WO2011015862A1

WO2011015862A1 - Cell support comprising dermal fibroblasts

Info

Publication number: WO2011015862A1
Application number: PCT/GB2010/051278
Authority: WO
Inventors: Colin Albert Buchanan Jahoda
Original assignee: University Of Durham
Priority date: 2009-08-03
Filing date: 2010-08-03
Publication date: 2011-02-10
Also published as: JP2013500738A; GB2483622A; EP2461817A1; GB0913469D0

Abstract

The invention provides an cell culture support comprising a fibroblast cell feeder layer, characterized in that said fibroblast cells are isolated from hair bearing skin tissue.

Description

CELL SUPPORT COMPRISING DERMAL FIBROBLASTS

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 wound repair.

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 are involved in providing the epidermal barrier and have 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.

Preferably said fibroblast cells isolated from hair bearing skin tissue are dermal fibroblasts.

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, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7.

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, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7. In one embodiment said fibroblast cells isolated from hair bearing skin tissue are characterized as expressing increased levels, when 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 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 wound.

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 details a method for the extraction and processing of RNA for microarray analysis.

Figure 2 illustrates the results of lmmunohistochemistry 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, specifically those isolated from hair bearing skin, have 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.

Methods of isolating fibroblasts from skin are well known in the art and the sub-population of dermal fibroblasts used in the present invention can be isolated using any of the general methods available.

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 , ITGA1 1 , POSTN, ISLR, DSP, ITGA8, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4,

CYP26B1 , WISP1 and IL7 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 , ITGA1 1 , POSTN, ISLR, DSP, ITGA8, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, F0XD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7. Preferably, a fibroblast isolated from hair bearing skin shows an increased expression of at

16881 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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 / or periostin protein expression, secretion or activity when compared to a fibroblast isolated from non-hair bearing skin.

SPARC (also known as osteonectin or BM40) 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) 1 10:122-126). 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, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7, 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 , ITGA1 1 , POSTN, ISLR, DSP, ITGA8, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7, 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. 1 130 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

104.873

206373_at 4 up ZIC1 Zic family member 1 (odd-paired homolog

8.08419

22361 1_s_at 6 up LNX1 ligand of numb-protein X 1

19.8108

235228_at 3 up CCDC85A coiled-coil domain containing 85A

18.4924

204779_s_at 6 up HOXB7 homeobox B7

16.0158

220559_at 4 up EN1 engrailed homeobox 1

15.8417 v-maf musculoaponeurotic fibrosarcoma

218559_s_at 8 up MAFB oncogene homolog B (avian)

14.3770

228904_at 3 up HOXB3 homeobox B3

235944_at 13.8787 up HMCN1 hemicentin 1

12.1886 LOC37529

228564 at 5 up 5 hypothetical gene supported by BC013438 2.35792 leucine-rich repeat-containing G protein-

218326_s_at 6 up LG R4 coupled receptor 4

12.4619 leucine-rich repeat-containing G protein-

213880_at 5 up LGR5 coupled receptor 5

1 1.3992

203180_at 7 up ALDH 1 A3 aldehyde dehydrogenase 1 family

1 1.2912

205357_s_at 9 up AGTR1 angiotensin Il receptor

9.98284

204776_at 1 up THBS4 thrombospondin 4

9.83816

205523_at 1 up HAPLN1 hyaluronan and proteoglycan link protein 1

9.49849

222899 at 5 up ITGA1 1 integrin

1555778 a a 6.43200

t 5 up POSTN periostin

9.36968 immunoglobulin superfamily containing

207191_s_at 5 up ISLR leucine-rich repeat

8.82503

1556209_at 7 up CLEC2B C-type lectin domain family 2

8.70770

203868_s_at 1 up VCAM 1 vascular cell adhesion molecule 1

8.48030

235301_at 7 up KIAA1324L KIAA1324-like

8.29004

205433_at 5 up BCHE butyrylcholinesterase

7.80574

206030_at 3 up ASPA aspartoacylase (Canavan disease)

7.68808

232523_at 9 up MEGF10 multiple EGF-like-domains 10

7.6571 1

200606_at 4 up DSP desmoplakin

7.56028

205568_at 8 up AQP9 aquaporin 9

7.39572

235666_at 9 up ITGA8 integrin

7.36267

212338_at 7 up MY01 D myosin ID

7.32884

228486 at 7 up SLC44A1 solute carrier family 44

1552398 a a 7.29815

t 4 up CLEC12A C-type lectin domain family 12

7.28304

209160_at 6 up AKR1 C3 aldo-keto reductase family 1

7.15270

1554062_at 1 up XG Xg blood group

219825_at 7.09656 up CYP26B1 cytochrome P450

7.09442

209732 at 8 up CLEC2B C-type lectin domain family 2

1556579_s_a 7.07023

t 2 up IGSF10 immunoglobulin superfamily

6.88210

206201_s_at 9 up MEOX2 mesenchyme homeobox 2

227461_at 6.82668 up STON2 stonin 2 8

6.75202

231906_at 9 up HOXD8 Homeobox D8

6.31728

210261_at 9 up KCN K2 potassium channel

6.24789

213994_s_at 1 up SP0N1 spondin 1

6.1071 1

206932_at 8_l _CN up CH25H cholesterol 25-hydroxylase

6.09470

2101 19_at 1 up KCNJ15 potassium inwardly-rectifying channel

6.02643

229127_at 7 up CDNA FLJ31517 fis

6.01353

206796_at 7 up WISP1 WNT1 inducible signaling pathway protein 1

5.94189

222860_s_at up PDGFD platelet derived growth factor D

5.91517 fibroblast growth factor receptor 2 (bacteria-

203638_s_at 5 up FGFR2 expressed kinase

5.86298

213415_at 4 up CLIC2 chloride intracellular channel 2

5.76012

207977_s_at 8 up DPT dermatopontin

5.74545

213993_at 5 up SP0N1 spondin 1

5.72012

244317_at 5 up KIAA1324L KIAA1324-like

5.02563

212187_x_at 1 up PTGDS prostaglandin D2 synthase 21 kDa (brain)

4.89466 dow

207155_at 8 n TBX5 T-box 5

219213_at 4.87695 up JAM2 junctional adhesion molecule 2

4.87272

214587_at 7 up COL8A1 collagen

4.85439 dow

222835_at 2 n THSD4 thrombospondin

4.84997

205932_s_at 4 up MSX1 msh homeobox 1

4.82746

205975_s_at 9 up HOXD1 homeobox D1

4.79362

204456_s_at 5 up GAS1 growth arrest-specific 1

4.76165

228262_at 2 up MAP7D2 MAP7 domain containing 2

206693_at 4.67829 up IL7 interleukin 7

4.24864

222450_at 1 up TMEPAI transmembrane

3.88967

206307_s_at 4 up FOXD1 forkhead box D1

44.0215 dow

206377_at 6 n F0XF2 forkhead box F2

67.0483 dow

206163 at 3 n MAB21 L1 mab-21-like 1 (C. eleqans) dow

212224_at 63.5042 n ALDH1A1 aldehyde dehydrogenase 1 family

44.0215 dow

206377_at 6 n F0XF2 forkhead box F2

dow

204584_at 24.71 18 n L1 CAM L1 cell adhesion molecule

18.5967 dow

221933_at 1 n 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 min at -2O⁰C. 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-alphaSMA 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 (λex=490nm and λem=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.

Keratinocvte 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/ml penicillin and 100 μg/ml streptomycin (Sigma) and 250 μg/ml 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 Epilife™ (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, DP 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 μg/mL hydrocortisone (Sigma), 10^~10 mol/L cholera toxin (Source), 1 .8^χ10^~4 mol/L adenine (Source), 5 μg/mL insulin (Sigma), 5 μg/mL transferrin (Source), 2 x 1 0^~3 mol/L glutamine (Sigma), 2 * 1 0^~7 mol/L triiodothyrionine, 0.625 μg/mL amphotericin B (Sigma), 100 IU/mL penicillin and 100 μg/mL streptomycin (Sigma).

Epidermal keratinocytes specifically stain with rhodamine B and the extent of staining can be quantified as previously described by Castro-Muήozledo (1997). Briefly, dermal cells (20,000 per 35 mm dish) were incubated for 3.5 hours with 8 μg/ml 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 ⁰C 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 DP 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 SKPS were generated from DF cells at early passage numbers (2 and 3) and late passage numbers (1 1 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% methycellulose (Sigma) to the SKP proliferation media showed no difference in SKP forming capacity.

Routine SKP formation from passaged 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 μm to 147.6 μm 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

1. A fibroblast cell isolated from hair bearing skin tissue for use as a medicament.

2. A fibroblast cell isolated from hair bearing skin tissue for use in skin wound healing.

3. A fibroblast according to claim 1 or claim 2, wherein said fibroblast is a dermal fibroblast.

4. The fibroblast according to claim 3, wherein said fibroblast is prepared for

administration with one or more epidermal cells.

5. The fibroblast according to claim 4, wherein said one or more epidermal cells are keratinocytes.

6. The fibroblast according to any one of claims 2 to 5, wherein said fibroblast cell is associated with a matrix.

7. The fibroblast according to claim 6, wherein said matrix is a collagen matrix.

8. The fibroblast according to any one of claims 2 to 7, wherein said fibroblast is prepared for topical administration.

9. A method of healing a skin wound comprising applying one or more fibroblast cells isolated from hair bearing skin tissue to the skin wound.

10. The method according to claim 9, wherein said fibroblast is a dermal fibroblast.

1 1. The method according to claim 10, wherein said one or more fibroblast cells is applied to a wound bed of the skin wound.

12. The method according to claim 10 or claim 1 1 , further comprising applying one or more epidermal cells to the skin wound or to the wound bed of the skin wound.

13. A wound healing composition comprising one or more fibroblast cells isolated from hair bearing skin tissue and a pharmaceutically acceptable carrier.

14. The wound healing composition according to claim 13 further comprising one or more epithelial cells for simultaneous, separate or sequential administration.

15. A wound healing composition consisting of one or more fibroblast cells isolated from hair bearing skin tissue and a pharmaceutically acceptable carrier.

16. A wound healing composition consisting of one or more fibroblast cells isolated from hair bearing skin tissue, a pharmaceutically acceptable carrier and one or more epithelial cells for simultaneous, separate or sequential administration.

17. The wound healing composition according to claim 14 or claim 16, wherein said one or more epidermal cells are keratinocytes.

18. The wound healing composition according to any one of claims 13 to 17, wherein said fibroblast is a dermal fibroblast.

19. The wound healing composition according to any one of claims 13 to 18 prepared for topical administration.

20. A wound dressing comprising a fibroblast according to any one of claims 2 to 8 or a composition according to any one of claims 13 to 19.

21. The fibroblast according to any one of claims 1 to 8, the method according to any one of claims 9 to 12, the composition according to any one of claims 13 to 19 or the wound dressing according to claim 20, wherein said fibroblast cell isolated from hair bearing skin tissue is 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, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7.

22. The fibroblast, the method, the composition or the wound dressing according to claim 21 , wherein said fibroblast cell isolated from hair bearing skin tissue is 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:

23. The fibroblast according to any one of claims 1 to 8, the method according to any one of claims 9 to 12, the composition according to any one of claims 13 to 19 or the wound dressing according to claim 20, wherein said fibroblast cell isolated from hair bearing skin tissue is 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.

24. The fibroblast, the method, the composition or the wound dressing according to any one of the preceding claims, wherein said hair bearing skin bears actively growing hair follicles.

25. The fibroblast, the method, the composition or the wound dressing 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.

26. A cell support comprising a fibroblast cell feeder layer, characterized in that said fibroblast cells are isolated from hair bearing skin tissue.

27. The support according to claim 26, further comprising an epidermal cell culture.

28. The support according to claim 27, wherein said epidermal cell culture is a keratinocyte cell culture.

29. The support according to claim 27, wherein said epidermal cell culture is an epidermal progenitor.

30. The support according to any one of claims 26 to 29, 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 ,

ITGA1 1 , POSTN, ISLR, DSP, ITGA8, MY01 D, KCNK2, CH25H, KCNJ15, CLIC2, DPT, TMEPAI, FOXD1 , LGR5, SPON1 , HAPLN1 , THBS4, CYP26B1 , WISP1 and IL7.

31. The support according to claim 30, 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:

32. The support according to any one of claims 26 to 31 , 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.

33. The support according to any one of claim 26 to 32, wherein said hair bearing skin bears actively growing hair follicles.

34. The support according to any one of claims 26 to 33, wherein said hear bearing tissue is selected from the group consisting of: scalp tissue, face tissue, pubic tissue.

35. The support according to any one of claims 26 to 34, wherein said culture support further comprises a feeder layer support.

36. The support according to claim 35, wherein said feeder layer support is a matrix, dish, a well, a flask or a plate.

37. The support according to claim 36, wherein said matrix is a collagen matrix.

38. 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.

39. The method according to claim 38, wherein said one or more fibroblast cells are provided as a fibroblast feeder layer.

40. The method according to claim 38 or claim 39, 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:

41. The method according to claim 40, 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:

42. The method according to any one of claims 38 to 41 , 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.

43. The method according to any one of claims 38 to 42, wherein said co-culture is in a serum free medium.

44. The method according to any one of claims 38 to 43, wherein said epidermal cell is a keratinocyte.

45. The method according to any one of claims 38 to 43, wherein said epidermal cell is an epidermal progenitor cell.

46. The epidermal culture support according to claim 45, wherein said epidermal progenitor is a keratinocyte progenitor.

47. The method according to any one of claims 38 to 46, wherein said epidermal cells are human epidermal cells.

48. The method according to any one of claims 38 to 47, wherein said fibroblast cells are human fibroblasts.

49. A co-culture vessel comprising a cell support according to any of claims 26 to 37 and one or more epidermal cells.

50. A co-culture vessel according to claim 49, wherein said one or more epidermal cells is a keratinocyte.

51. A co-culture vessel according to claim 49, wherein said one or more epidermal cells is an epidermal progenitor cell.

52. A co-culture vessel according to claim 51 , wherein said one or more epidermal progenitor cell is a keratinocyte progenitor cell.

53. A co-culture vessel according to any one of claim 49 to 52, wherein said vessel further comprises a serum free medium.

54. 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.

55. A culture support substantially as described herein with reference to the accompanying drawings.

56. A method of epidermal cell culture as described herein with reference to the

accompanying drawings.

57. A co-culture vessel as described herein with reference to the accompanying drawings.

58. A method of healing a skin wound as described herein with reference to the accompanying drawings.

59. A wound healing composition as described herein with reference to the accompanying drawings.

60. A wound dressing as described herein with reference to the accompanying drawings.