased levels compared to a fibroblast cell isolated from non-hair bearing tissue of a protein marker selected from the group consisting of: SPARC and periostin.
18. The method according to any one of claims 13 to 17, wherein said co-culture is in a serum free medium.
19. The method according to any one of claims 13 to 18, wherein said epidermal cell is a keratinocyte.
20. The method according to any one of claims 13 to 18, wherein said epidermal cell is an epidermal progenitor cell.
21. The epidermal culture support according to claim 20, wherein said epidermal progenitor is a keratinocyte progenitor.
22. The method according to any one of claims 13 to 21, wherein said epidermal cells are human epidermal cells.
23. The method according to any one of claims 13 to 22, wherein said fibroblast cells are human fibroblasts.
24. A co-culture vessel comprising a fibroblast feeder layer according to any of claims 1 to 12 and one or more epidermal cells.
25. A co-culture vessel according to claim 24, wherein said one or more epidermal cells is a keratinocyte.
26. A co-culture vessel according to claim 24, wherein said one or more epidermal cells is an epidermal progenitor cell.
27. A co-culture vessel according to claim 26, wherein said one or more epidermal progenitor cell is a keratinocyte progenitor cell.
28. A co-culture vessel according to any one of claim 24 to 27, wherein said vessel further comprises a serum free medium.
29. A fibroblast cell isolated from hair bearing skin tissue for use as a medicament.
30. Use of a fibroblast cell isolated from hair bearing skin tissue in the manufacture of a medicament for skin wound healing.
31. The use according to claim 30, wherein said medicament is prepared for administration with one or more epidermal cells.
32. The use according to claim 30 or claim 31, wherein said one or more epidermal cells are keratinocytes.
33. The use according to any one of claims 30 to 32, wherein said fibroblast cell is associated with a matrix.
34. The use according to claim 33, wherein said matrix is a collagen matrix.
35. The use according to any one of claims 30 to 34, wherein said medicament is prepared for topical administration.
36. A method of healing a skin wound comprising applying one or more fibroblast cells isolated from hair bearing skin tissue to the skin wound.
37. The method according to claim 36, wherein said one or more fibroblast cells is applied to a wound bed of the skin wound.
38. The method according to claim 36 or claim 37, further comprising applying one or more epidermal cells to the skin wound or to the wound bed of the skin wound.
39. A wound healing composition comprising one or more fibroblast cells isolated from hair bearing skin tissue and a pharmaceutically acceptable carrier.
40. The wound healing composition further comprising one or more epithelial cells for simultaneous, separate or sequential administration.
41. The wound healing composition according to claim 40, wherein said one or more epidermal cells are keratinocytes.
42. The wound healing composition according to any one of claims 39 to 41 prepared for topical administration.
43. A wound dressing comprising a medicament according to any one of claims 30 to 35 or a composition according to any one of claims 39 or 42.
44. Use of a culture of fibroblasts isolated from hair bearing skin to obtain a skin derived precursor (SKP), wherein said culture of fibroblasts has been subject to at least one passage.
45. A culture support substantially as described herein with reference to the accompanying drawings.
46. A method of epidermal cell culture as described herein with reference to the accompanying drawings.
47. A co-culture vessel as described herein with reference to the accompanying drawings.
48. A method of healing a skin wound as described herein with reference to the accompanying drawings.
49. A wound healing composition as described herein with reference to the accompanying drawings.
50. A wound dressing as described herein with reference to the accompanying drawings.