WO2018078031A1

WO2018078031A1 - Methods of derivation and/or propagation of epithelial cells

Info

Publication number: WO2018078031A1
Application number: PCT/EP2017/077479
Authority: WO
Inventors: Andrea ZAFFALON; Yann Barrandon
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2016-10-28
Filing date: 2017-10-26
Publication date: 2018-05-03

Abstract

The present invention relates to methods of derivation and/or propagation of epithelial cells from tissue comprising culturing said tissue in media containing an effective amount of a proprotein convertase inhibitor for a period of time allowing the derivation and/or propagation of epithelial cells.

Description

Methods of derivation and/or propagation of epithelial cells

FIELD OF THE INVENTION The present invention relates to methods of derivation and/or propagation of epithelial cells from tissue comprising culturing said tissue in media containing an effective amount of a proprotein convertase inhibitor for a period of time allowing the derivation and/or propagation of epithelial cells.

BACKGROUND OF THE INVENTION

Today, there is a need in regenerative medicine to improve the therapies for epithelial cell associated diseases and injuries. The use of these genes products or small molecules, independently or in combination, will improve the process of ex-vivo amplification for autologous and homologous cell based therapies. It will also be of interest for the development of therapeutic agents to enhance healing of epithelia such as the skin, cornea, oral and nasal mucosa etc.

There is also a need for the development of robust in vitro systems to study normal epithelial cells (ex: skin keratinocytes, corneal epithelial cells etc.). The current state of the art for the culture of human epithelial stem cells (skin, cornea and other stratified epithelia) involves the use of feeder cells (3T3 cells). Those cells were derived from mouse embryo in the laboratory of Howard Green (Todaro and Green, 1963). When cell cycle arrested (by irradiation or mitomycin c treatment), feeder cells can support the growth of human epithelial cells and allow to produce enough autologous material to treat large burn wounds (Gallico et al., 1984; Rheinwald and Green, 1975). However, the regulatory authorities constantly push towards the development of alternatives to get rid of the "xeno" component of the system (Feeder cells and serum). BRIEF DESCRIPTION OF THE FIGURES Figure 1 represents 3T3-J2 feeder cells (MEFs) and the Cultured Epidermal

Autografts (CEA) procedure.

Figure 2 depicts the screening procedure.

Figure 3 shows the effects produced by inhibition of FURIN with a small molecule (Proprotease convertase inhibitor). FURIN inhibition reduces the signal of the rhodamine B fluorescence assay.

Figure 4 shows the results of the QPCR analysis of the expression of DNp63, HOPX, LEKTI, IVL and KRT1. It also shows the results of the quantification of KI67 positive cells by immunofluorescence analysis. DESCRIPTION OF THE INVENTION

To identify the genes involved in the cross-talk between 3T3-J2 cells and human keratinocytes stem cells, the Inventors decided to screen the 3T3 cells with the mouse druggable genome siRNA library (QIAGEN). The library covered only 8'320 genes from the mouse genome. To validate the results of the primary screen, a secondary screen for the same 126 genes was performed. Other genes expected to have an effect in the culture system (Table 1 ) were also included. For the secondary screen, both individual ("deconvolved"), and pools of siRNAs were tested in duplicates for each targeted gene. The secondary screen confirmed the effect for 70 genes (53 %) (Table 2).

Table 1

Cdk2 12566

Ces3b 13909

Clec2d 93694

Csnklal 93687

Ctdsp2 52468

Dctnl 13191

Ddx39 68278

Ddx54 71990

Ddx58 230073

DII3 13389

DII4 54485

Dmbtl 12945

Dokl 13448

Dok2 13449

Dok5 76829

Efnal 13636

Egf 13645

Eglnl 112405

Ehd4 98878

Eif3c 56347

Emd 13726

Epb4.2 13828

Ercc2 13871

Ewsrl 14030

Exosc8 69639

Eya3 14050

Ezh2 14056

F10 14058

Fbxo31 76454

Fbxo34 78938

Fbxwll 103583

Fcrl5 329693

Fgf7 14178

Fhl5 57756

Fkbpla 14225

Fkbp8 14232

Flcn 216805

Ftmt 67634

Furin 18550

Fut8 53618

Fzd3 14365

GaBstl 53897

Gcdh 270076

Ggal 106039 Gjb2 14619

Gm5867 545756

Gnb5 14697

Gne 50798

Gpd2 14571

Gpnmb 93695

Gpx7 67305

Grial 14799

Gsn 227753

Hs3stl 15476

Htr4 15562

Htr6 15565

II If 9 215257

1120 58181

Il21r 60504

1125 140806

Itgav 16410

Kcnj9 16524

Lifr 16880

Lmo2 16909

Mapkl5 332110

Mapkapk2 17164

Mknkl 17346

Mllt6 246198

Mmplb 83996

Necab2 117148

Nefh 380684

Nfkbie 18037

Nrlh2 22260

Nsmf 56876

Olfrl301 258889

Olfr32 18331

Olfr684 244187

Olfr731 258360

Olfr888 258416

Olfr922 258777

Olfr95 258506

Pabpnl 54196

Papln 170721

Pccb 66904

Pdlim7 67399

Pfnl 18643

Pgam2 56012

Pkia 18767 Pkn3 263803

Pla2g2d 18782

Pld3 18807

Pole 18973

Porcn 53627

Ppan 235036

Ppil2 66053

Prss22 70835

Prss48 368202

Prtn3 19152

Psma6 26443

Psmb4 19172

Psmcl 19179

Psmc2 19181

Psmc5 19184

Psmd2 21762

Ptk2 14083 pl35 66489

Rpl38 67671

Rpl4 67891

Rrml 20133

Rrm2 20135

Sars 20226

Smc3 13006

Snrpdl 20641

Stat4 20849

Styxll 76571

Tbck 271981

Tgfbl 21803

Tsen2 381802

Tssk2 22115

Vmnlr45 22297

Wdr92 103784

Wnk4 69847

Wnt3 22415

Xab2 67439

Xpol 103573

Xpo7 65246 Table 2

Then, a functional annotation analysis with the DAVID bioinfornnatics resources (Huang et al., 2009) on the list of putative hits has been performed. Only 3 pathways were enriched (Table 3). Most of genes identified in the secondary screen are involved in different cellular processes. To sort the putative hits in a comprehensive list, the subcellular annotations from GeneCards® (http://www.genecards.org/) were retrieved. This allowed to identify putative "feeder" genes (transmembrane receptors, growth factors, transmembrane ligands and other signaling molecules that influence epithelial stem cells growth in vitro). Several genes associated with epidermal homeostasis and wound repair are represented. For example, IL20 is a cytokine that is upregulated in psoriasis (a disease characterized by a hyper proliferative epidermis) (Ouyang et al., 201 1 ). MMP1 , a collagenase, is also upregulated in wounded skin and facilitates the migration of keratinocytes (Rohani et al., 2014). FURIN is also known to play a key role during wound repair (Gurtner et al., 2008). Table 3

In view of these findings, the present invention thus relates to methods of derivation and/or propagation of epithelial cells from tissue comprising culturing said tissue in media containing an effective amount of a modulator of i) one or more genes involved in epithelial cells propagation and/or differentiation, or of a ii) product of said genes for a period of time allowing the derivation and/or propagation of epithelial cells. Preferably, the one or more genes involved in epithelial cells propagation and/or differentiation will be selected from the group of gene listed in Table 1 . Most preferably the one or more genes involved in epithelial cells

propagation and/or differentiation will be selected from the group comprising DII3, DII4, Dmbtl , Efnal , Furin, Gpnmb, II20, Itgav, Lifr, Mapk15, Mmpl b, Papln and Smc3, or from a combination of one of more of these genes.

Generally, the modulator will be selected from the group comprising a chemical agent, an antibody, an engineered protease, and enzymatically active RNA. The modulator can either activate or silence the genes or inhibit or activate the product of said genes.

Most preferably, the enzymatically active RNA is selected from the group comprising a miRNA, a siRNA, a piRNA, a hnRNA, a snRNA, esiRNA, shRNA, decoys, RNA aptamers and an antisense oligonucleotide. One will appreciate that any compound with different formulations capable to inhibit or activate one or more physiological actions effected by a gene involved in epithelial cells propagation and/or

differentiation is encompassed by the present invention. Also provided is a cell culture media containing an effective amount of a modulator of i) one or more genes involved in epithelial cells propagation and/or differentiation, or of a ii) product of said genes for derivation and/or propagation of epithelial cells.

The present invention also provides a pharmaceutical composition comprising a modulator of i) one or more genes involved in epithelial cells propagation and/or differentiation, or of a ii) product of said genes for use in the treatment of epithelial cell associated diseases, disorders and injuries. Preferably, the pharmaceutical composition comprises a pharmaceutically effective amount of the active compound, i.e. a modulator of i) one or more genes involved in epithelial cells propagation and/or differentiation, or of a ii) product of said genes, optionally with one or more pharmaceutically acceptable carriers, diluents and adjuvants. The present invention also provides a pharmaceutical composition comprising a modulator of the invention for use in the treatment of epithelial cell associated diseases, disorders and injuries. Preferably, the pharmaceutical composition comprises a pharmaceutically effective amount of the active compound, i.e. a modulator of the invention, optionally with one or more pharmaceutically acceptable carriers, diluents and adjuvants.

Also provided herein is a kit comprising i) a pharmaceutical composition comprising a therapeutically effective amount of a modulator of the invention and/or a ii) a therapeutically effective amount of epithelial cells obtained according to the method of derivation and/or propagation of epithelial cells as described herein, pharmaceutically acceptable salts, solvates or esters thereof, for the treatment of epithelial cell associated diseases, disorders and injuries.

Among the hits identified as target genes that showed a significant impact on human keratinocyte's proliferation when silenced, the inventors identified the gene Furin. When an inhibitor of the Furin gene product was added to the culture medium, the inventors surprisingly observed an increase in keratinocyte proliferation and an inhibition of keratinocyte differentiation. FURIN belongs to the proprotein convertase family that is a family of enzymes catalysing the proteolytic maturation of prohormones and proproteins (G. Thomas, 2002; Seidah and Prat, 2012). To date, the proprotein convertase family comprises FURIN, PC1/3, PC2, PC4, PACE4, PC5/PC6, PC7/LPC/PC8, SKI/S1 P and PCSK9.

In one instance, the present invention also concerns a method of derivation and/or propagation of epithelial cells from tissue comprising culturing said tissue in media containing an effective amount of a proprotein convertase inhibitor for a period of time allowing the derivation and/or propagation of epithelial cells.

The tissue can be cultured in the presence of fibroblast feeder cells. Feeder cells support the growth of epithelial and epithelial stem cells by producing growth factors and providing adhesion molecules and ECM components for cell

attachment. Preferably, the fibroblast feeder cells are selected from the group comprising mouse embryonic and human fibroblasts, either primary cells or immortalized cells (such as 3T3 cells), or a combination thereof. Human fibroblast feeder cells are usually derived from several human cell types, such as human foreskin fibroblasts (hFFs) or adult Fallopian tube epithelial cells. Not wishing to be bound to any theory, it is believed that the use of such feeder cells, or conditioned media derived from such feeder cells, provides one or more substances necessary to promote the growth of the epithelial and epithelial stem cells and/or prevent or decrease the rate of differentiation of such cells. Such substances are believed to include membrane-bound and/or soluble cell products that are secreted into the surrounding medium by the cells. In addition, those of skill will also recognize that one or more substances produced by the feeder cells, or contained in the

conditioned media, can be identified and added to the cell culture media of the invention to obviate the need for such feeder cells and/or such conditioned media.

Usually, the epithelial cells comprise epithelial stem cells. Preferably, the method of the invention improves the number of proliferative epithelial stem cells, preferably by inhibiting their terminal differentiation.

The proprotein convertase inhibitor is selected from the group comprising a FURIN inhibitor, a PC1/3 inhibitor, a PC2 inhibitor, a PC4 inhibitor, a PACE4 inhibitor, a PC5/PC6 inhibitor, a PC7/LPC/PC8 inhibitor, an SKI/S1 P inhibitor and a PCSK9 inhibitor, or a combination of one or more of these inhibitors. Preferably, the proprotein convertase inhibitor is selected from the group comprising a FURIN inhibitor, a PC4 inhibitor, a PACE 4 inhibitor, a PC5/6 inhibitor, and a PC1/3 inhibitor. More preferably, the proprotein convertase inhibitor is a FURIN inhibitor or a combination of one or more of these inhibitors.

When the proprotein convertase inhibitor is a FURIN inhibitor, the effective amount of the FURIN inhibitor is about 1 to about 100 μΜ, or about 10 to about 75 μΜ, or about 50 μΜ.

PCT applications No. WO 2009/023306, WO2013029182 and WO2013138665 A1 , as well Becker et al., 2012 in Journal of Biological Chemistry, which are hereby incorporated by reference in their entirety, disclose FURIN inhibitors.

The proprotein convertase inhibitor can also be selected among the group

comprising Hexa-D-arginine (potent inhibitor of FURIN, PACE4 and PC1 ) and Decanoyl-Arg-Val-Lys-Arg-CMK (potent inhibitor of FURIN, SPC3/PC1/PC3, PACE4, SPC6/PC5/PC6 and SPC7/LPC/PC7/PC8).

The tissue is usually selected among the group comprising a stratified epithelium comprising keratinocytes and a pseudostratified epithelium (simple epithelium) comprising keratinocytes. Preferably, the stratified epithelium is selected from the group comprising cornea, skin, esophagus, oral mucosa, conjunctiva, vagina, and cervix (ectocervix) whereas the pseudo-stratified or simple epithelium is selected from the group comprising trachea, lung stomach, intestine, pancreas, thymus and bladder.

The keratinocytes can be any type of preferably primary keratinocyte. In some examples, the primary keratinocyte is a foreskin keratinocyte, vaginal keratinocyte or cervical keratinocyte. In some embodiments, the primary keratinocytes are obtained by a tissue biopsy. In some examples, the tissue biopsy is taken from the skin (e.g., the cutaneous and/or mucosal squamous epithelium). The stratified or pseudostratified epithelium comprising keratinocytes are cultured in the presence of a proprotein convertase inhibitor for any suitable period of time to allow for derivation and/or propagation of epithelial cells. In some aspects, the keratinocytes are cultured in the presence of a proprotein convertase inhibitor for at least 15 days, at least 20 days, at least 40 days, at least 60 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, at least 350 days, at least 400 days, at least 450 days, or at least 500 days.

Usually, the epithelial cells that have been derived and/or propagated from any of the above-mentioned tissue are recovered using techniques known in the art.

Also provided is a cell culture media containing an effective amount of a proprotein convertase inhibitor for derivation and/or propagation of epithelial cells. Any culture media suitable for derivation and/or propagation of epithelial cells from tissue in the presence of fibroblast feeder cells or not, envisioned by a person skilled in the art is encompassed.

In case of derivation and/or propagation of epithelial cells from Human keratinocytes, the cells were usually propagated on top of irradiated 3T3-J2 cells with cFAD culture medium, a 3:1 ratio of DMEM and Ham's F12 culture medium, supplemented with insulin (5 g/mL), Triiodothyronine (2 x 10^"9 M, Sigma), hydrocortisone (0.4 pg/mL, Calbiochem) and cholera toxin (10^"1° M, ICN). Cells were incubated in 10% CO2 humid atmosphere at 37°C.

The present invention also provides a pharmaceutical composition comprising a proprotein convertase inhibitor for use in the treatment of epithelial cell associated diseases, disorders and injuries. Preferably, the pharmaceutical composition comprises a pharmaceutically effective amount of the active compound, i.e.

proprotein convertase inhibitor, optionally with one or more pharmaceutically acceptable carriers, diluents and adjuvants. "A pharmaceutically effective amount" refers to a chemical material or compound which, when administered to a human or animal organism induces a detectable pharmacologic and/or physiologic effect. The respective pharmaceutically effect amount can depend on the specific patient to be treated, on the disease, disorder or injury to be treated and on the method of administration. Further, the pharmaceutically effective amount depends on the specific peptide used. The treatment usually comprises a multiple administration of the pharmaceutical composition, usually in intervals of several hours, days or weeks. The pharmaceutically effective amount of a dosage unit of the peptide of the invention usually is in the range of 0.1 ng to 100 mg per kg of body weight of the patient to be treated. However, it is understood that the suitable dosage of the pharmaceutical composition of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.

Acceptable carriers, diluents and adjuvants which facilitates processing of the active compound into preparation which can be used pharmaceutically are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine;

monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as

TWEEN®, PLURONICS® or polyethylene glycol (PEG). The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules.

The form of administration of the pharmaceutical composition may be systemic or topical. For example, administration of such a composition may be various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular,

intraperitoneal, intranasal, transdermal, buccal routes or via an implanted device, and may also be delivered by peristaltic means. The pharmaceutical composition comprising a pharmaceutically effective amount of the proprotein convertase inhibitor, as described herein, as an active compound may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a gel or a solid support. In addition the matrix may be comprised of a biopolymer.

Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi permeable matrices of solid hydrophobic polymers containing the proprotein convertase inhibitor, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished for example by filtration through sterile filtration membranes. One aspect of the present invention also concerns a method of treating an epithelial cell associated disease, disorder or injury in a patient in need thereof characterized in that it comprises

(i) obtaining a tissue from said patient in need thereof, or from an individual, (ii) subjecting the tissue to the method of derivation and/or propagation of epithelial cells as described herein,

(iii) recovering the epithelial cells derived and/or propagated from the tissue, and

(iv) contacting said epithelial cells with the patient in need thereof.

Alternatively or additionally, the method of treating an epithelial cell associated disease, disorder or injury may comprise a step of administering a therapeutically effective amount of proprotein convertase inhibitor, after or concomitantly to contacting the epithelial cells with the patient in need thereof.

Preferably, the epithelial cell associated disease, disorder or injury is selected among the non-limiting group comprising a wound such a burn or ulcer, bed sores, a skin disease, chronic wound, lacerations, abrasions, contusions, skin cuts, surgical wounds, chemical and/or thermal wounds, bites, stings and corneal wound.

In some aspects, the method comprises treating the patient in need thereof with an organotypic tissue equivalent prepared according to the methods disclosed herein. The primary keratinocytes are then obtained by a tissue biopsy of the patient to be treated with the organotypic tissue equivalent (thus, the organotypic tissue equivalent is an autograft).

Further provided is the use of a pharmaceutical composition of the invention in the preparation of a medicament for the treatment of an epithelial cell associated disease, disorder or injury in a patient in need thereof.

Also provided herein is a kit comprising i) a pharmaceutical composition comprising a therapeutically effective amount of proprotein convertase inhibitor and/or a ii) a therapeutically effective amount of epithelial cells obtained according to the method of derivation and/or propagation of epithelial cells as described herein, pharmaceutically acceptable salts, solvates or esters thereof, for the treatment of epithelial cell associated diseases, disorders and injuries. EXAMPLES

Material & Methods Culture of 3T3-J2 (Feeder cells)

3T3-J2 cells were propagated in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 8% Bovine Serum (BS, Thermo Scientific) and incubated in 10% CO2 atmosphere at 37°C. For serial amplification, 3T3 cells were inoculated every 7 days at low density (1 to 5 x 105 cells per 162 cm2 flasks. The culture medium was replaced every 3 to 4 days. For keratinocytes propagation, 3T3- J2 cells were irradiated with a dose of 60 Gy and then plated at a density of 2.5 x 104 cells/cm2.

Culture of Human keratinocytes (Identical protocol for all epithelial cells derived from stratified, pseudostratified or simple epithelia, ex: cornea, skin, esophagus, oral mucosa, conjunctiva, prostate, bladder, vagina, thymus)

Human keratinocytes were propagated on top of the irradiated 3T3-J2 cells with cFAD culture medium, a 3:1 ratio of DMEM and Ham's F12 culture medium (Amimed), supplemented with insulin (5 pg/mL, Sigma), Triiodothyronine (2 x 10^"9 M, Sigma), hydrocortisone (0.4 pg/mL, Calbiochem) and cholera toxin (10^~10 M, ICN). Cells were incubated in 10% CO2 humid atmosphere at 37°C. For serial amplification, pre- confluent keratinocytes were trypsinized (0.05% trypsin and 0.1 % EDTA) and seeded at appropriate density once a week. The culture medium was changed every 2 to 3 days for mass culture or every 4 days for colony forming efficiency. EGF was added at each feeding (10 ng/mL, Upstate Biotechnology Inc.). For FURIN inhibition experiments, the Proprotein Convertase Inhibitor (50 μΜ, Calbiochem) was added daily in cFAD medium.

High throughput screening of 3T3-J2 (Discovery strategy) The mouse druggable genome siRNA library was first pooled (4 siRNAs per gene) by the Biomolecular Screening Facility (BSF) and then spotted on 96 well plates (BD Falcon) in duplicates. Briefly, 10 μί of pooled siRNAs (QIAGEN) were spotted (40 nM final concentration) with 0.4 μΙ_ of RiboCellln transfection reagent (BioCellChallenge) and 10 μΙ_ of OptiMem (Life Technologies) with the Caliper Sciclone platform (PerkinElmer). The first and last columns were spotted with scramble siRNA (negative control) and mouse specific cell death cocktail siRNAs (positive control) (QIAGEN) respectively. After 15 min incubation at RT, 10Ό00 irradiated 3T3-J2 cells (in 80 μΙ_ of DME/BS) were seeded in each well using a Multiflow liquid dispenser (Biotek). Assay plates were then incubated overnight in 10% CO2 atmosphere at 37°C. The next day, the medium was removed using an ELx405 (Biotek) and 600 normal human epidermal cells (strain YF29) in 100 μΙ_ of cFAD were dispensed in each well with the Multiflow. The plates were then incubated for one week in 10% CO2 atmosphere at 37°C with one medium change (cFAD without EGF) on the third day after seeding using the ELx405 and the Multiflow (Biotek). After 7 days of culture, the plates were fixed for 15 min with 3.4% formaldehyde (Sigma-Aldrich) and stained for 10 min with 3% rhodamine B (Sigma). Next, the plates were washed 9 times with PBS using the ELx406 (Biotek) and the remaining rhodamine B was then re-suspended in 100 μΙ_ of PBS by 20 min incubation at RT on an orbital shaker at 90 rpm. Finally, the total fluorescence of the rhodamine B was measured using an Infinite F500 plate reader (Tecan). A Z' was computed to assess the assay's quality both during the assay development and the screening campaign (Zhang et al., 1999).

Immunocytochemistry

Cell samples were fixed with 4% paraformaldehyde (PFA) for 15 min. The samples were washed three times with PBS and permeabilized with 0.3% Triton X-100 (Sigma) in PBS for 10min. After another three consecutives washes with PBS, the samples were blocked with 2% bovine serum albumin (BSA) in PBS for 45 min and then incubated overnight at 4°C with primary antibody diluted in blocking solution. After three washes with PBS, the samples were incubated with corresponding conjugated secondary antibodies diluted in PBS for 45 min at RT. DNA was counterstained with DAPI for 10 min. Coverslips were washed twice with PBS and mounted on microscope slides. REFERENCES

Becker et al., 2012. Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. J Biol Chem. 2012 Jun 22;287(26):21992-2003

Gallico, G.G., O'Connor, N.E., Compton, C.C., Kehinde, O., and Green, H. (1984). Permanent coverage of large burn wounds with autologous cultured human epithelium. N. Engl. J. Med. 311, 448-451.

Gordon, K., Kochkodan, J.J., Blatt, H., Lin, S.Y., Kaplan, N., Johnston, A., Swindell, W.R., Hoover, P., Schlosser, B.J., Elder, J.T., et al. (2013). Alteration of the EphA2/Ephrin-A signaling axis in psoriatic epidermis. J. Invest. Dermatol. 133, 712-722.

Gurtner, G.C., Werner, S., Barrandon, Y., and Longaker, M.T. (2008). Wound repair and regeneration. Nature 453, 314-321.

Huang, D.W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44-57.

Muller, H., Hu, J., Popp, R., Schmidt, M.H.H., Muller-decker, K., Fisslthaler, B., Eble, J.A., Fleming, I.,

Mu, H., and Mollenhauer, J. (2012). Deleted in malignant brain tumors 1 is present in the vascular extracellular matrix and promotes angiogenesis. Arterioscler. Thromb. Vase. Biol. 32, 442-448.

Ouyang, W., Rutz, S., Crellin, N.K., Valdez, P. a, and Hymowitz, S.G. (2011). Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annu. Rev. Immunol. 29, 71-109.

Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975)

Rheinwald, J.G., and Green, H. (1975). Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331-343.

Rohani, M.G., Pilcher, B.K., Chen, P., and Parks, W.C. (2014). Cdc42 inhibits ERK-mediated collagenase-1 (MMP-1) expression in collagen-activated human keratinocytes. J. Invest. Dermatol. 134, 1230-1237.

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Claims

1 . A method of derivation and/or propagation of epithelial cells from tissue comprising culturing said tissue in media containing an effective amount of a proprotein convertase inhibitor for a period of time allowing the derivation and/or propagation of epithelial cells.

2. The method of claim 1 characterized in that the tissue is culture in the presence of fibroblast feeder cells.

3. The method of claim 1 or 2 characterized in that the epithelial cells comprise epithelial stem cells.

4. The method of anyone of the preceding claims characterized in that the number of proliferative epithelial cells is improved.

5. The method of anyone of the preceding claims characterized in that the terminal differentiation of the epithelial cells is inhibited.

6. The method of anyone of the preceding claims characterized in that the proprotein convertase inhibitor is selected from the group comprising a FURIN inhibitor, a PC4 inhibitor, a PACE 4 inhibitor, a PC5/6 inhibitor, and a PC1 /3 inhibitor.

7. The method of anyone of the preceding claims characterized in that the tissue is a stratified epithelium comprising keratinocytes, a pseudostratified epithelium or a simple epithelium.

8. The method of claim 7 characterized in that the stratified epithelium is selected from the group comprising cornea, skin, esophagus, oral mucosa, conjunctiva, vagina, and cervix (ectocervix).

9. The method of anyone of the preceding claims characterized in that the epithelial cells are recovered.

10. A cell culture media containing an effective amount of a proprotein convertase inhibitor for derivation and/or propagation of epithelial cells.

1 1 . A proprotein convertase inhibitor for use in the treatment of epithelial cell associated diseases, disorders and injuries.

12. A method of treating an epithelial cell associated disease, disorder or injury in a patient in need thereof characterized in that it comprises

(i) obtaining a tissue from said patient in need thereof, or from an individual,

(ii) subjecting the tissue to the method of derivation and/or propagation of epithelial cells according to anyone of claims 1 to 9,

(iv) contacting said epithelial cells with the patient in need thereof.

13. The method of claim 12 characterized in that it further comprises

administering a therapeutically effective amount of proprotein convertase inhibitor, after or concomitantly to contacting the epithelial cells with the patient in need thereof.

14. A kit comprising

i) a pharmaceutical composition comprising a therapeutically effective amount of proprotein convertase inhibitor and/or a

ii) a therapeutically effective amount of epithelial cells obtained according to anyone of claims 1 to 9,

pharmaceutically acceptable salts, solvates or esters thereof, for the treatment of epithelial cell associated diseases, disorders and injuries.