WO2017060240A1 - Method and culture medium for ex vivo culturing of epidermis-derived stem cells - Google Patents

Method and culture medium for ex vivo culturing of epidermis-derived stem cells Download PDF

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WO2017060240A1
WO2017060240A1 PCT/EP2016/073675 EP2016073675W WO2017060240A1 WO 2017060240 A1 WO2017060240 A1 WO 2017060240A1 EP 2016073675 W EP2016073675 W EP 2016073675W WO 2017060240 A1 WO2017060240 A1 WO 2017060240A1
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epidermis
stem cells
cells
derived stem
fgf
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PCT/EP2016/073675
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French (fr)
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Sara Annika WICKSTRÖM
Carlos Andrés CHACÓN MARTINEZ
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to US15/765,779 priority Critical patent/US20180282690A1/en
Priority to EP16787352.0A priority patent/EP3359645A1/en
Publication of WO2017060240A1 publication Critical patent/WO2017060240A1/en

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Definitions

  • the present invention relates to a method for culturing epidermis-derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three- dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising: Epidermal Growth Factor (EGF); and/or a Vascular Endothelial Growth Factor (VEGF); and/or a Fibroblast Growth Factor (FGF); and further a Rho-kinase (ROCK) inhibitor.
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the present invention further relates to a method for ex vivo de novo generation of epidermis-derived stem cells.
  • the present invention relates to an epidermis-derived stem cell that is obtainable by a method according to the present invention.
  • the present invention further relates to a cell culture medium that is employed in the context of a method of the present invention.
  • SCs are designed to fuel tissue renewal, repair, and remodeling in mature organs. By tuning their proliferation rate to match the changing needs of their resident tissues, SCs maintain organ form and function.
  • Mammalian skin is characterized by a hair coat that maintains body temperature, homeostasis and serves a protective function.
  • SCs residing in the epidermis such as interfollicular epidermal SCs or hair follicle stem cells (HFSCs) ensure the maintenance of adult skin homeostasis and/or trigger hair regeneration.
  • both SCs residing in the epidermis and within the hair follicles (HFs) participate in the repair of the epidermis after injuries (Bianpain & Fuchs, 2014, Science 344, 1242281 ).
  • HFSCs one important class of epidermis-derived stem cells
  • the constant renewal of the hair coat occurs by renewing the lower portion of the existing HFs in cycling phases of growth (anagen), regression (catagen), and rest (telogen) (Paus & Cotsarelis, 1999, N Engl J Med 341 : 491-497).
  • the continuous self-renewal of the HF is fueled by HFSCs residing in the specific niche referred to as bulge region that locates to the base of the non- cycling part of the HF.
  • bulge region that locates to the base of the non- cycling part of the HF.
  • telogen these HFSCs exist in a quiescent, slow-cycling state.
  • CSCs epidermis-derived cancer stem cells
  • intestinal SC cultures are known as mini-gut or organoid cultures and can be established from a single Lgr5-expressing SC.
  • SCs generate intestinal crypt-like structures that resemble the functionality and cellular heterogeneity of a normal gut. Similar to tissues/organs, these organoids contain a large proportion of differentiated cells in addition to SCs and they form anatomically distinct structures such as crypt-like structures.
  • WO 2010/090513 A2 describes a method for culturing of epithelial stem cells and isolated tissue fragments comprising epithelial SCs.
  • the method described in WO 2010/090513 A2 is a method for culturing epithelial stem cells or tissue fragments comprising epithelial stem cells, wherein said stem cells or tissue fragments originate from the intestine, colon, stomach or pancreas but not from the epidermis.
  • the disclosed culturing method involves the use of an extracellular matrix and a special cell culture medium, which comprises inter alia a Bone Morphogenic Protein (BMP) inhibitor and a mitogenic growth factor.
  • BMP Bone Morphogenic Protein
  • the culturing method results in organoid structures.
  • this method is a method for in vitro generation and/or culturing of organoids that comprise stem cells and a number of differentiated cells that form distinct anatomical structures.
  • the culturing conditions are adapted to epithelial stem cells from the intestine, colon, stomach or pancreas but have not been used for culturing epidermis-derived stem cells.
  • these methods for culturing epithelial stem cells other than epidermis-derived stem cells also methods for culturing epidermis-derived SCs have been described in the art.
  • known culturing methods have certain disadvantages and/or limitations as further discussed below.
  • epidermis-derived SCs are cultured with in vitro culturing methods that have been described for culturing primary epidermal keratinocytes and also HF-derived keratinocytes (Barrandon & Green, 1987, Cell 50: 1131-1137, Blanpain et al., 2004, Cell 1 8: 635-648, Lien et al., 2014, Nat Cell Biol 16: 179-190, Ouji et al, 2015, J Invest Dermatol 135: 1598-1608, Trempus et al., 2003, J Invest Dermatol 120: 501 -51 1 , Bilousova & Roop, 2013, Methods Mol Biol, 961 : 337-350, Jensen et al., 2010, Nat Protoc 5: 898-91 1 , Lichti et al., 2008, Nat Protoc 3: 799-810, Oh et al., 2013, J Invest Dermatol 133: e14 doi:10.
  • Such methods typically involve culturing primary epidermal keratinocytes and also HF-derived keratinocytes comprising epidermis-derived stem cells on a fibroblast feeder-layer in a growth medium with low serum content, in low calcium content and in presence of growth factors, most commonly a combination of epidermal growth factor (EGF) and insulin (Shevchenko et al., 2010 J R Soc Interface 43, 229-258).
  • EGF epidermal growth factor
  • insulin Shevchenko et al., 2010 J R Soc Interface 43, 229-258
  • HFSCs that were initially FACS-purified from a freshly isolated mixture of epidermis-derived cells and afterwards grown using such culture conditions known for primary epidermal keratinocytes and also HF-derived keratinocytes comprising epidermis-derived SCs, form tight colonies known as holoclones (Barrandon & Green, 1987, Cell 50: 1131 -1 137), that contained HFSCs (Blanpain et al., 2004, Cell 1 18: 635-648, Kandyba et al., 2013, Proc Natl Acad Sci USA, 1 10: 1351-1356).
  • Such holoclones could be clonally passaged up to 10 (Blanpain et al., 2004, Cell 118: 635-648) and 20 (Kandyba et al., 2013, Proc Natl Acad Sci USA, 1 0: 1351-1356) times while preserving their typical morphology.
  • the capacity of holoclones to give rise to higher number of colonies decreases (Blanpain et al., 2004, Cell 118: 635-648), indicating that the HFSCs do not self-renew in this system and therefore the number of HFSC cannot be increased using this culture method.
  • This inability to self-renew and therefore the inability to expand cultured HFSCs are major limitations of this method.
  • Another limitation of this method is the need of employing a heterologous feeder cell layer, most commonly fibroblasts, for culturing. Such feeder cell layers are particularly disadvantageous for a number of downstream applications of cultured cells.
  • epidermal SCs from newborn mice were cultured on collagen I and fibronectin-coated 2D-surfaces using newborn fibroblast-conditioned medium, i.e. growth media preincubated with fibroblasts, as growth medium (Reiisi et al, 2010, In vitro Cell Dev. Biol. 46:54-59).
  • newborn fibroblast-conditioned medium i.e. growth media preincubated with fibroblasts, as growth medium
  • conditioned media from feeder cells.
  • Such conditioned media is similarly to the alternative heterologous feeder cells (most commonly fibroblasts) also disadvantageous as regarding down-stream analysis of cultured cells, but needed to maintain keratinocytes and epidermal SC growth.
  • HFSCs Another method for culturing epidermis-derived stem cells, in particular HFSCs involves the reprogramming multipotent keratinocytes from induced pluripotent stem cells (iPS cells) and subsequent maintenance of FACS-purified HFSCs (Bilousova & Roop, 2013, Methods Mol Biol 961 : 337-350; Lichti et al., 2008, Nat Protoc 3: 799-810; Oh et al., 2013, J Invest Dermatol 133: e14; Ouji et al, 2015, J Invest Dermatol 135: 1598-1608).
  • iPS cells induced pluripotent stem cells
  • certain committed adult cell types is reprogrammed into a iPS cell by introducing a set of transcription factors (KLF4, c-MYC, OCT4, and SOX2, also known as the core Yamanaka factorsj.
  • the iPS cells are subsequently differentiated into keratinocytes using retinoic acid and BMP-4 to induce differentiation toward a keratinocyte lineage, which is then followed by the growth of differentiated iPS cells on collagen type I- and collagen type IV-coated dishes to enrich for iPS cell-derived keratinocytes.
  • the technical problem underlying the present invention is the provision of improved means and methods for ex vivo culturing and/or expansion of epidermis- derived stem cells.
  • the present invention relates to a method for culturing epidermis-derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three- dimensional extracellular matrix (3D-ECM) and a basal cell culture medium, said cell culture medium comprising:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the present inventors could demonstrate that the above-mentioned method allows for improved culturing of epidermis-derived stem cells ex vivo while preserving an undifferentiated phenotype, self-renewing capabilities and/or multipotency.
  • epidermis-derived stem cells e.g. hair follicle stem cells
  • a basal cell culture medium comprising e.g.
  • a ROCK inhibitor referred to as "Y-E”, see Figures and Examples
  • a EGF and a ROCK inhibitor referred to as "Y”
  • KGM 2D coated cell culture plates
  • An even improved survival and/or even a expansion of epidermis-derived stem cells during culturing could, for example, be achieved by a culturing method according to the present invention that employs a 3D-ECM and a basal cell culture medium that comprises EGF, a FGF and a ROCK inhibitor (referred to as "YF”, see Figures and Examples); EGF, a VEGF and a ROCK inhibitor (referred to as "YV”, see Figures and Examples); or a FGF, a VEGF and a ROCK inhibitor (referred to as "3C-E”, see Figures and Examples).
  • the method for culturing epidermis-derived stem cells involves culturing of these cells in so-called "3D" cultures that are characterized by the presence of a three-dimensional extracellular matrix (3D-ECM) and a specially adopted cell culture medium that consists of a basal cell culture medium comprising one or more mitogenic growth factor(s) selected from the group consisting of EGF, a VEGF and a FGF; and further comprising a ROCK inhibitor.
  • the provided ECM forms a three-dimensional structure (3D-ECM) in which the cultured epidermis-derived stem cells are embedded. This is achieved by mixing the cells and the ECM components before the ECM components solidify.
  • 3D culturing is crucial for the method of culturing epidermis-derived stem cells according to the present invention.
  • 3D- ECMs generated with commercially available ECMs such as basement membrane extracts (Matrigel, BME) or gels comprising a collagen (e.g. collagen I) or 3D-ECM gels comprising a collagen (e.g. collagen I) and a laminin (e.g. laminin-332; laminin-511 ; or laminin-332 and laminin-511 )
  • 3D-ECMs generated with commercially available ECMs
  • 3D-ECMs comprising a collagen (e.g. collagen I)
  • 3D-ECM gels comprising a collagen (e.g. collagen I) and a laminin e.g. laminin-332; laminin-511 ; or laminin-332 and laminin-511
  • a basal cell culture medium for human and/or animal cells which comprises the above mentioned mitogenic growth factor(s) and a ROCK inhibitor, is particularly advantageous for culturing epidermis-derived stem cells, in particular in the presence of 3D-ECM (see Figures and Examples).
  • the method for culturing epidermis-derived stem cells preferably comprises the step of culturing epidermis-derived stem cells in the presence of a three- dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • 3C three-dimensional extracellular matrix
  • 3C conditions 3C culturing conditions
  • 3C medium refers to the cell culture medium composition of these conditions.
  • the 3C culturing conditions allow for culturing of epidermis-derived stem cells in the sense of maintenance/survival of epidermis-derived stem cells and additionally also trigger expansion/growth and/or enrichment of epidermis-derived stem cells in cell mixtures.
  • the present invention also provides for a method for expanding epidermis-derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising Epidermal Growth Factor (EGF), a Vascular Endothelial Growth Factor (VEGF), a Fibroblast Growth Factor (FGF) and a Rho-kinase (ROCK) inhibitor.
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • expansion means that the number of cells, e.g. of a specific cell type (e.g. epidermis- derived stem cells) increases during the time of in vitro culturing by mitotic cell division.
  • expansion of the cultivated cells during culturing can be unlimited.
  • expansion means to increase the number of epidermis-derived stem cells by at least 1 ,5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or 200-fold during the time of culturing.
  • Culturing in this context relates to maintaining cells in vitrolex vivo.
  • the present invention provides an improved method for culturing epidermis-derived stem cells which allows to expand epidermis-derived stem cells in vitro in an in principle unlimited manner. Also provided is a method that allows expansion of epidermis-derived stem cells. Thus, epidermis-derived stem cells can be expanded in large quantities amenable for high-throughput applications.
  • epidermis-derived SCs such as HFSCs or CSCs (originating from epidermis-derived papilloma or carcinoma) can be maintained without losing the surface proteins that are characteristic markers for their stem cell identity.
  • the cultured epidermis-derived SCs maintain their multipotency and/or self-renewing capability.
  • the epidermis- derived stem cells remain their capability of regenerating epidermal and/or hair follicle tissue (see Figures and Examples).
  • the epidermis-derived stem cells cultured by the method of the present invention can give rise to intact epidermal tissue and/or hair follicles and thus can be used, for example, in tissue engineering and or tissue transplantation.
  • HFSCs cultured with the method according to the invention can give rise to hair growth even more efficiently than freshly isolated keratinocytes comprising HFSCs (see Figures and Examples).
  • Other methods for ex vivo culturing of epidermis-derived stem cells, such as HFSCs, known in the art involve repetitive rounds of enriching for stem cells which maintained the respective stem cell surface markers, because otherwise those cells would be depleted during culturing.
  • the method for culturing epidermis-derived stem cells according to the present invention is much less time and resource consuming, i.e. it is more time- and/or resource-efficient.
  • the present invention therefore provides an improved, highly reliable and easy to handle method for culturing epidermis-derived stem cells. Also provided is a method that allows expansion of epidermis-derived stem cells.
  • a particular further advantage of the method for culturing epidermis-derived stem cells according to the present invention is that the method does not necessarily involve feeder cells and is therefore more robust, more reproducible, more easy to manipulate and more amenable to scale-up applications than the culturing methods known in the art that make use of feeder cells or conditioned medium from feeder cells.
  • Such a feeder-free culturing system is more precisely defined and can, for example, be particularly advantageous for studying basic cellular processes such as SC fate determination, receptor-ligand responses and cell signaling.
  • a particular advantage of the method for culturing epidermis-derived stem cells according to the present invention is that this method unlike other available culturing methods can not only be applied to previously purified epidermis-derived stem cells but can also easily be established from bulk keratinocytes comprising said epidermis- derived stem cells (e.g. see Figures and Examples).
  • the method provides the possibility to avoid the laborious FACS-sorting needed to purify particular epidermis- derived stem cells prior to culturing that is an essential step of most culturing methods described in the prior art.
  • the method for culturing of epidermis-derived SCs does not require any steps of FACS-purification of epidermis-derived stem cells during culturing. This is a step that some known culturing methods employ in order to avoid the depletion of epidermis-derived SCs from the culture.
  • the method according to the present invention establishes, however, a stable equilibrium between epidermis-derived SCs and non- SCs without any additional FACS purification of epidermis-derived stem cells during culturing. W
  • the method for culturing epidermis-derived stem cells according to the present invention also provides the advantage of preserving the karyotypic integrity, the multipotency and the self-renewal capability of epidermis-derived stem cells such as e.g. HFSCs, even after long-term culturing.
  • Long-term in this context means at least 360 days, at least 250 days, at least 100 days, at least 50 days, at least 20 days, at least 14 days, at least 10 days, at least 8 days, at least 5 days, at least 3 days or at least 2 days.
  • the epidermis-derived stem cells resulting from the method according to the present invention can be freeze-thawn and therefore stored during culturing for later applications.
  • a freeze-thaw cycle does not result in an evident loss of epidermis-derived stem cells and their multipotency (see Figures and Examples).
  • the increased freeze-thaw resistance is in particular of commercial relevance, as it, for example allows for shipping of the epidermis-derived stem cells.
  • a further advantage resulting from the increased freeze-thaw resistance is that it allows for long-term storage of epidermis-derived stem cells after which these cells can be re- cultured with a method according to the present invention. None of the previously reported methods for culturing epidermis-derived SCs has reportedly permitted freezing and subsequent thawing and repropagation of SCs without loss of their SC properties.
  • the present inventors identified that the method for culturing epidermis- derived stem cells according to the present invention always results in a mixture of epidermis-derived stem cells and differentiated progeny cells independent of the starting material (see Figures and Examples).
  • the cultivation will result in a mixture of cells comprising epidermis-derived stem cells and differentiated progeny cells (see Figures and Examples).
  • a stable equilibrium of stem cells and differentiated cells is reached within only 12 days and is stably maintained.
  • the resulting cell mixture is particularly advantageous to efficiently maintain and/or expand/proliferate the epidermis-derived stem cells in vitro.
  • keratinocyte cell mixtures are used for transplantation experiments. These freshly isolated mixtures, however, comprise a much lower proportion of epidermis-derived stem cells and actually also are less efficient than epidermis-derived stem cells comprised in a cell mixture that arises from the culturing method of the present invention (e.g. under 3C conditions) as, for example shown in Figure 16. Accordingly, the method according to the present invention can even provide more potent cell mixtures for medical or cosmetic transplantation.
  • the method according to the present invention may further comprise a step of isolating said epidermis-derived stem cells; i.e. isolating the cells e.g. by FACS-sorting based on the cellular markers of the respective epidermis-derived stem cells as further specified herein elsewhere.
  • the method for culturing epidermis-derived stem cells employs an ECM, in particular a 3D-ECM, and a basal cell culture medium comprising EGF, a VEGF, a FGF and a ROCK inhibitor/antagonist, wherein said basal cell culture medium further comprises a Sonic Hedgehog (SHH) inhibitor/antagonist.
  • ECM in particular a 3D-ECM
  • basal cell culture medium comprising EGF, a VEGF, a FGF and a ROCK inhibitor/antagonist
  • said basal cell culture medium further comprises a Sonic Hedgehog (SHH) inhibitor/antagonist.
  • SHH Sonic Hedgehog
  • the epidermis-derived stem cells may also be first cultured under 3C culturing conditions and after some passages under 3C culturing conditions further comprising a SHH inhibitor.
  • a SHH inhibitor for culturing epidermis-derived SCs, even if starting from nearly pure, FACS-purified epidermis-derived stem cells result in cell mixtures with much lower SC content.
  • organoid cultures that have been described for epithelial stem cells other than epidermis-derived SCs are not capable of generating cultures with such a high SC proportion.
  • the invention also relates to a method that allows for ex vivo de novo conversion of differentiated epidermal cells (that lack epidermis-derived SCs) into epidermis-derived SCs with undifferentiated phenotype and self-maintenance capabilities.
  • the epidermis- derived stem cells generated with this method can, for example, also be subsequently used in the method for cuituring epidermis-derived SCs.
  • the method for ex vivo de novo conversion of differentiated epidermal cells can be performed as described herein for the method for cuituring epidermis-derived stem cells.
  • 3C cuituring conditions are employed.
  • de novo generation of epidermis- derived stem cells can subsequently also be cultured (also in long-term) by the method for cuituring epidermis-derived stem cells according to the present invention.
  • the method for de novo generation of epidermis-derived stem cells also results in a cell mixture, which comprises epidermis-derived stem cells and differentiated cells (see Figures and Examples).
  • a cell mixture which comprises epidermis-derived stem cells and differentiated cells.
  • an equilibrium between epidermis-derived stem cells and differentiated cells e.g. with about 40-60% epidermis- derived stem cells; see Figures and Examples
  • the ex vivo de novo conversion of HFSCs from non-HFSCs could exemplary be demonstrated.
  • the method for ex vivo de novo conversion of differentiated epidermal cells according to the present invention is particularly beneficial if an organism/individual that lacks certain epidermis-derived stem cells.
  • epidermis-derived stem cells can be de novo generated in vitro from differentiated cells and used for the treatment of such diseases/scenarios.
  • such a treatment can be envisaged in the treatment of Alopecia, a disease where patients lose hair due to destruction of HFs and/or loss of HFSCs, thereby resulting in inability to regenerate hair.
  • epidermis-derived stem cells such as HFSCs originating from a method of cuituring and/or a method for de novo generation of HFSCs can, for example be applied in the field of regenerative medicine, in particular, for example, for treatment of skin diseases, skin injuries and/or alopecia.
  • the present invention relates to a method for cuituring epidermis- derived SCs comprising the step of cuituring epidermis-derived SCs in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising: - Epidermal Growth Factor (EGF); and/or
  • 3D-ECM three-dimensional extracellular matrix
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the present invention also relates to a method for culturing epidermis-derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising a ROCK (Rho-kinase) inhibitor.
  • a ROCK inhibitor improves the survival of epidermis-derived stem cells (preferably HFSCs) compared to standard 2D culturing methods.
  • the present invention further relates to a method for culturing epidermis-derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the present invention relates to a method for culturing epidermis-derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • epidermis-derived stem cell means a stem cell (SC) that is contained in the epidermis/epidermal tissue of mammals and/or can be derived from the epidermis/epidermal tissue of mammals (e.g. by method known in the art to isolate epidermal cells as described further below), including also the epidermis/epidermal tissue of mammals that comprises papilloma or carcinoma.
  • SC stem cell
  • an epidermis-derived stem cell is characterized by high expression of integrins a6 and/or ⁇ 1.
  • Specific antibodies that recognize these cell surface proteins can be used to identify these cells (e.g. by FACS) and/or to isolate them from the tissue using for example FACS purification.
  • antibodies that can be used in FACS analysis and FACS purification are anti-CD34 (clone RA 34, for example from eBioscience or BD Biosciences), anti-a6 integrin (clone GoH3, for example from eBioscience, Abeam, Novus Biologicals), anti- CD200 (clone OX90, for example from eBioscience, Abeam, Thermo Fischer), anti- EpCAM (clone G8.8, for example from eBioscience, Abeam, Thermo Fischer), anti- CD140a (clone APA5, for example from eBioscience, Abeam, Thermo Fischer); anti- CD45 (clone 30-F11 , for example from eBioscience, Novus Biologicals, Thermo Fischer), anti-CD31 (clone MEC13.3, for example from eBioscience, Novus Biologicals).
  • anti-CD34 clone RA 34, for example from eBioscience or BD Biosciences
  • the following protocol can be used.
  • Single cell suspensions are incubated for example with the above-mentioned antibodies for 30 min at +4°C.
  • FACS Buffer 2% FCS, 2 mM EDTA in phosphate-buffered saline
  • cells are analyzed in a flow cytometer such as the FACSCanto II (BD Biosciences) or purified in a flow cytometer such as FACSAria II or a FACSAria Fusion (both from BD Biosciences).
  • FACS Buffer 2% FCS, 2 mM EDTA in phosphate-buffered saline
  • a series of sensors detect the types of light that are refracted or emitted from the cells. Data acquired by the sensors is compiled and integrated to build a dataset where the measured intensities of each cell are represented allowing quantification of staining intensities or purification of cell populations according to specific staining intensities. Expression of cell surface markers is analyzed on live cells after exclusion of cell doublets and dead cells using DAPI (2-(4-amidinophenyl)-1 H -indole-6-carboxamidine), 7AAD (eBioscience) or Fixable Viability Dye eFluor405 (eBioscience).
  • epidermis-derived stem cells/epidermal SCs are, for example, mammalian HFSCs, mammalian interfollicular epidermis SCs, and mammalian cancer stem cells (CSCs), wherein said CSCs originate from the skin and/or epidermis.
  • CSCs originating from the skin and/or epidermis are, for example, SCs that reside in a skin papilloma and/or carcinoma.
  • epidermis-derived stem cell or “epidermal stem cell” according to the present invention also includes a stem cell with high expression of integrins a6 and ⁇ 1 generated in vitro from stem cells, preferably induced pluripotent stem cells (iPS cells).
  • iPS cells induced pluripotent stem cells
  • iPS cells are differentiated into keratinocytes using retinoic acid and bone- morphogenetic protein-4 to induce differentiation toward a keratinocyte lineage, which is then followed by the growth of differentiated iPS cells on collagen type I- and collagen type IV-coated dishes to enrich for iPS cell-derived keratinocytes as described in Kogut et al, 2014, Methods Mol Biol, 1 195: 1 -12.
  • the epidermis comprises multiple, independent epidermis-derived stem cell populations that exist in various specialized microenvironments termed niches within the epidermis and the hair follicle.
  • One of the most important SCs reside in the hair follicle and are termed hair follicle stem cells (HFSCs).
  • HFSCs have the capacity to differentiate along multiple lineages and give rise to hair follicles, interfollicular epidermis and sebaceous glands.
  • HFSCs can be identified by the expression of high levels of a6 integrins and/or ⁇ 1 integrins and CD34 on their cell surface. In addition, these cells are marked by Sox9, K15, Nfatd , Lhx2, Lgr5, and Lgr6 expression.
  • a6 integrins, CD34, Lgr5, and Lgr6 are cell surface markers that particularly can be used to isolate these cells, e.g. by FACS as described above.
  • the following antibodies can be employed; anti-CD34 (clone RAM34, eBioscience, BD Biosciences), anti-a6 Integrin (clone GoH3, eBioscience, Abeam, Novus Biologicals). W
  • Quiescent stem cells express high levels of cell cycle inhibitors and downregulate cell cycle promoters, thus maintaining quiescence. These quiescent stem cells can be primed, leading to their translocation downwards into the hair germ of the HF. The primed stem cells give rise to the activated transit amplifying cells of the hair matrix, at the onset of the anagen growth phase.
  • Lgr6 + and Lrigl + SCs have been shown to be multipotent during development and in skin reconstitution experiments, respectively.
  • Lrigl + stem cells have been shown to be bipotent and only contribute to the regeneration of interfollicuiar epidermis and the sebaceous gland.
  • the interfollicuiar epidermis also contains of stem or progenitor cells. These keratinocytes, when plated at low clonal density, can give rise to highly proliferative colonies (holoclones), aborted colonies (paraclones) and relatively small heterogeneous colonies (meroclones). The fact that the in vitro progeny of a single cell from such holoclone can give rise to a functional self-renewable epidermis in vivo, suggests that these cells are indeed stem cells or progenitors. Also when grafted on the body of burn patients, interfollicuiar epidermal cells can produce viable and functional epidermal sheets. Like hair follicle stem cells, the stem cells of the interfollicuiar epidermis express high levels of a6 and/or ⁇ 1 integrins.
  • the mammalian epidermis/epidermal tissue is composed of a pilosebaceous unit that consists of the hair follicle (HF), the sebaceous gland and the surrounding interfollicuiar epidermis.
  • HF hair follicle
  • Each pilosebaceous unit contains multiple distinct SC populations that fuel the constant renewal of the interfollicuiar epidermis and HFs during postnatal tissue homeostasis and regeneration.
  • the epidermis comprising of keratinocytes, is a multilayered stratified epithelium consisting of a basal, undifferentiated layer of stem cells and several suprabasal, differentiated cell layers.
  • the stem cells of the basal layer are mitotically active and characterized by the expression of certain intermediate filament proteins called keratins.
  • keratins As basal to suprabasal fate switch occurs, cells switch off the expression of integrins with a concomitant switch of keratin expression.
  • Suprabasal cells contribute to three distinct layers of a stratified epithelium. The first layer is the spinous layer where the cells generate even more robust network of intermediate filament proteins.
  • the epidermis also comprises hair follicles, organs that are generated from the interfollicular epidermis and specialize to generate hair.
  • Hair follicle morphogenesis is initiated by an inductive bi-directional signaling crosstalk between keratinocytes that eventually form the HF and a specialized population of dermal fibroblasts that constitute the dermal papilla. This crosstalk that involves multiple signaling pathways ultimately results in the down growth of the hair follicle through a combination of cell migration and proliferation.
  • the hair coat is constantly renewed. This occurs by renewing the lower portion of the hair follicle in cycling phases of growth (anagen), regression (catagen), and rest (telogen).
  • the bulge is the niche for quiescent SCs that during anagen are activated to leave the niche to fuel the growing hair follicle. Tight regulation of stem cell quiescence and activation is required to maintain a stable pool of stem cells enabling HF cycling throughout the lifetime of an individual.
  • epidermis-derived Cells that are contained in the epidermis and/or can be derived from the epidermis are termed "epidermis-derived" in the context of the present invention.
  • stem cells relates to undifferentiated biological cells that can differentiate into specialized cells and can divide (through mitosis) to produce more stem cells (a process referred to as self-renewal). They are found in multicellular organisms. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult somatic stem cells (SCs), which are found in various tissues. In the context of the present invention the term stem cells refers to the group of adult somatic stem cells. Adult somatic stem cells maintain organ form and function.
  • stem cells vary according to tissue of origin, but they share the following properties: an undifferentiated phenotype; potency to differentiate toward all lineages of the tissue of origin (multipotency); capacity to self-renew; capacity to regenerate the tissue upon injury.
  • the homeostatic and reparative functions of stem cells require that their behavior is adjusted to the needs of the tissue.
  • stem cells must be able to gain inputs on the state of the tissue and organism and transform these inputs into fate decisions.
  • the stem cells reside in specific anatomical locations termed niches that integrate information from the local tissue environment as well as from systemic signals. To be able to execute these functions, niches are complex system composing of multiple cell types and a specialized extracellular matrix.
  • Stem cells are typically identified by one cellular marker or a combination of cellular markers. These markers or marker combinations are in most cases proteins, which are expressed in stem cells, in particular on their cell surface. Typically one or several of such marker proteins (combinations of markers), which can e.g. be detected by FACS/flow cytometry analysis as described further below, can clearly identify a stem cell and/or stem cell family/class. The markers typically differ between different types of stem cells. As metioned above, epidermis-derived stem cells are in particular characterized by high expression of integrins a6 and ⁇ 1.
  • any epidermis-derived stem cell can be employed.
  • a epidermis-derived stem cell selected from the group consisting of mammalian hair follicle stem cells (HFSCs), mammalian interfollicular epidermal stem cells, and mammalian CSCs wherein said CSCs originate from the epidermis (in particular the epidermis of papilloma- or carcinoma-comprising skin) are, for example epidermis-derived stem cells known in the art.
  • epidermis-derived stem cells/epidermal stem cells are selected from the group consisting of HFSCs and epidermis-derived cancer stem cells.
  • Said epidermal-derived cancer stem cells are preferably selected from the group consisting of papilloma-derived and carcinoma-derived stem cells.
  • the method for culturing epidermis-derived stem cells is a method for culturing epidermis- derived stem cells selected from the group consisting of hair follicle stem cells and epidermis-derived cancer stem cells.
  • the method for culturing epidermis-derived stem cells is a method for culturing hair follicle stem cells.
  • the epidermis-derived stem cells employed in the context of the present invention are preferably of human or murine origin, most preferably human origin.
  • epidermis-derived stem cells cultured with the method for culturing epidermis-derived stem cells according to the present invention are comprised in an isolated tissue fragment; are isolated/pure, i.e. the cells cultured do not comprise significant amounts of other cell types; or preferably are comprised in a mixture of cell types, which further comprises at least one differentiated epidermal cell type (e.g. such mixture can be bulk keratinocytes/bulk skin cell mixtures).
  • the term "not comprise significant amounts" in the context of isolated/pure cells means less than 1 %, 2%, 3%, 5% or 10% of cells other than epidermis-derived stem cells.
  • the remaining amount of other epidermal cell types can, for example, be determined by FACS-analysis.
  • Such FACS-analysis allows to determine the proportion of epidermis- derived stem cells based on the markers mentioned above for epidermis-derived stem cells in general and certain types epidermis-derived stem cells. Respective antibodies for the FACS-analysis are preferably selected depending on the type of epidermis- derived stem cells. The amount of other epidermal stem cell can be calculated by subtracting the number of epidermis-derived stem cells from 100%. If epidermis-derived stem cells are cultured in said mixture of cell types, such mixture of cell types preferably comprises isolated keratinocytes, epidermal papilloma cells or epidermal carcinoma cells.
  • Keratinocytes such as bulk keratinocytes preferably comprise one or more cell types of the epidermis. These are among others: epidermal progenitor cells, transit amplifying cells, outer root sheath cells, hair germ cells, hair matrix cells and keratinocytes.
  • cell mixtures comprising epidermis-derived stem cells are crude single-cell suspension derivable from tissue biopsies as described further below.
  • the method according to the present invention has the particular advantage of being able to start from such crude keratinocyte samples and therefore does not necessarily require further purification (e.g. by FACS) of epidermis-derived stem cells.
  • the preferred number of cells used to initiate a culture employing isolated/pure epidermis-derived stem cells or a cell mixture is between 1x10 4 and 3x10 5 cells or at least 2x10 4 and not higher than 3x10 5 cells.
  • a preferred number is at least 1x10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , cells and not higher than 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 cells.
  • a more preferred number is at least 7x10 4 and not higher than 9x10 4 cells.
  • An even more preferred number is about 8x10 4 cells or 8x10 4 cells.
  • the initial cells comprise at least 1 %, preferably at least 2%, most preferably at least 4% epidermis-derived stem cells.
  • tissue fragments preferably tissue fragments comprising epidermis- derived stem cells, wherein said tissue is skin are cultured with the method of the present invention.
  • Skin fragments can be isolated by protocols that are known to the person skilled in the art. For example, skin fragments may be isolated by obtaining a punch biopsy of the skin or excising a piece of skin using a surgical scalpel.
  • Cell mixtures/populations comprising epidermis-derived stem cells can be isolated/gained by protocols that are known to the person skilled in the art.
  • skin fragments may be isolated from a tissue biopsy using trypsin or other proteolytic enzymes to degrade the basement membrane, thereby separating the epidermis from the underlying connective tissue cells in a form of a single-cell suspension.
  • skin biopsies can be obtained by incubating skin biopsies in 0.8% Trypsin (for example from Gibco) for 50 min at 37°C after which the epidermis can be mechanically separated from the dermis by scraping.
  • Single cells can then be separated by passing the epidermis tissue through 70 or 45 pm cell strainers (for example from BD Biosciences).
  • cell strainers for example from BD Biosciences.
  • epidermis-derived stem cells or only particular epidermis- derived stem cell types e.g. HFSCs
  • HFSCs epidermis-derived stem cell types
  • cell mixtures/populations containing interfollicular epidermal stem cells can be obtained by protocols that are known to the skilled person.
  • cells can be isolated by incubation of isolated tissue with proteases such as trypsin that release cells from the basement membrane. After separation, the keratinocyte cell layer along with the hair follicle cells is mechanically separated from the dermis and minced. This is followed by extraction of single cells using, for example, filtration and/or centrifugation steps.
  • proteolytic enzymes such as dispase I, can be used instead of or in combination with trypsin.
  • Hair follicle stem cells can also be isolated/separated/purified from other cell types prior to culturing. Methods to specifically separate hair follicle stem cells from other skin cells are known in the art. A preferred method is based on the fact that hair follicle stem cells express CD34 and a6 integrin on their surface. A preferred method comprises preparing a cell suspension from the epithelial tissue, contacting the cell suspension with CD34/a6 integrin binding compound, removing the non-bound, excess of the CD34 and a6 integrin binding compound by washing, and isolating the stem cells that bind the binding compound. In another alternative method, stem cell surface markers are Lrigl , Lgr5, and Lgr6.
  • stem cells can further be isolated from genetically engineered mice where promoters of known stem cell markers such as Lgr5, Lgr6, Sox9, Keratin15, Lrig5 control the expression of a reporter protein such as, for example, GFP, YFP, RFP, and LacZ.
  • promoters of known stem cell markers such as Lgr5, Lgr6, Sox9, Keratin15, Lrig5 control the expression of a reporter protein such as, for example, GFP, YFP, RFP, and LacZ.
  • Fluorescence-activated cell sorting and magnetic bead-based cell separation make use of labeled-binding compounds e.g. antibodies that specifically recognize cell surface proteins. After labeling epidermal-derived cell mixtures with said binding compounds stem cells can be isolated with high purity (up to 99.9%) from the non-stem cells, which do not bind said binding compounds. FACS makes use of lasers to excite fluorophores attached to the binding compounds and then specifically selects/purifies cells that are bound by said binding compounds. Magnetic bead-based cell separation also isolates cells that specifically bind binding compounds with the help of antibodies attached to magnetic beads. These antibodies recognize the fluorophores or labels (e.g. biotin) attached to the binding compounds.
  • labeled-binding compounds e.g. antibodies that specifically recognize cell surface proteins.
  • CSCs Epidermis-derived cancer stem cells
  • derived from for example papilloma or carcinoma cells can be isolated from tumor tissue by methods known in the art.
  • methods comprising the use of dissociating agents such as dispase I, collagenase, trypsin and EDTA and/or mechanical disruption may be employed to obtain single cell suspensions containing CSCs.
  • cell suspensions can be generated from tumor biopsies by first mechanically mincing them using a surgical scalpel and subsequently incubating them in 0.25% collagenase (for example from Sigma), 62.5 U/mL DNasel (Roche) in Hank's Balanced Salt Solution, (for example from Gibco) for 60 min with gentle agitation at 37°C.
  • Single cell suspensions can be obtained by passing the cell suspension through a 45 pm cell strainer (for example from BD Biosciences).
  • CSCs can be further purified from the single cell suspension by methods known in the art. These methods include but are not limited to FACS and magnetic bead separation, as describe above for other epidermis-derived stem cells. In particular, for separating CSCs from papilloma or/carcinoma the cell surface markers CD34 and a6 integrin can be employed as some CSCs express CD34 and a6 and/or ⁇ 1 integrin. These cells should be further express EpCAM and be negative for CD31 , CD140a and CD45.
  • CSCs from papilloma and/or carcinoma also additional cell surface markers others than CD34 and a6 integrin are employed and are known in the art.
  • a surface marker set to isolate CSCs from papilloma or/carcinoma contains at least the following markers: CD140a, CD31 , CD45, EpCAM, CD34 and a6 integrin, wherein CSCs are EpCAM-, CD34- and a6 integrin-positive and negative for CD140a, CD31 and CD45.
  • Cells expressing these markers can be isolated from the papilloma or carcinoma using CD140a-, CD31 -, CD45-, EpCAM-, CD34- and a6-binding compounds, followed by magnetic bead separation and/or FACS as described for other epidermis-derived stem cells.
  • Another example of a surface marker set to isolate CSCs from papilloma or/carcinoma contains at least the following markers: CD140a, CD31 , CD45, EpCAM, CD34, ⁇ 1 integrin and a6 integrin, wherein CSCs are EpCAM-, CD34-, ⁇ 1 integrin- and a6 integrin-positive and negative for CD140a, CD31 and CD45.
  • culture or “culturing”, which is also referred to as “cultivating” elsewhere herein, particularly relates to maintaining and/or preferably expanding/growing cells of multicellular organisms, in particular mammals ex vivo.
  • Said culturing is a method which is well known in the art and is, for example described in Phelan, 2007, Curr Protoc Cell Biol, 36: 1.1.1-1.1 .18.
  • culturing involves the provision of cell culture medium to the cells (which is described further below) and exchanging this cell culture medium in regular time frames by fresh cell culture medium. Cultures are preferably incubated/cultured at 34-37 ° C, preferably 37°C.
  • cells are preferably cultured in an atmosphere comprising between 5% and 10%, preferably 5% C0 2 .
  • the cell culture medium is preferably exchanged every 2, 3, 4, 5, 6, or 7 days. Most preferably every 2 days.
  • During culturing cells are preferably also regularly removed from the cultivation vessel and all or some of these cells are transferred again in the same or a fresh cultivation vessel to allow for further maintenance and/or expansion/growth of the cells.
  • This is referred to as cell passaging and after each round of passage the cells are typically referred to a corresponding passage, i.e. for example cells that have been passaged twice are referred to as passage two cells.
  • said passaging in the context of the presence invention typically also comprises the isolation of the cultured cells from the 3D-ECM. Passaging of cells may, for example, be performed every 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 days. Most preferably every 14 days.
  • epidermis-derived SCs are cultured in the presence of a three-dimensional extracellular matrix (3D-ECM).
  • a "three-dimensional extracellular matrix (3D-ECM)" refers to a three-dimensionally shaped microenvironment or structure comprising at least one ECM component.
  • the 3D-ECM microenvironment is preferably constituted in a manner that cells, preferably single cells can be/are embedded in a way that they are surrounded by 3D-ECM components. Accordingly, in other words a 3D-ECM is preferably an ECM that surrounds the embedded cells.
  • a 3D-ECM This can, for example be achieved by mixing cells and liquid ECM components before such ECM components solidify and build a gel-like ECM.
  • the purpose of forming a 3D-ECM is to mimic in vitro the conditions in vivo, in which cells are typically embedded inside an extracellular matrix. "In the presence of a 3D-ECM” means that the cells are embedded within such 3D- ECM.
  • a preferred method for culturing epidermis-derived stem cells is further a method, wherein said epidermis-derived stem cells are embedded in said 3D- ECM.
  • ECM or one or more ECM component(s) is/are provided and mixed with the cells intended to be embedded (e.g.
  • a 3D shape is generated by forming a droplet as described below and/or by using a structured shape in which the mixture is embedded.
  • a method for culturing and/or generating cells according to the present invention employed "in presence of a 3D-ECM” may also be expressed as a method employed in the presence of ECM, wherein said cells are embedded in the ECM.
  • this alternative expression for "in presence of a 3D-ECM” may further comprise: "wherein the ECM forms a three-dimensional structure".
  • cells can be embedded within an extracellular matrix, or in other words a 3D-ECM (comprising cells) may be generated by methods known in the art as described for example in Haycock, 201 1 , Methods Mol Biol 695: 1-15.
  • epidermis-derived stem cells e.g. gained by one of the methods described above
  • a buffer solution such as a keratinocyte growth medium (e.g. MEM Spinner's modification (Sigma) containing 8% calcium-depleted fetal calf serum) containing an extracellular matrix protein (e.g. collagen I) or a mixture of extracellular matrix proteins (e.g.
  • This mixture of epidermis-derived stem cells and extracellular matrix is dispensed as a drop on the bottom of a tissue culture petri dish or other appropriate receptacle.
  • the extracellular matrix solution/mixture will subsequently solidify to a 3D-ECM when the pH is increased (for example from pH 3.0 to 7.5 in the case of collagen I) and/or when the temperature is increased (for example from 4°C to 25°C in the case of an extracellular matrix extract such as Matrigel). After solidification the droplet is covered by a cell culture media for culturing epidermis-derived stem cells according to the present invention.
  • 3D-ECM For passaging cells are typically isolated from the 3D-ECM, for example, using the following protocol: The 3D-ECM mixture is mechanically disrupted by repeated aspiration with a pipet tip in a solution containing 0.5% Trypsin, 0.5 mM EDTA in PBS or alternatively in Accutase (Gibco). This is followed by further 10 min incubation at 37°C in the said solutions. Cells are subsequently harvested by centrifugation, diluted in a ratio of 1 :3 - 1 :5 and re-suspended in a new mixture of ECM, which is subsequently transformed to a 3D-ECM by methods as described elsewhere herein. Preferably, cells are passaged every 10-14 days.
  • Culturing of epidermis-derived stem cells in particular refers to maintenance of these cells without losing typical features of epidermis-derived stem cells such as multipotency and/or the capacity to self-renew.
  • these cells will be able to generate new HFSCs through cell division as well as to give rise to all differentiated cell types of the hair follicle and the interfollicular epidermis.
  • interfollicular epidermal stem cells it is particularly envisaged that these cells will be able to generate new interfollicular epidermal stem cells through cell division as well as to give rise to all differentiated cell types of the hair follicle and the interfollicular epidermis.
  • epidermis-derived cancer stem cells it is particularly envisaged that these cells will be able to generate new cancer stem cells through cell division as well as to give rise to all differentiated cell types of the tumor.
  • culturing relates to maintaining epidermis-derived stem cells, which means the survival of said cells ex vivo in cell culture.
  • Maintenance relates to a survival of the cells initially subjected to in vitro culturing according to the method of the current invention of at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 100%.
  • the number of surviving epidermis-derived stem cells can be determined by flow cytometry.
  • epidermis-derived stem cells cultured according to the present invention are extracted from the 3D-ECM as described above and analyzed by a respective FACS analysis.
  • single cell suspensions are then labeled with fluorescently labeled binding compounds that bind HFSCs, such as CD34 and a6 integrin.
  • single cell suspensions are labeled with fluorescently-labeled binding compounds that bind, for example, CD140a, CD31 , CD45, EpCAM, CD34 and a6 integrin. Labeled cells are analyzed using a flow cytometer to determine the proportion of epidermis-derived stem cells in respect to other cell populations.
  • culturing epidermis-derived stem cells not only includes maintenance/survival of the cells subjected to culturing, but also relates to an expansion/growth of said cells.
  • Expansion/growing means that the number of cells is increased during the time of in vitro culturing by mitotic cell division.
  • expansion of the cultivated cells during culturing can be unlimited.
  • expansion means to increase the number of epidermis-derived stem cells by at least 1 ,5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or 200-fold during the time of culturing.
  • extracellular matrix is a collection of extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells.
  • extracellular matrix means a collection of one or more extracellular molecules (as e.g, secreted by cells) that provides structural and biochemical support to the surrounding cells (in particular the cultured cells.
  • Components/Extracellular molecules of an extracellular matrix that are known in the art are, for example, a variety of polysaccharides, water, elastin and glycoproteins, wherein the glycoproteins comprise collagens, entactin(s) (also referred to as nidogen(s)), fibronectins, perlecan and/or laminins.
  • ECM extracellular matrix
  • an ECM in the context of the present invention may comprise and/or consist of: Collagen type I, Collagen type II, Collagen type III, Collagen type IV; Laminin-1 11 , Laminin-211 , Laminin-121 , Laminin-221 , Laminin-332, Laminin-3B32, Laminin-31 1 , Laminin-321 , Laminin-41 1 , Laminin-421 , Laminin-51 1 , Laminin-521 , Laminin-213, Laminin-423, Laminin-522, Laminin-523, an Entactin/Nidogen (such as Nidogen 1 , Nidogen 2), Perlecan, Vitronectin, and/or another Heparan sulfate proteoglycan than Perlecan such as agrin or Collagen XVIII.
  • an Entactin/Nidogen such as Nidogen 1 , Nidogen 2
  • Perlecan Perlecan
  • Vitronectin and/
  • 3D-ECM three- dimensional ECM
  • 3D-ECM cultures or 3D cultures single cell suspensions are embedded within the ECM mixture prior to its solidification, allowing the cells to be embedded within the ECM from all sides.
  • 3D-ECM can, for example, be generated by utilizing the natural properties of ECM components, in particular ECM proteins to self-assemble and to polymerize.
  • 3D-ECM can also be produced by using synthetic polymers to which ECM components, in particular ECM proteins, or synthetic reactive groups of ECM components, in particular ECM proteins, are subsequently incorporated to as for example described in Rimann & Hauser, 2012, Curr Opin Biotech, 23: 1-7; Prestwich, 2007, J Cell Biochem 101 : 1370-1383.
  • 2D-ECM or 2D-culture conditions are achieved by coating a tissue culture petri dish with ECM components as listed above, allowing them to form a thin layer on top of the surface of the tissue culture receptacle and subsequently plating the cells on top of the ECM surface.
  • 2D-ECM does therefore not comprise embedded cells. Further, it typically rather forms a thin 2D-layer on which cells are subsequently plated.
  • any three-dimensional extracellular matrix (3D-ECM) can be employed.
  • 3D-ECM three-dimensional extracellular matrix
  • Different types of 3D-ECM with different compositions in particular including different types of glycoproteins and/or different combinations of glycoproteins, are known in the art. Non-limiting examples are, for example, described in Hynes, R. O. and Naba, A. 2012. Cold Spring Harb. Perspect. Biol. 4, a004903.
  • an ECM/ECM components can be produced by culturing ECM-producing cells, such as, for example, fibroblast cells or various cancer-derived cell lines, such as, but not limited to M1536B3, PYS, HT1080, PF-HR9.
  • ECM-producing cells are cultured in a receptacle such as tissue culture petri dishes using standard culture conditions known in the art.
  • ECM component production occurs throughout the period of culture, most typically between 2 and 20 days.
  • the ECM components are secreted into the culture medium as soluble proteins from where they can be deposited as a soluble or insoluble protein meshwork, termed the extracellular matrix (ECM), into the bottom of the petri dish/receptacle.
  • ECM extracellular matrix
  • ECM proteins can be harvested directly from the culture medium as soluble proteins or by solubilizing the ECM meshwork using detergents such as Triton-X 100 or urea. ECM proteins can be further purified using methods known in the art for protein purification such as precipitation, ultracentrifugation, or chromatography (Yurchenco et al, 2002, Methods Cell Biol, 69:1 11 -44). Such ECM protein(s), ECM meshwork, or further purified ECM protein(s) can subsequently be employed in the method for culturing epidermis-derived SCs according to the present invention. For this purpose, the cells are embedded in the ECM to form a 3D-ECM as described above in the context of culturing/passaging of cells.
  • the 3D-ECM can be assembled from purified proteins that have been purified from biological materials such as tissues or produced using methods of recombinant protein production in E. coii, P. pastoris, or other organisms suitable for protein production as known to a skilled person.
  • These recombinant ECM proteins can, for example, be purchased commercially (e.g. provided by Invitrogen, Biolamina, or Millipore), or are recombinantly expressed and purified according to protocols well known in the art. Embedding of cells in such an ECM to form a 3D-ECM is achieved as described above in the context of culturing/passaging of cells.
  • a collagen type I- based 3D-ECM to culture epidermis-derived stem cells is prepared by diluting commercially available, purified rat tail collagen type I (Millipore) in the desired growth medium (preferably one of the culturing media described by the current invention), mixing this dilution with epidermis-derived stem cells and then dispensing the mixture as a droplet on the bottom of a tissue culture petri dish, where it will subsequently solidify.
  • the solidified 3D-ECM-cell mixture is subsequently covered by the culture medium described in the present invention. This way of culturing is typically also referred to as 3D-culturing.
  • a commercially available ECM mixture may be employed, in particular for generating a 3D-ECM.
  • ECMs are, for example, MatrigelTM (BD Biosciences), CultrexTM (Amsbio), EHS matrixTM (Sigma) or GeltrexTM (LifeTechnologies). All these commercially available ECMs are soluble forms of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor and comprise: between 60% and 85% of laminins, between 5% and 30% collagen IV, between 1 % and 10% nidogen, and 1 between 1 % and 10% heparan sulfate proteoglycan.
  • EHS Engelbreth-Holm-Swarm
  • a 3D-ECM comprises or consists of basement membrane proteins, preferably for example purified from Engelbreth-Holm- Swarm (EHS) tumor and composing between 60% and 85% of laminins, between 5% and 30% collagen IV, between 1 % and 10% nidogen, and between 1 % and 10% heparan sulfate proteoglycan.
  • EHS Engelbreth-Holm- Swarm
  • Such 3D-ECM is preferably generated by solidifying a initially soluble form of basement membrane proteins, preferably for example purified from Engelbreth-Holm-Swarm (EHS) tumor and composing between 60% and 85% of laminins, between 5% and 30% collagen IV, between 1 % and 10% nidogen, and between 1 % and 10% heparan sulfate proteoglycan to a 3D structure under defined conditions as described elsewhere herein.
  • EHS Engelbreth-Holm-Swarm
  • a synthetic hydrogel ECM/3D-ECM or a naturally occurring ECM/3D-ECM can be employed.
  • a synthetic 3D-ECM is generated with a commercially available ECM mixture as described above, or is assembled by one or more purified or recombinantly produced ECM components as described above.
  • the most preferred synthetic 3D-ECMs are generated with commercially available ECM components, e.g. soluble forms of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor.
  • Naturally occurring ECMs are preferably ECMs that are produced by ECM-producing cells as described above.
  • a 3D-ECM is employed that comprises one or more of the components selected from the group consisting of Collagen type I, Collagen type II, Collagen type III, Collagen type IV; Laminin- 11 , Laminin-211 , Laminin-121 , Laminin-221 , Laminin-332, Laminin-3B32, Laminin-311 , Laminin-321 , Laminin-411 , Laminin-421 , Laminin-511 , Laminin-521 , Laminin-2 3, Laminin-423, Laminin-522, Laminin-523, a Entactin/nidogen (Nidogen 1 or Nidogen 2), Perlecan, Vitronectin, and/or another Heparan sulfate proteoglycan.
  • Laminin- 11 Laminin-211 , Laminin-121 , Laminin-221 , Laminin-332, Laminin-3B32, Laminin-311 , Laminin-321
  • a 3D-ECM comprising at least one ECM glycoprotein
  • ECM glycoproteins are, for example, collagens, entactin(s) (nidogen(s)), fibronectins, perlecan, and/or laminins.
  • the preferred ECM glycoprotein is at least one collagen or laminin. Most preferred the at least one ECM glycoprotein is a collagen.
  • a method for culturing epidermis-derived stem cells employs a 3D-ECM that comprises at least two distinct glycoproteins.
  • a 3D-ECM comprising a laminin and a collagen.
  • a 3D-ECM comprising a laminin, a collagen and an entactin.
  • a 3D-ECM comprising a laminin, a collagen and an entactin, wherein said laminin is laminin-51 1 or laminin-332.
  • a 3D-ECM comprising a laminin, a collagen and an entactin, wherein said collagen is collagen IV.
  • a 3D-ECM that comprises: laminin-51 1 , collagen IV and an entactin; or laminin-332, collagen IV and an entactin (also referred to as nidogen).
  • an entactin/nidogen is nidogen 1 or nidogen 2.
  • basal cell culture medium relates to a liquid medium that is known in the art for being suited for culturing human and/or animal cells.
  • basal cell culture medium can be a commercially available cell culture medium such as MEM (Spinners modification, Sigma) or DMEM.
  • a preferred cell culture medium is a defined synthetic medium that is buffered at a pH of 7.4 (preferably between 7.2 and 7.6 or at least 7.2 and not higher than 7.6) with a carbonate-based buffer, while the cells are cultured in an atmosphere comprising between 5% and 10% C0 2 , or at least 5% and not more than 10% C0 2 , preferably 5% C0 2 .
  • a preferred cell culture medium can for example be selected from MEM that does not contain calcium, such as the Spinner's modification.
  • the basal cell culture medium comprises L-glutamine, insulin, hydrocortisone, penicillin, streptomycin, calcium-depleted fetal calf serum and/or calcium-depleted fetal bovine serum.
  • Fetal bovine serum or fetal calf serum are preferably depleted of calcium, for example by chelating agents or dialysis.
  • Supplements such as, for example, Ethanolamine and/or Phosphoethanolamine as supplements for phospholipid synthesis can further be added to the medium.
  • the basal cell culture medium may further comprise epidermal growth factor (EGF).
  • EGF is a mitogenic growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR.
  • EGFs from different mammalian species are well known in the art and are for example described in (Edwin et al, 2006, Methods Mol Biol, 327:1 -24).
  • EGF(s) of any mammalian organisms may be employed.
  • EGF(s) from the same organism from which the cultured epidermis-derived stem cells originate are employed.
  • EGF(s) of other organisms which are known by the person skilled in the art to be biologically active in the organism from which the cultured epidermis-derived stem cells originate, are preferably employed. Accordingly, for example, in the context of culturing human epidermis-derived stem cells with the method of the current invention, human and/or murine EGF(s) originating from humans and/or mouse and/or most preferably resulting from recombinant protein production are employed.
  • LONG® EGF also (mutant) variants of EGF(s) known in the art such as LONG® EGF may be employed.
  • LONG® EGF a recombinant analogue of human that comprises the human EGF amino acid sequence plus a 53 amino acid N-terminal extension peptide. It is particularly preferred as it is animal-free, recombinantly expressed in E-coli. It can, for example, replace human, murine or recombinant EGF and it is already used in approved and marketed cell-based therapies.
  • LONG® EGF is commercially available, for example from Repligen Inc.
  • EGF is preferably comprised in the basal cell culture medium employed in the method for culturing epidermis-derived stem cells according to the present invention at a concentration of between 2 ng/ml and 500 ng/ml or of at least 2 ng/ml and/or not higher than 500 ng/ml.
  • a preferred concentration is at least 5, 10, 20, 25, 30, 40, 45, or 50 ng/ml and/or not higher than 500, 450, 400, 350, 300, 250, 200, 150, or 100 ng/ml.
  • a more preferred concentration is between 5 ng/ml and 100 ng/ml.
  • a more preferred concentration is also at least 5 ng/ml and/or not higher than 100 ng/ml.
  • An even more preferred concentration is between 5 ng/ml and 100 ng/ml.
  • An even more preferred concentration is about 20 ng/ml, or exactly 20 ng/ml.
  • the basal cell culture medium may comprise Vascular Endothelial Growth Factor(s) (VEGF(s)).
  • VEGF Vascular Endothelial Growth Factor
  • the VEGF family comprises of five members in mammals (VEGF- A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D) and VEGFs are produced by cells that stimulate vasculogenesis and angiogenesis.
  • VEGF in particular refers to the VEGF-A. Accordingly, if the term VEGF is used elsewhere herein, VEGF-A is preferably meant.
  • VEGF's normal function is to create new blood vessels in different occasions such as for example during embryonic development, after injury, in the process of muscle formation following exercise and the bypass of blocked vessels.
  • VEGF is also used by tumor cells to induce neovascularization within tumors.
  • VEGF stimulates cellular responses by binding to VEGF receptors (the VEGFRs), which are tyrosine kinase receptors on the cell surface.
  • the VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains, a single transmembrane spanning region, and an intracellular portion containing a split tyrosine-kinase domain.
  • VEGF-A binds to VEGFR- 1 (Flt-1 )) and VEGFR-2 (KDR/Flk-1 ), however, VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF.
  • VEGF vascular endothelial growth factor
  • VEGF-A exists in multiple isoforms that result from alternative splicing of mRNA from the VEGF (VEGF-A) gene.
  • VEGFs and their isoforms from different mammalian species are well known in the art and are for example described in Chung & Ferrara, 2011 , Annu Rev Cell Dev Biol 27: 563-584.
  • any VEGF isoform(s) originating from any mammalian organisms may be employed.
  • VEGF(s) from the same organism from which the cultured epidermis-derived stem cells originate are employed.
  • VEGF(s) of other organisms which are known to be biologically active in the organism from which the cultured epidermis-derived stem cells originate, are preferably employed. Accordingly, for example, in the context of culturing human epidermis-derived stem cells with the method of the current invention, human and/or murine VEGF(s) originating from humans and/or mouse or most preferably from recombinant protein production are employed.
  • VEGF(s) which is/are selected from the group of VEGF isoforms consisting of VEGF-12 , VEGF- 20, VEGF-145, VEGF-165, VEGF-164, VEGF-183, VEGF-188, VEGF-189 and VEGF-206 (Holmes and Zachary. 2005. Genome Biol 6:209), is (are) preferably employed.
  • VEGF- 188, VEGF-164 and/or VEGF-120 is/are murine.
  • VEGF-121 , VEGF-145, VEGF-165, VEGF-183, VEGF-189 and/or VEGF-206 is/are human.
  • VEGF(s) selected from the group of VEGF isoforms consisting of VEGF-164, VEGF- 165, VEGF-121 , VEGF-120 and VEGF-188 is/are employed.
  • the isoform(s) VEGF-164 and VEGF-121 is (are) employed as VEGF(s).
  • VEGF(s) known in the art such as chimeric molecules consisting of mosaic of VEGF-A VEGF-C or VEGF-A / ENZ7 may be employed (Jeltsch et al, 2006, J Biol Chem, 281 :12187-12195; Zheng et al, 2007, Aterioscler Thromb Vase Biol, 27: 503-51 1 ).
  • a VEGF is preferably comprised in the basal cell culture medium employed in the method for culturing epidermis-derived stem cells according to the present invention at a total concentration of between 2 ng/ml and 500 ng/ml or of at least 2 and/or not higher than 500 ng/ml.
  • a preferred concentration is at least 2, 5, 10, 20, 25, 30, 40, 45, or 50 ng/ml and/or not higher than 500, 450, 400, 350, 300, 250, 200, 150, or 100 ng/ml.
  • a more preferred concentration is between 5 ng/ml and 100 ng/ml.
  • a more preferred concentration is also at least 5 ng/ml and/or not higher than 100 ng/ml.
  • An even more preferred concentration is between 5 ng/ml and 100 ng/ml.
  • An even more preferred concentration is about 20 ng/ml, or even more preferred exactly 20 ng/ml.
  • the concentration of a VEGF is as defined above and refers to the total concentration of VEGFs used, i.e. the total sum of VEGF concentrations (e.g. if VEGF-164 and VEGF-165 are employed, the sum of the concentrations of VEGF-164 and VEGF-165).
  • the above-mentioned values apply only to the sum of VEGF concentrations and do not mean that all of the VEGFs need to be present in the above mentioned concentration ranges. For example, at least 5 ng/ml of total VEGFs would be present if the basal cell culture medium comprises 3 ng/ml VEGF-164 and 2 ng/ml VEGF-165.
  • the basal cell culture medium may comprise Fibroblast Growth Factor(s) (FGF(s)).
  • FGFs are a family of mitogenic growth factors, with members involved in angiogenesis, wound healing, embryonic development and various endocrine signaling pathways.
  • the FGFs are heparin-binding proteins and interactions with cell-surface-associated heparan sulfate proteoglycans have been shown to be essential for FGF signal transduction.
  • FGFs are key players in the processes of proliferation and differentiation of wide variety of cells and tissues.
  • FGF-1 FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-10, FGF-22, FGF-8, FGF-17, FGF- 18, FGF-24, FGF-9, FGF-16, FGF-20, FGF-1 1 , FGF-12, FGF-13, FGF-14, FGF-19, FGF-21 , and FGF-23.
  • FGFs from different mammalian species are well known in the art and are, for example, described in Ornitz & Itoh, 2001 , Genome Biol 2: 3005.1 - 3005.12..
  • FGF(s) function is mediated by its (their) binding to so called fibroblast growth factor receptor(s) (FGFRs).
  • FGFRs fibroblast growth factor receptor
  • the mammalian fibroblast growth factor receptor family has 4 members, FGFR1 , FGFR2, FGFR3, and FGFR4.
  • any FGF(s) of any mammalian organism may be employed.
  • FGF(s) is/are preferably selected from the group consisting of FGF-1 , FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-10, FGF-22, FGF-8, FGF-17, FGF-18, FGF-24, FGF-9, FGF-16, FGF-20, FGF-1 1 , FGF-12, FGF-13, FGF-14, FGF-19, FGF-21 , and FGF-23.
  • FGF-2, FGF-7, FGF-10 and/or FGF-18 are employed.
  • FGF-2 and/or FGF-18 are employed.
  • FGF(s) from any mammalian organisms may be employed.
  • FGF(s) from the same organism from which the cultured epidermis-derived stem cells originate is/are employed.
  • FGF(s) of other organisms, which is/are known to be biologically active in the organism from which the cultured epidermis- derived stem cells originate is/are preferably employed.
  • human and/or murine FGF(s) originating from humans or mouse or most preferably from recombinant protein production are employed.
  • FGF(s) known in the art
  • FGF(s) include, but are not restricted to a heat-stable chimeric variant of FGF, termed FGFC.
  • FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF-1 and FGF-2 (Onuma et al., 2015, Plos One 10:e0118931 ).
  • a FGF is preferably comprised in the basal cell culture medium employed in the method for culturing epidermis-derived stem cells according to the present invention at a concentration of between 2 ng/ml and 500 ng/ml or of at least 2 ng/ml and/or not higher than 500 ng/ml.
  • a preferred concentration is at least 2, 5, 10, 20, 25, 30, 40, 45, or 50 ng/ml and/or not higher than 500, 450, 400, 350, 300, 250, 200, 150, or 100 ng/ml.
  • a more preferred concentration is between 5 ng/ml and 100 ng/ml.
  • a more preferred concentration is also at least 5 ng/ml and/or not higher than 100 ng/ml.
  • An even more preferred concentration is between 5 ng/ml and 100 ng/ml.
  • An even more preferred concentration is about 20 ng/ml, or even more preferred exactly 20 ng/ml.
  • the concentration of a FGF is as defined above and refers to the total concentration of FGFs used, i.e. the total sum of FGF concentrations (e.g. if FGF-2 and FGF-7 are used, the sum of the concentrations of FGF-2 and FGF-7).
  • the above mentioned values apply only to the sum of FGF concentrations and do not mean that all of the FGFs need to be present in the above mentioned concentration ranges. For example, at least 5 ng/ml of total FGFs would be present if the basal cell culture medium comprises 3 ng/ml FGF-2 and 2 ng/ml FGF-7.
  • a basal cell culture medium comprising an inhibitor of the Rho-kinase (ROCK), referred to as ROCK inhibitor/antagonist is employed.
  • ROCK inhibitor/antagonist an inhibitor of the Rho-kinase
  • Rho-kinase is a kinase of the serine-threonine kinase family. It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton. ROCK exists in two homologous, ROCK1 and ROCK2. ROCKs (ROCK1 and ROCK2) occur in mammals (human, rat, mouse, cow), zebrafish, Xenopus, invertebrates (C. elegans, Mosquito, Drosophila) and chicken and are, for example described in Julian & Olson, 2014, Small GTPases e29846. doi: 10.4161 /sgtp.29846. The addition of a Rock inhibitor/antagonist was found to prevent anoikis that is a form of programmed cell death that is induced by anchorage-dependent cells detaching from the surrounding extracellular matrix (ECM).
  • ECM extracellular matrix
  • a "ROCK inhibitor”, “ROCK antagonist” or “ROCK inhibitor/antagonist” is a chemical substance, which inhibits the kinase activity of ROCK1 (also known as p160ROCK) and/or ROCK2. Most preferred a “ROCK inhibitor”, “ROCK antagonist” or “ROCK inhibitor/antagonist” inhibits ROCK1. Preferably, the kinase activity of ROCK 1 and/or ROCK2 is inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or at least 99%.
  • the inhibition/reduction in activity of ROCK1 and/or ROCK2 is high, but only as high as it does not have significant negative impacts on cell viability.
  • the inhibition of ROCK1 and/or ROCK2 can be measured by methods known in the art, like e.g. in vitro kinase activity assays or in vivo measurement of the phosphorylation status of one or more ROCK substrates.
  • An example for a ROCK substrate is the myosin light chain.
  • myosin light chain phosphor-specific antibodies may be used in assays like western blotting to detect the reduction of phosphorylation, i.e.
  • ROCK1 and/or ROCK2 the inhibition/reduction in activity of ROCK1 and/or ROCK2 (Julian & Olson, 2014, Small GTPases e29846. doi: 10.4161 /sgtp.29846).
  • any ROCK inhibitor can be employed.
  • the ROCK inhibitor(s) is (are) selected from the group consisting of (R)-(+)-trans-4-(1 -aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (referred to as Y-27632), 5-(1 ,4-diazepan-1-ylsulfonyl) isoquinoline (referred to as fasudil or HA 1077), and (S)-(+)-2-methyl-1-[(4-methyl-5- isoquinolinyl)sulfonyl]-hexahydro-1 H-1 ,4-diazepine dihydrochloride (referred to as H- 1152).
  • Y-27632 is employed in the method of culturing epidermal- derived stem cells as mentioned above.
  • a ROCK inhibitor is comprised in the basal cell culture medium at a total concentration of between 2 ⁇ and 20 ⁇ , preferably of between 3 ⁇ and 15 ⁇ or most preferably of between 5 to 10 ⁇ or most preferably 5 ⁇ .
  • the concentration of a ROCK inhibitor is as defined above and refers to the total concentration of ROCK inhibitors used, i.e. the total sum of ROCK inhibitor concentrations (e.g. if Y-27632 and HA 1077 are used, the sum of the concentrations of Y-27632 and HA 1077).
  • the above mentioned values apply only to the sum of ROCK inhibitor concentrations and do not mean that all of the ROCK inhibitors need to be present in the above mentioned concentration ranges. For example, at least 5 ⁇ of total ROCK inhibitors would be present if the basal cell culture medium comprises 3 ⁇ Y-27632 and 2 ⁇ HA 077.
  • Rho-kinase (ROCK) inhibitors may be added to the culture medium in regular time frames, e.g. every, every third, every forth or preferably every second day. Preferably, it/they are added together with fresh cell culture medium, which is, as described above, regularly (e.g. every second day) provided.
  • Rho-kinase (ROCK) inhibitors Rho-kinase (ROCK) inhibitors
  • EGF EGF
  • VEGF(s) VEGF(s)
  • FGF(s) FGF(s)
  • any other supplements of the basal cell culture medium are present in the culture medium during the complete culturing.
  • epidermis-derived stem cells in the absence of ROCK inhibitors after the cells have been cultured and/or expanded (also referred to as pre-culturing or initial culturing) for at least 2 days, preferably at least 3 days, more preferably at least 4 days, even more preferably at least 5 days, even more preferably at least 6 days, and most preferably at least 7 days according to the methods of the present invention using a basal cell culture medium comprising a Rho-kinase (ROCK) inhibitors) and EGF, VEGF(s), FGF(s), and/or any other supplements.
  • a basal cell culture medium comprising a Rho-kinase (ROCK) inhibitors) and EGF, VEGF(s), FGF(s), and/or any other supplements.
  • ROCK Rho-kinase
  • Y-27632 is employed as ROCK inhibitor. Most preferably, Y-27632 is employed in the concentrations as defined elsewhere herein for the ROCK inhibitor(s) and/or Y27632. As shown in the appended Examples and Figures, the presence of a ROCK inhibitor is only absolutely required during the first two days of culturing and/or expanding epidermis-derived stem cells comprised in freshly isolated keratinocytes. After the initial two days of culturing the presence of ROCK inhibitor(s) is/are not absolutely required to maintain or increase the proportion of epidermis-derived stem cells in the cultured cells.
  • ROCK inhibitor in the absence of ROCK inhibitor(s) after the initial culturing for at least 2 days (in presence of ROCK inhibitor(s)) the absolute numbers of epidermis- derived stem cells decreases over time.
  • An advantage of the absence of ROCK inhibitors) after two days of culturing may nevertheless be advantageous as regarding reducing costs and efforts for providing ROCK inhibitor(s) or in case the cultures are being used to study the actomyosin cytoskeleton, whereby inhibiting ROCK might not be desired.
  • the present invention also relates to a method for culturing epidermis-derived stem cells comprising the step of culturing epidermis- derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the present invention also provides for a method for culturing epidermis- derived stem cells comprising: a first step of culturing epidermis-derived stem cells for at least 2 days in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase inhibitor
  • Y-27632 a Rho-kinase inhibitor
  • a subsequent second step of culturing epidermis-derived stem cells after said least 2 days in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • the second step may be no longer than 10 days, preferably no longer than 9 days, more preferably no longer than 8 days, more preferably no longer than 7 days, more preferably no longer than 6 days, more preferably no longer than 5 days, more preferably no longer than 4 days, more preferably no longer than 3 days and most preferably no longer than 2 days.
  • ROCK inhibitor(s) are only absolutely required for the first 2 days of culturing and can subsequently be omitted from the employed basal cell culture medium can also be applied mutatis mutandis in the methods for de novo generation of epidermis-derived stem cells disclosed in the present invention. Accordingly, also corresponding methods for de novo generation of epidermis-derived stem cells employing either cells pre-cultured for at least 2 days as described above or having the above-mentioned first and second steps are disclosed herein.
  • the method for culturing epidermis-derived stem cells of the present invention employs, inter alia, a basal cell culture medium comprising EGF and/or a VEGF and/or a FGF and a ROCK inhibitor.
  • EGF, VEGF(s) and FGF(s) are known to be mitogenic growth factors.
  • the basal cell culture medium employed in the method of the current invention preferably comprises: a) at least one mitogenic growth factor selected from the group consisting of EGF, a VEGF and a FGF; and b) a ROCK inhibitor.
  • the method of culturing epidermis-derived stem cells may employ a basal cell culture medium, which comprises a VEGF and a ROCK inhibitor, a FGF and a ROCK inhibitor or preferably EGF and a ROCK inhibitor.
  • the cell culture medium preferably comprises: a) one mitogenic growth factor selected from the group consisting of EGF, a VEGF, and a FGF; and b) a ROCK inhibitor.
  • the method of culturing epidermis-derived stem cells employs a basal cell culture medium, which comprises EGF, a VEGF and a ROCK inhibitor; EGF, a FGF and a ROCK inhibitor; or a VEGF, a FGF and a ROCK inhibitor.
  • the basal cell culture medium employed in the method of the current invention preferably comprises: a) at least two mitogenic growth factors selected from the group consisting of EGF, a VEGF, and a FGF; and b) a ROCK inhibitor.
  • the method for culturing epidermis-derived stem cells preferably employs a basal cell culture medium comprising: EGF and a VEGF and/or a FGF and a ROCK inhibitor.
  • the basal cell culture medium employed in the method of the current invention preferably comprises: EGF; at least one mitogenic growth factor selected from the group consisting of the VEGFs VEGF-121 , VEGF-120, VEGF-145, VEGF-165, VEGF-164, VEGF-183, VEGF-188, VEGF-189 and VEGF-206 and the FGFs FGF-1 , FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-10, FGF-22, FGF-8, FGF-17, FGF-18, FGF-24, FGF-9, FGF-16, FGF-20, FGF-11 , FGF-12, FGF-13, FGF- 14, FGF-19, FGF-21 , and FGF-23; and
  • the group of VEGFs consists of VEGF-121 and VEGF-164.
  • the group of FGFs preferably consists of FGF-2, FGF-7, FGF-10 and FGF-18. Even more preferably, the group of FGFs consists of FGF-2 and FGF-18.
  • the basal cell culture medium preferably comprises: EGF, a VEGF selected from the group consisting of VEGF-164 and VEGF-121 , and further a ROCK inhibitor (preferably Y-27632); or EGF, a FGF selected from the group consisting of FGF-2, FGF-7, FGF-10 and FGF-18, and further a ROCK inhibitor (preferably Y-27632); or a VEGF selected from the group consisting of VEGF-164 and VEGF-121 , a FGF selected from the group consisting of FGF-2, FGF-7, FGF-10 and FGF-18, and further a ROCK inhibitor (preferably Y-27632).
  • the group of FGFs consists even more preferably of FGF-2 and FGF-18.
  • the method for culturing epidermis-derived stem cells preferably employs a basal cell culture medium comprising: EGF, FGF-2, and a ROCK inhibitor (preferably Y- 27632); or EGF, FGF-7, and a ROCK inhibitor (preferably Y-27632); or EGF, FGF-10, and a ROCK inhibitor (preferably Y-27632); or EGF, FGF-18, and a ROCK inhibitor (preferably Y-27632); or EGF, VEGF-164, and a ROCK inhibitor (preferably Y-27632); or EGF, VEGF-121 , and a ROCK inhibitor (preferably Y-27632); or FGF-2, VEGF-121 and a ROCK inhibitor (preferably Y-27632).
  • a basal cell culture medium comprising: EGF, FGF-2, and a ROCK inhibitor (preferably Y- 27632); or EGF, FGF-7, and a ROCK inhibitor (preferably Y-27632); or EGF, FGF-10,
  • the method for culturing epidermis-derived stem cells employs a basal cell culture medium, which comprises: EGF; a VEGF; a FGF and a ROCK inhibitor.
  • the basal cell culture medium employed in the method of the current invention comprises: EGF; a VEGF selected from the group consisting of VEGF-121 , VEGF-120, VEGF-145, VEGF-165, VEGF-164, VEGF-183, VEGF-188 and VEGF-206; a FGF selected from the group consisting of FGF-1 , FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-10, FGF-22, FGF-8, FGF-17, FGF-18, FGF-24, FGF-9, FGF-16, FGF-20, FGF-11 , FGF-12, FGF-13, FGF-14, FGF-19, FGF-21 , and FGF-23; and
  • the method for culturing epidermis-derived stem cells preferably employs a basal cell culture medium comprising: EGF; a VEGF selected from the group consisting of VEGF-164 and VEGF-121 ; and a FGF selected from the group consisting of FGF-2, FGF-7, FGF-10 and FGF-18; and a ROCK inhibitor (preferably Y-27632).
  • a FGF selected from the group consisting of FGF-2 and FGF-18 is employed.
  • the method for culturing epidermis-derived stem cells employs a basal cell culture medium, which comprises: EGF, VEGF-164, FGF-2 and a ROCK inhibitor (preferably Y-27632); or EGF, VEGF-164, FGF-18 and a ROCK inhibitor (preferably Y-27632); or EGF, VEGF-121 , FGF-2 and a ROCK inhibitor (preferably Y-27632); or EGF, VEGF-121 , FGF-18 and a ROCK inhibitor (preferably Y- 27632); or EGF, VEGF-164, FGF-7 and a ROCK inhibitor (preferably Y-27632); or EGF, VEGF-164, FGF-10 and a ROCK inhibitor (preferably Y-27632); or EGF, VEGF-121 , FGF-7 and a ROCK inhibitor (preferably Y-27632); or EGF, VEGF-121 , FGF-10 and a ROCK inhibitor (preferably Y-27632); or EGF, VE
  • the method for culturing epidermis-derived stem cells employs a basal cell culture medium, which comprises: EGF, VEGF-164, FGF-2 and the ROCK inhibitor Y- 27632; or EGF, VEGF-164, FGF-18 and the ROCK inhibitor Y-27632; or EGF, VEGF- 121 , FGF-2 and the ROCK inhibitor Y-27632; or EGF, VEGF-121 , FGF-18 and the ROCK inhibitor Y-27632; or EGF, VEGF-164, FGF-7 and the ROCK inhibitor Y-27632; or EGF, VEGF-164, FGF-10 and the ROCK inhibitor Y-27632; or EGF, VEGF-121 , FGF-7 and the ROCK inhibitor Y-27632; or EGF, VEGF-121 , FGF-10 and the ROCK inhibitor Y-27632.
  • a basal cell culture medium which comprises: EGF, VEGF-164, FGF-2 and the ROCK inhibitor
  • the method for culturing epidermis-derived stem cells employs a basal cell culture medium, which comprises: EGF, VEGF-164, FGF-2 and the ROCK inhibitor Y-27632. It is also particularly preferred that the method for culturing epidermis-derived stem cells, as mentioned above, employs a basal cell culture medium, which comprises: EGF, VEGF-121 , FGF-2 and the ROCK inhibitor Y-27632.
  • a basal cell culture medium as described herein which further comprises a Sonic Hedgehog (SHH) inhibitor/antagonist.
  • SHH Sonic Hedgehog
  • SHH Sonic Hedgehog
  • the term "Sonic Hedgehog (SHH)" in the context of SHH inhibitor/antagonist refers to the Sonic Hedgehog (SHH) signaling pathway. Dysregulation of this pathway is usually lethal in early embryonic stages. Mutations in the SHH pathway have been identified in a large variety of malignant tumors.
  • the protein Hedgehog is the extracellular component of the pathway and activates intracellular signals after binding to its specific receptor "Patched" (Ptch), a protein located on the cellular membrane.
  • SMO smoothened
  • SHH inhibitor/antagonist in the context of the present invention is defined as a chemical compound targeting components of the SHH signaling pathway, thus inhibiting its activity/signaling activity.
  • the activity/signaling activity is inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.
  • higher values i.e. stronger inhibition/reduction in activity/signaling activity of the SHH signaling pathway, are preferred.
  • the inhibition of the SHH signaling pathway can be measured by methods known in the art, like e.g.
  • any SHH inhibitor/antagonist can be employed in the method for culturing epidermis-derived stem cells according to the present invention.
  • a SHH inhibitor/antagonist is employed, which is selected from the group consisting of (2 , R,3S,3' 3'aS,6 , S,6aS,6bS,7'a 1 1aS,1 1 bR)- 1 ,2,3,3'aAA',5',6,&,6a,6b ,T,Ta,QA 1 ,1 1 a,11 b-Octadecahydro-3',6',10,11 b- tetramethylspiro[9 - -benzo[a]fluorene-9,2'(3'H)-furo[3,2-/b]pyridin]-3-ol (referred to as Cyclopamine); 2-chloro-N-(4-chloro-3-pyridin-2-ylphenyl)-4
  • a SHH inhibitor that results in inhibition of the SHH signaling pathway, as defined and measured by the methods described above, can be used.
  • a SHH inhibitor is comprised in the basal cell culture medium at a total concentration of between 1 ⁇ and 20 ⁇ , preferably of between 5 ⁇ and 15 ⁇ , more preferably of between 5 ⁇ and 10 ⁇ and most preferably of about 10 ⁇ or 10 ⁇ .
  • concentration range(s) or concentration(s) is/are particularly preferred if cyclopamine is used as a SHH inhibitor.
  • the concentration of a SHH inhibitor is as defined above and refers to the total concentration of SHH inhibitors used, i.e. the total sum of SHH inhibitor concentrations (e.g. if Cyclopamine and HhAntag691 are used, the sum of the concentrations of Cyclopamine and HhAntag691 ).
  • the above mentioned values apply only to the sum of SHH inhibitor concentrations and do not mean that all of the SHH inhibitors need to be present in the above mentioned concentration ranges. For example, at least 5 ⁇ of total SHH inhibitors would be present if the basal cell culture medium comprises 3 ⁇ Y-27632 and 2 ⁇ HA 1077.
  • the present invention also relates to a method for culturing epidermis- derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the basal cell culture medium in this context comprises:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the basal cell culture medium in this context comprises:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the present invention also relates to a method for culturing epidermis- derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising: a) at least one mitogenic growth factor selected from the group consisting of EGF, a VEGF and a FGF; and b) a ROCK inhibitor; and c) a SHH inhibitor.
  • 3D-ECM three-dimensional extracellular matrix
  • a basal cell culture medium comprising: a) at least one mitogenic growth factor selected from the group consisting of EGF, a VEGF and a FGF; and b) a ROCK inhibitor; and c) a SHH inhibitor.
  • a basal cell culture medium comprising: a) at least two mitogenic growth factors selected from the group consisting of EGF, a VEGF and a FGF; and b) a ROCK inhibitor; and c) a SHH inhibitor.
  • the present invention also provides a method for culturing epidermis-derived stem cells according to the present invention, wherein a basal cell culture medium as described anywhere elsewhere herein is employed which does not comprise a Bone Morphogenetic Protein (BMP) inhibitor/antagonist. In principle, it is preferred to employ a cell culture medium that does not comprise any BMP inhibitor/antagonist.
  • BMP Bone Morphogenetic Protein
  • no BMP inhibitor(s) selected from the group consisting of Noggin, chordin, gremlin, crossveinless, USAG-1 , follistatin, Dorsomorphin, K02288, DMH.1 , ML 347, LDN-193189 is comprised in the basal cell culture medium in the context of the method for culturing epidermis-derived stem cells according to the present invention.
  • Bone morphogenetic proteins are a group of growth factors also known as cytokines and as metabologens. Originally discovered by their ability to induce the formation of bone and cartilage, BMPs are now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body.
  • Known members of the BMP protein family are for example: BMP1 , BMP2, BMP2a, BMP3, BMP3b, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15.
  • BMP inhibitor/antagonist in the context of the present invention is defined as a chemical compound and/or a protein with inhibitory activity towards the activity/signaling activity of one or more BMP proteins.
  • Such an inhibitory protein could, for example be Noggin, chordin, gremlin, crossveinless, USAG-1 and follistatin (Walsh et al., 2010 Trends Cell Biol 20:244-256).
  • a BMP inhibitor is a compound that inhibits the activity/signaling activity of one or more BMP by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.
  • higher values i.e. stronger inhibition/reduction in activity/signaling activity of the BMP activity/signaling activity, are a preferred definition.
  • the inhibition of the BMP activity/signaling activity can be measured by methods known in the art, like e.g. measurement of the expression levels of the target genes and the corresponding encoded proteins by methods that are well known in the art, and/or by measuring the activity status of the pathway proteins.
  • BMP target genes such as Id1 , Id2, and Id3
  • Smad 1 , 5, and 8 Walsh et al., 2010, Trends Cell Biol 20:244-256
  • the transcriptional activity of BMP as for example exemplified in Zilberberg et al. 2007, BCM Cell Biol 8:41.
  • the present invention also relates to a method for culturing epidermis-derived stem cells according to the present invention, wherein a basal cell culture medium as described anywhere elsewhere herein is employed which does not comprise a Bone Morphogenetic Protein (BMP) inhibitor/antagonist and/or a Wnt agonist.
  • BMP Bone Morphogenetic Protein
  • the present invention further relates a method for culturing epidermis-derived stem cells according to the present invention, wherein a basal cell culture medium as described anywhere elsewhere herein is employed which does not comprise a Wnt agonist.
  • the Wnt signaling pathway is defined by a series of events that occur when a Wnt protein binds to a cell-surface receptor of a Frizzled receptor family member. This results in the activation of Dishevelled family proteins which inhibit a complex of proteins that includes axin, GSK-3, and the protein APC to degrade intracellular ⁇ -catenin. The resulting enriched nuclear ⁇ -catenin enhances transcription by TCF/LEF family transcription factors.
  • a Wnt agonist is defined as an agent that activates TCF/LEF- mediated transcription in a cell.
  • Wnt agonists are therefore selected from true Wnt agonists that bind and activate a Frizzled receptor family member including any and all of the Wnt family proteins, an inhibitor of intracellular ⁇ -catenin degradation, and activators of TCF/LEF.
  • Said Wnt agonist stimulates a Wnt activity in a cell by at least 10%, more preferred at least 20%, more preferred at least 30%, more preferred at least 50%, more preferred at least 70%, more preferred at least 90%, more preferred at least 99%, relative to a level of said Wnt activity in the absence of said molecule.
  • a Wnt activity can be determined by measuring the transcriptional activity of Wnt, for example by pTOPFLASH and pFOPFLASH Tcf luciferase reporter constructs (Korinek et al , 1997 Science 275 1784-1787).
  • a Wnt agonist is or comprises a secreted glycoprotein such as: Wnt- l/lnt- 1 , Wnt-2/lrp (InM -related Protein), Wnt-2b/13, Wnt-3/lnt-4, Wnt-3a (R&D sytems), Wnt- 4, Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi H et al 2001 Biochem Biophys Res Com 283 798- 805), Wnt-7a (R&D sytems), Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14, Wnt-9b/14b/15, Wnt- lOa, Wnt-10b/12, WnM I, and/or Wnt-16.
  • Wnt- l/lnt- 1 secreted glycoprotein
  • Wnt-2/lrp InM -related Protein
  • Wnt agonists are or include the R-spondin family of secreted proteins, which is implicated in the activation and regulation of Wnt signaling pathway and which is comprised of 4 members (R-spondin 1 (NU206, Nuvelo, San Carlos, CA), R-spondin 2 ((R&D sytems), R-spondin 3, and R-spondin-4), and Norrin (also called Nome Disease Protein or NDP) (R&D sytems), which is a secreted regulatory protein that functions like a Wnt protein in that it binds with high affinity to the Frizzled-4 receptor and induces activation of the Wnt signaling pathway (Kestutis Planutis et al (2007) BMC Cell Biol 8 12).
  • a small-molecule agonist of the Wnt signaling pathway was recently identified and is also expressly included as a Wnt agonist (Lm et al (2005) Angew Chem Int Ed Engl 44, 1987-90).
  • a basal cell culture medium as described in any one of the embodiments herein is employed which further comprises ethanolamine, phospho- ethanolamine and/or transferrin.
  • the method for culturing epidermis-derived stem cells according to the present invention can in principle be performed for an unlimited time. As mentioned elsewhere herein it is, however, a particular advantage of the method according to the present invention to allow for long-term culturing. Accordingly the method is preferably a method for long-term culturing. In particular, the method is a method for culturing epidermis- derived stem cells, wherein said culturing is performed for at least 360 days, at least 250 days, at least 150 days, at least 100 days, at least 50 days, at least 20 days, at least 14 days, at least 10 days, at least 8 days, at least 5 days, at least 3 days, at least 2 days. Although the method is particularly suited for long-term culturing, in principle in this context lower minimal culturing times are preferred.
  • the method for culturing epidermis-derived stem cells according to the present invention is preferably a method for expanding said epidermis-derived stem cells (in vitro/ ex vivo).
  • the method for culturing epidermis-derived stem cells according to the present invention is a method for generating a mixture of cell types comprising epidermis-derived stem cells and at least one differentiated cell type.
  • the method for culturing epidermis-derived stem cells according to the present invention is further a method for enriching said epidermis-derived stem cells provided in said mixture of cell types or said isolated tissue fragment relative to the other cell type(s) comprised in a mixture of cell types or a isolated tissue fragment subjected to the method of culturing according the method provided herein.
  • the method of culturing epidermis-derived stem cells as described in any one of the embodiments herein may further be a method for maintaining the multipotency, proliferative potential and/or capacity to self-renew of epidermis-derived stem cells.
  • Multipotency is the ability of a cell to give rise to other cell types.
  • multipotency describes the potential of stem cells to differentiate into multiple different, but a limited number of different cell types.
  • epidermis-derived stem cells describes the potential to generate both the differentiated cell types in the interfollicular epidermis as well as in the hair follicles. This property can be, for example, be assessed in full thickness skin reconstitution assays, also referred to as transplantation assays as described in detail further below where the ability of a given epidermis-derived stem cell to generate interfollicular epidermis and/or hair follicles can be assayed.
  • HFSCs and/or interfollicular epidermal stem cells preferably the ability to generate both hair follicles (as detected for example by hair growth) and interfollicular epidermis is measured.
  • epidermis-derived cancer stem cells the ability of these cells to generate a tumor consisting of all differentiated cell types of the tumor of origin is measured.
  • the capacity of self-renew describes the ability of a stem cell to go through numerous cycles of cell division while maintaining the undifferentiated stem cell state. This property can be assessed by defining the amount of stem cells present in a culture or tissue over a period of time.
  • the amount of epidermis-derived stem cells can, for example, be determined by the FACS analysis as described elsewhere herein.
  • Stem cells are characterized by their ability to generate a large progeny of differentiated cells. This is in contrast to differentiated cells that can undergo a limited number of cell divisions and thereby produce only limited amount of progeny. This characteristic is referred to as proliferative potential and can be assessed in a colony-forming assay.
  • a colony-forming assay can, for example be conducted as previously described by Jensen and colleagues (Jensen et al., 2010, Nat Protoc 5: 898-91 1 ). For example, 2000-4000 cells are plated on 6-well plates containing Mitomycin C-treated feeder cells (J2 fibroblasts). Cultures are incubated at 37°C, 5% CO2 for 12-14 days during which the medium is replaced every 2 days.
  • the method for culturing epidermis-derived stem cells as described in herein is further a method for amplifying epidermis-derived stem cells, wherein said method further comprises a step of isolating said epidermis-derived stem cells from said cell mixtures after said culturing.
  • the isolation/purification of epidermis- derived stem cells can be achieved by methods known in the art and as described herein elsewhere.
  • the same methods as used for isolating/purifying/separating epidermis-derived stem cells prior to culturing starting from a single cell suspension may be employed.
  • the step of isolation/purification/separation results in reducing the content of cells other than epidermis-derived stem cells below 1 %, below 2%, below 3%, below 5% or below 10% of cells other than epidermis-derived stem cells.
  • the method for culturing epidermis-derived stem cells is employed without employing "feeder cells” or conditioned medium produced by feeder cells.
  • feeder cells or as sometimes interchangeably referred to as “feeders” are cells, typically of fibroblastic origin, which are placed in layer in a cell culture dish and are simultaneously cultured with stem cells. Feeder cells in this context serve as a substrate for the stem cells to grow on. Inter alia, they may secrete a number of essential growth factors that are important for maintaining, for example, the multipotency of the cultured stem cells. Feeder cells can alternatively be cultured to produce a medium termed conditioned medium.
  • feeders are cultured for a period of time, typically 2-7 days, during which the cells secrete growth factors and other bioactive compounds into the culture medium.
  • This culture medium now termed condition medium, can subsequently be transferred to cells to promote their survival and growth.
  • condition medium can be transferred to cells to promote their survival and growth.
  • the precise composition of the condition medium is not defined.
  • the culturing of epidermis-derived stem cells can also be performed with epidermis- derived SCs, preferably hair follicle stem cells or cancer stem cells that are de novo generated ex vivo. Methods for de novo generating epidermis-derived SCs, preferably hair follicle stem cells or cancer stem cells, ex vivo are not known in the art but are also provided herein below.
  • said epidermis-derived stem cells are preferably epidermis- derived SCs, preferably hair follicle stem cells or cancer stem cells that are de novo generated by the method for ex vivo de novo generation of epidermis-derived SCs, preferably hair follicle stem cells or cancer stem cells, as described in any embodiment of the present invention.
  • the present invention also provides for a method for expanding epidermis-derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising Epidermal Growth Factor (EGF), a Vascular Endothelial Growth Factor (VEGF), a Fibroblast Growth Factor (FGF) and a Rho-kinase (ROCK) inhibitor.
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • expansion means that the number of cells, e.g. of a specific cell type (e.g. epidermis- derived stem cells) increases during the time of in vitro culturing by mitotic cell division.
  • expansion of the cultivated cells during culturing can be unlimited.
  • expansion means to increase the number of epidermis-derived stem cells by at least 1 ,5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or 200-fold during the time of culturing.
  • Culturing in this context relates to maintaining cells in vitro/ex vivo.
  • epidermis-derived stem cells are HFSCs, interfollicular epidermal stem cells and/or epidermis-derived CSCs. Even more preferably, epidermis-derived stem cells are HFSCs and/or epidermis-derived CSCs. Most preferably epidermis-derived stem cells are HFSCs.
  • the definitions and preferences as regarding the 3D-ECM/ECM and the basal cell culture medium as described in the context of the method for culturing epidermis-derived stem cells further below also apply mutatis mutandis for the method for expanding epidermis- derived stem cells.
  • the present invention further relates to a method for ex vivo de novo generation of epidermis-derived stem cells comprising the step of culturing epidermal cells that lack epidermis-derived stem cells intended to be de novo generated in the presence of a three-dimensional extracellular matrix (3D-ECM) as defined mutatis mutandis in any embodiment relating to 3D-ECM in the context of the method of culturing epidermis- derived stem cells elsewhere herein and a basal cell culture medium as defined mutatis mutandis in any embodiment relating to basal cell culture in the context of the method of culturing epidermis-derived stem cells elsewhere herein, wherein said culturing is performed for at least 2 days, at least 5 days, at least 12 days, at least 14 days or at least 360 days.
  • 3D-ECM three-dimensional extracellular matrix
  • epidermis-derived stem cells in this context are hair follicle stem cells, interfollicular epidermal stem cells and/or epidermis-derived cancer stem cells. More preferably, epidermis-derived stem cells in this context are hair follicle stem cells and/or epidermis- derived cancer stem cells. Most preferably epidermis-derived stem cells are HFSCs.
  • a shorter culturing time is preferred.
  • a higher yield of epidermis-derived stem cells can, however, be achieved by culturing said epidermal cells for at least at least 2 days, at least 6 days, at least 12 days, at least 18 days, at least 24 days or at least 30 days. The longer the culturing time, the higher is the yield.
  • the epidermal cells are preferably selected from a group consisting of the following cell types: epidermal cells of the interfollicular epidermis or of the hair follicle that are depleted of HFSCs as defined by the set of cell surface markers mentioned.
  • Epidermal cells of the interfollicular epidermis are particularly preferred in this context.
  • Preferred is also the usage of epidermal cells originating from a mammal that lack HFSCs, e.g. due to a disease and/or genetic defect.
  • non-HFSCs characterized in that they are CD34 " a6 + are employed.
  • the epidermal cells are preferably selected from a group consisting of HFSCs (characterized in that they are CD34+a6 + ) and epidermal cells of the interfollicular epidermis (characterized in that they are CD34 " a6 + ).
  • the epidermal cells are epidermal cells from epidermis-derived tumors (e.g. papilloma and/or carcinoma) that are depleted of CSCs as defined by the set of cell surface markers mentioned above are employed in this context.
  • epidermis-derived tumors e.g. papilloma and/or carcinoma
  • non-CSCs characterized in that they are CD140a ⁇ , CD31 " , CD45 “ , EpCAtvf, CD34 " a6 + are employed.
  • the present invention also relates to an epidermis-derived stem cell, wherein said epidermis-derived stem cell is obtainable by a method for culturing epidermis-derived stem cells according to the present invention or a method for de novo ex vivo generation of epidermis-derived stem cells.
  • the present invention further relates to a cell mixture comprising said epidermis-derived stem cell.
  • the present invention relates to a tissue fragment comprising said epidermis-derived stem cell.
  • said epidermis-derived stem cell according to the present invention is a HFSC, an interfollicular epidermis stem cell, or an epidermis-derived CSC (preferably a papilloma-derived or a carcinoma-derived epidermis-derived CSC).
  • the epidermis-derived stem cell is a HFSC.
  • the epidermis-derived stem cell and/or the cell mixture comprising said epidermis- derived stem cell and/or the tissue fragment comprising said epidermis-derived stem cell according to the present invention is preferably characterized by a higher potential of hair formation and/or interfollicular epidermis formation after transplantation.
  • Such higher potential may, for example, be measured by transplantation experiments into immunocompromised hairless mice where maintenance of multipotency can be measured by the ability of the said stem cells to regenerate hair and/or interfollicular epidermis.
  • transplantation assays are also known in the art as "full thickness skin reconstitution assays" as described for example in Jensen et al., 2010, Nat Protoc, 5: 898-91 1.
  • epidermis-derived cells containing stem cells are mixed with dermal fibroblasts (preferentially around 5 x 10 6 fibroblasts) and grafted onto the back of immunocompromised hairless mice.
  • dermal fibroblasts preferentially around 5 x 10 6 fibroblasts
  • epidermis-derived stem cells When epidermis-derived stem cells are present in the transplanted cell mixture, they will generate hair follicles that will subsequently grow hair and/or interfollicular epidermis de novo.
  • a higher/increased potential of hair formation can, for example be measured by comparing the amount of hair follicles generated by the cultured and freshly isolated epidermis-derived stem cells. In principle, also the morphology of the grown hair may be compared.
  • a higher/increased potential of interfollicular epidermis formation can, for example, be measured by comparing the area (e.g. in cm 2 ) of newly grown interfollicular epidermis between cultured and freshly isolated epidermis-derived stem cells. Alternatively, also the speed of interfollicular epidermis growth may be compared by comparing the times in which a distinct area of interfollicular epidermis has been generated.
  • HFSCs are characterized by a higher/increased potential of hair follicle/hair formation.
  • interfollicular epidermal stem cells are preferably characterized by a higher/increased potential of interfollicular epidermis formation. Bona fide multipotent HFSCs and interfollicular epidermis stem cells are capable of generating both hair follicles and interfollicular epidermis. These transplantation experiments can be performed with either mouse or human cells.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a epidermis-derived stem cell according to the present invention, a cell mixture according to the present invention, or tissue fragment according to the present invention along with other cell types such as fibroblasts and/or an ECM scaffold.
  • the present invention further provides an epidermis-derived stem cell according to the present invention, a cell mixture comprising said epidermis-derived stem cell according to the present invention, a tissue fragment comprising said epidermis-derived stem cell or a pharmaceutical composition according to the present invention for use as a medicament.
  • the invention also relates to corresponding method of treatments.
  • the invention also relates to an epidermis-derived stem cell according to the present invention, a cell mixture comprising said epidermis-derived stem cell according to the present invention, a tissue fragment comprising said epidermis-derived stem cell or a pharmaceutical composition according to the present invention for use in tissue transplantation.
  • the tissue in this context is skin or even more preferably skin with hairs.
  • said epidermis-derived stem cell is a hair follicle stem cell and said tissue transplantation reconstitutes hair growth.
  • an epidermis-derived stem cell according to the present invention a cell mixture comprising said epidermis-derived stem cell according to the present invention, a tissue fragment comprising said epidermis-derived stem cell or a pharmaceutical composition according to the present invention for use in treatment of dermal burn, treatment of conditions where areas of skin have been removed due to surgical operation, biopsy or trauma, chronic wounds, baldness is provided.
  • baldness is selected from the group consisting of male pattern baldness, nutrition-induced baldness, infection-induced baldness, drug-induced baldness, traction alopecia or other trauma-induced baldness, pregnancy-induced baldness, alopecia areata and/or alopecia mucinosa.
  • the present invention also relates to a method of treating baldness.
  • baldness is preferably selected from the group consisting of male pattern baldness, nutrition- induced baldness, infection-induced baldness, drug-induced baldness, traction alopecia or other trauma-induced baldness, pregnancy-induced baldness, alopecia areata and/or alopecia mucinosa.
  • the method of treating baldness is exclusively used for cosmetic purposes.
  • the present invention further provides uses of an epidermis-derived stem cell, a cell mixtures comprising said epidermis-derived stem cell or a tissue fragment comprising said epidermis-derived stem cells according to the present invention.
  • the present invention also relates to an epidermis-derived stem cell, a cell mixtures comprising said epidermis-derived stem cell or a tissue fragment comprising said epidermis-derived stem cells according to the present invention for in vitro tissue production.
  • the tissues generated are skin or skin comprising hair follicle (preferably also hairs).
  • Said in vitro tissue production may, for example be achieved by isolating epidermal cells from a tissue biopsy. These cells can be cultured using the method of the invention allowing expansion of multipotent epidermis-derived stem cells or alternatively generation of hair follicle stem cells de novo from hairless epidermis and their subsequent expansion.
  • This method would thus allow generation of epidermis- derived stem cells in large quantities for transplantation, or alternatively for generation of epidermal organs in vitro (so called organotypic cultures; Oh et al., 2013, J Invest Dermatol 133: e14 doi:10.1038/jid.2013.387) that can be grafted or transplanted to recipients at a later time point.
  • the present invention relates to an epidermis-derived stem cell, a cell mixtures comprising said epidermis-derived stem cell or a tissue fragment comprising said epidermis-derived stem cells according the present invention for in vitro drug discovery screening.
  • in vitro drug discovery screening refers to a method which comprises the step of contacting said epidermis-derived stem cells with one or more molecules that are intended as a medicament.
  • a method may further comprise a step that allows for determining whether the intended medical effect is achieved.
  • said drug discovery screening is performed to identify molecules/compounds that affect stem cell function, quiescence, growth and/or differentiation.
  • said drug discovery screening is performed to identify molecules/compounds that modulate hair growth. Modulating hair growth includes increasing or decreasing hair growth upon application of said molecule/compound (e.g. systematically or locally) to an organism (preferably a mammal, most preferably a human).
  • An increase or decrease in hair growth may, for example, be an increase in the number of hairs of at least 2%, at least 4%, at least 6%, at least 10% or at least 20%, whereas a decrease in hair growth may, for example, be an decrease in the number of hairs of at least 2%, at least 4%, at least 6%, at least 10% or at least 20%.
  • the present invention further relates to a method for culturing epidermis-derived stem cells, wherein said method additionally comprises a step of in vitro generating a tissue from the cultured epidermis-derived stem cells.
  • the invention also provides a method for ex vivo de novo generation of epidermis-derived stem cells according to the present invention, wherein said method additionally comprises a step of in vitro generating a tissue from the cultured epidermis-derived stem cells.
  • the present invention provides a method for culturing epidermis-derived stem cells, wherein said method additionally comprises a step of incubating said cultured epidermis-derived stem cells with a drug candidate.
  • said drug candidate is a molecule as described above for the use of an epidermis-derived stem cell according to the present invention for in vitro drug discovery screening. Most preferably said drug candidate modulates stem cell function, quiescence, growth and/or differentiation and/or hair growth.
  • the invention also provides a method for ex vivo de novo generation of epidermis-derived stem cells according to the present invention, wherein said method additionally comprises a step of incubating said cultured epidermis-derived stem cells with a drug candidate.
  • the same preferences for the drug candidate as described above for the method of culturing epidermis-derived stem cells apply.
  • the present invention also relates to a cell culture medium as defined mutatis mutandis in the sections describing a method for culturing epidermis-derived stem cells or a method for ex vivo de novo generation of epidermis-derived stem cells. Accordingly, the present invention, for example, relates to a cell culture medium which is a basal cell culture medium that further comprises:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the invention provides a cell culture medium which is a basal cell culture medium that further comprises:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • the present invention also relates to a cell culture medium which is a basal cell culture medium that further comprises:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • a preferred cell culture medium is a basal cell culture medium that comprises:
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • said basal cell culture medium further comprises a SHH inhibitor.
  • the present invention also relates to uses of a method for culturing epidermis-derived stem cells and/or a method for ex vivo de novo generation of epidermis-derived stem cells according to the present invention.
  • Everything mentioned in the context of the methods of the present invention applies here mutatis mutandis.
  • expansion/growing means that the number of cells is increased during the time of in vitro culturing by mitotic cell division.
  • expansion of the cultivated cells during culturing can be unlimited.
  • expansion means to increase the number of epidermis-derived stem cells by at least 1 ,5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or 200-fold during the time of culturing.
  • a differentiated epidermal cell type can be a differentiated epidermal cell type as defines elsewhere herein.
  • Another use of method for culturing epidermis-derived stem cells and/or a method for ex vivo de novo generation of epidermis-derived stem cells according to the present invention is the use for enriching said epidermis-derived stem cells provided in said mixture of cell types or said isolated tissue fragment relative to the other cell type(s) comprised in said mixture of cell types or said isolated tissue fragment.
  • the present invention relates to the following items:
  • a method for culturing epidermis-derived stem cells comprising the step of culturing epidermis-derived stem cells in the presence of a three-dimensional extracellular matrix (3D-ECM) and a basal cell culture medium comprising: - Epidermal Growth Factor (EGF); and/or
  • 3D-ECM three-dimensional extracellular matrix
  • EGF Epidermal Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • FGF Fibroblast Growth Factor
  • ROCK Rho-kinase
  • said basal cell culture medium comprises said EGF, said VEGF, said FGF and said ROCK inhibitor.
  • VEGF Vascular Endothelial Growth Factor
  • VEGF is preferably selected from the group consisting of VEGF-164, VEGF-165, VEGF-121 , VEGF-120 and VEGF-188. More preferably, VEGF is selected from the group consisting of VEGF-164, VEGF-165 and VEGF-121. Most preferably, VEGF is selected from the group consisting of VEGF-164 and VEGF-121.
  • Fibroblast Growth Factor is selected from the group consisting of : FGF-1 , FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-10, FGF-22, FGF-8, FGF-17, FGF-18, FGF-24, FGF-9, FGF-16, FGF-20, FGF-11 , FGF-12, FGF-13, FGF-14, FGF-19, FGF-21 , and FGF-23.
  • FGF is preferably selected from the group consisting of FGF-2, FGF-7, FGF-10 and FGF-18. Even more preferably FGF is from the group consisting of FGF-2 and FGF-18.
  • ROCK inhibitor is selected from the group consisting of (R)-(+)-trans-4-(1-aminoethyl)-N-(4- Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1 ,4- diazepan-1-ylsulfonyl) isoquinoline (fasudil or HA 1077), and (S)-(+)-2-methyl-1- [(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1 H-1 ,4 diazepine dihydrochloride (H-1152).
  • ROCK inhibitor is preferably selected from the group consisting of (S)-(+)-2-methyl-1 -[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1 H-1 ,4 diazepine dihydrochloride (H-1152) and (R)-(+)-trans-4-(1-aminoethyl)-N-(4-
  • ROCK inhibitor is (R)-(+)-trans-4-(1-aminoethyl)-N-(4- Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632).
  • EGF is comprised in the basal cell culture medium at a concentration of between 2 ng/ml and 500 ng/ml, preferably of between 5 ng/ml and 100 ng/ml or most preferably of between 10 ng/ml and 50 ng/ml.
  • VEGF is comprised in the basal cell culture medium at a concentration of between 2 ng/ml and 500 ng/ml, preferably of between 5 ng/ml and 100 ng/ml or most preferably of between 10 ng/ml and 50 ng/ml.
  • SHH inhibitor is selected from the group consisting of (2 , R,3S,3 , 3 , aS,6'S,6aS,6bS,7 , a 11aS,11 bR)- 1 I 2 > 3 > 3 , a,4 ⁇ 5 , ,6,6 , I 6a,6b > 7,7 , ,7 , a,8,11 ,11a,11b-OctadecahydiO-3 , ,6 , ,10,11b- tetramethylspiro[9H-benzo[a]fluorene-9,2'(3'/-/)-furo[3,2-ib]pyridin]-3-ol
  • SHH inhibitor is comprised in the basal cell culture medium at a concentration of between 1 ⁇ and 20 ⁇ , preferably between 5 ⁇ and 15 ⁇ , most preferably between 5 ⁇ and 10 ⁇ .
  • said basal culture medium does not comprise a Bone Morphogenetic Protein (BMP) inhibitor.
  • BMP Bone Morphogenetic Protein
  • the BMP inhibitor is selected from the group consisting of Noggin, 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4- ylpyrazolo[1 ,5-a]pyrimidine;dihydrochloride (Dorsomorphin), 3-[6-amino-5-(3,4,5- trimethoxyphenyl)pyridin-3-yl]phenol (K02288), 4-[6-(4-propan-2- yloxyphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl]quinoline (DMH.1 ), 2-(hydroxymethyl)- 6-[3-(hydroxymethyl)phenoxy]oxane-3,4,5-triol (ML 347), 4-[6-(4-piperazin-1- ylphenyl)pyrazolo[1 ,5-a]pyrimidin-3-yl]quinoline (LDN-193189).
  • said basal cell culture medium further comprises ethanolamine, phospho-ethanolamine and/or transferrin.
  • said basal cell culture medium further comprises L-glutamine, insulin, hydrocortisone, penicillin, streptomycin, calcium-depleted fetal calf serum and/or calcium-depleted fetal bovine serum.
  • said method is further a method for expanding said epidermis-derived stem cells.
  • HFSCs hair follicle stem cells
  • CSCs cancer stem cells
  • the method of item 22 wherein said cancer stem cells are papilloma-derived or carcinoma-derived stem cells.
  • said epidermis-derived stem cells are papilloma-derived or carcinoma-derived stem cells.
  • - are comprised in a mixture of cell types, which further comprises at least one differentiated epidermal cell type;
  • said mixture of cell types comprises isolated keratinocytes, epidermal papilloma cells or epidermal carcinoma cells; or
  • any one of items 1 to 25 which is a method for generating a mixture of cell types comprising epidermis-derived stem cells and at least one differentiated cell type.
  • the method of item 25 or 26 wherein said method is a method for enriching said epidermis-derived stem cells provided in said mixture of cell types or said isolated tissue fragment relative to the other cell type(s) comprised in said mixture of cell types or said isolated tissue fragment.
  • any one of items 1 to 28, wherein said 3D-ECM is a synthetic hydrogel 3D-ECM or a naturally occurring 3D-ECM.
  • said 3D-ECM comprises one or more component selected from the group consisting of collagen type I, collagen type II, collagen type III, collagen type IV, laminin-111 , laminin-211 , laminin-121 , laminin-221 , laminin-332, laminin-3B32, laminin-311 , laminin-32 , laminin-411 , laminin-421 , laminin-511 , laminin-521 , laminin-213, laminin-423, laminin-522, laminin-523, entactin, perlecan, vitronectin and heparan sulfate proteoglycan.
  • the ECM is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. In a solidified form it forms a 3D-ECM.
  • EHS Engelbreth-Holm-Swarm
  • ECM comprises or consists of 60% to 85% laminin, 5 to 30% collagen IV, 1 to 10% entactin, and 1 to 10% heparan sulfate proteoglycan.
  • a method for ex vivo de novo generation of epidermis-derived stem cells comprising the step of culturing epidermal cells lacking epidermis-derived stem cells in the presence of an three-dimensional extracellular matrix (3D-ECM) as defined by any one of items 1 and 29 to 39 and a basal cell culture medium as defined in any one of items 1 to 19 for at least 2 days, at least 4 days, at least 6 days, at least 8 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, at least 20 days, at least 100 days, at least 200 days, at least 360 days.
  • 3D-ECM three-dimensional extracellular matrix
  • epidermis-derived stem cells are preferably HFSCs and/or epidermis- derived CSCs, most preferably HFSCs.
  • said de novo generated epidermis-derived stem cells are HFSCs and the epidermal cells comprise or consist of: epidermis-derived stem cells other than HFSCs, differentiated epidermal cells or mixtures with all possible combinations of said stem cells and differentiated cells;
  • said de novo generated epidermis-derived stem cells are epidermal stem cells other than HFSCs and the epidermal cells comprise or consist of: HFSCs, differentiated epidermal cells or mixtures with all possible combinations of said stem cells and differentiated cells; or
  • said de novo generated epidermis-derived stem cells are CSCs and the epidermal cells comprise or consist of: epidermal cells from premalignant, inflamed or otherwise diseased skin, epidermal cells from epidermal tumor tissue, or mixtures with all possible combinations of said stem cells, differentiated cells, premalignant cells and/or tumor cells.
  • An epidermis-derived stem cell that is obtainable by a method as defined in any one of the items 1 to 44.
  • a cell mixture comprising said epidermis-derived stem cell of item 45.
  • a tissue fragment comprising said epidermis-derived stem cell of item 45.
  • a pharmaceutical composition comprising an epidermis-derived stem cell of item 45, a cell mixture of item 46, or a tissue fragment of item 47.
  • the epidermis-derived stem cell, the cell mixture, the tissue fragment or the pharmaceutical composition according to item 51 for the use according to item 5 wherein said epidermis-derived stem cell is a hair follicle stem cell and said tissue transplantation reconstitutes hair growth.
  • Figure 1 Standard keratinocyte culture conditions do not support growth of CD34 + a6 + HFSCs.
  • Y EGF + Y27632
  • YV Y27632 + EGF + VEGF
  • YF Y27632 + EGF + FGF-2
  • 3C Y27632 + EGF + VEGF + FGF-2.
  • Epi dO Freshly isolated epidermal cells, KGM 2D: basal medium in 2D; 3C 2D: Y27632 + EGF + VEGF + FGF-2 in 2D; Y-E: Y27632 without EGF in the basal medium in 3D-ECM; Y: EGF + Y27632 in 3D-ECM, YV: Y27632 + EGF + VEGF in 3D-ECM; YF: Y27632 + EGF + FGF-2 in 3D-ECM; 3C: Y27632 + EGF + VEGF + FGF-2 in 3D-ECM.
  • Y Y27632 + EGF
  • YV Y27632 + EGF + VEGF
  • YF Y27632 + EGF + FGF-2
  • 3C Y27632 + EGF + VEGF + FGF-2. All conditions are in 3D-ECM.
  • CD34 + a6 + cells normalized numbers of CD34 + a6 + cells from day 14 HFSC cultures in 3C media with (3C) and without (3C-E) EGF.
  • 3C-E Y27632 + VEGF-164 + FGF-2 without EGF in basal medium. All conditions are in 3D-ECM.
  • D Representative FACS plots of freshly isolated (day 0; dO) and keratinocytes cultured in 3C conditions for 2 weeks (day 14; d14). Gates were drawn according to the respective unstained and isotype-stained controls. Percentages are indicated per quadrant.
  • CD34 + a6 + cells were quantified by flow cytometry from day 14 Matrigel cultures containing various growth factor combinations. Normalized CD34 + a6 + content of cultures is shown.
  • A Direct comparison of different VEGF and FGF isoforms in 3C conditions (3D-ECM; basal cell culture medium comprising Y27632+EGF+FGF+VEGF).
  • VEGF-121 performs equally well as VEGF-164 (set to 1 ).
  • FGF-12, FGF-10, and FGF-7 perform equally well as FGF-2 (set to 1).
  • B Direct comparison of VEGF-121 and VEGF- 164 in YV conditions (3D-ECM; basal cell culture medium comprising Y27632+EGF+VEGF).
  • VEGF-121 performs slightly better than VEGF-164 (set to 1 ).
  • C Direct comparison of different FGF isoforms in YF conditions (3D-ECM; basal cell culture medium comprising Y27632+EGF+FGF).
  • FGF-12, FGF-10, and FGF-7 perform equally well as FGF-2 (set to 1 ).
  • CD34 + a6 + cells were quantified by flow cytometry from day 14 3C cultures in various ECM component-containing 3D-ECM gels. CD34 + a6 + content normalized to 3C Matrigel cultures is shown.
  • Col I Collagen type I
  • L332 laminin 332
  • L511 laminin 511
  • BME basement membrane extract.
  • Figure 6 3C cultures enrich for CD200 + cells
  • Figure 7 Morphology of cells cultured under 3C conditions
  • FIG. 10 3C cultures contain exclusively epithelial cells Representative FACS histogram of day 14 (d14) HFSC cultures stained for EpCAM and quantified by flow cytometry. Unstained cells are shown as control. Percentage of EpCAIVf cells ⁇ SD from 6 independent cultures is shown.
  • qPCR Quantitative PCR analysis of Epi dO and 3C shows that cells in 3C conditions upregulate HFSC identity genes (e.g. Cd34, Sox9, Tcf3, Wnt7a).
  • HFSCs cultured under 3C conditions can be freeze-thaw Representative FACS plots of HFSC cultures before (day 14; d14) and 14 days after freeze-thaw. Cells were frozen for at least a month before thawing them. Thawed cells grew for additional 14 days before analysis.
  • Colony forming assays were performed with 3000 cells/well derived from three mice. Representative results (A) and quantification (B) are shown. Freshly isolated epidermal cells were used as a control (mean ⁇ SEM; *>p 0.05, Mann-Whitney U-test). YF: Y27632 + EGF + FGF-2; 3C: Y27632 + EGF + VEGF + FGF-2. All conditions were performed in triplicates.
  • Figure 15 Morphology of colonies from colony forming assay of HFSCs cultured under 3C conditions
  • Figure 16 HFSCs cultured under 3C conditions maintain their multipotency and self-renewal potential in a skin reconstitution assay
  • A Representative images of recipient mice transplanted with either freshly isolated epidermal cells (Epi dO) or cultured cells cultured under 3C conditions (3C). Note that animals transplanted with cells cultured in 3C developed more hair than Epi dO controls indicating their multipotency and self-renewal capacity after 14 days of culture.
  • B H&E staining from transplant's biopsies.
  • C Hair follicle quantification from Hematoxylin & Eosin staining.
  • CD34 + a6 + HFSCs and CD34 " a6 + non-HFSCs were purified from total epidermis into at least 98% purity and cultured under 3C conditions for 14 days. FACS plots of day 14 cultures established from the indicated purified cell populations show that both cultures consist of a significant population of CD34 + a6 + HFSCs and CD34 " a6 + non-HFSCs.
  • B. CD34 + a6 + HFSCs were completely depleted by culturing total epidermal cells in 2D growth conditions for 14 days (2D 14d). The resulting CD34 " a6 + non-HFSCs were subsequently passaged and cultured under 3C conditions for additional 14 days (3D-3C d14). Shown are FACS blots of the indicated cells and the percentage of CD34 + a6 + HFSCs is indicated.
  • CD34 + a6 + and CD34 " a6 + cells in 3C cultures were monitored every 2 days for a period of 14 days.
  • Cell populations (A) and absolute numbers (B) were quantified by flow cytometry.
  • Cells were grown for 9 days under 3C conditions and treated with the SHH inhibitor cyclopamine (10 ⁇ ) for 5 days. Treated cultures and untreated 3C control cultures were analyzed by flow cytometry on day 14.
  • Figure 21 Papillomas contain phenotypically defined CSCs in a mouse model of skin cancer
  • Papillomas arising in the K14rTA tet-O-Kras mouse model were stained for CSCs markers defined as Lin " EpCAM + CD34 + a6 + .
  • Representative FACS plots from unaffected skin and tumor material. Gates were drawn according to the respective unstained and isotype-stained controls. Percentages are indicated per quadrant. n>12 tumors; n 5 mice. SEM is shown.
  • FIG. 22 Papilloma CSCs can be grown and maintained in vitro under different culture conditions Single cell suspensions prepared from papillomas of the K14rTA tet-O-Kras mouse model were grown using different media that support HFSC growth.
  • Tumor cells isolated from tumors; KGM: keratinocyte growth medium.
  • YV Y27632 + EGF + VEGF; YF: Y27632 + EGF + FGF-2; 3C: Y27632 + EGF + VEGF + FGF-2.
  • Figure 23 3C culture conditions support enrichment of papilloma CSCs in long- term cultures
  • A FACS plots of papilloma cells before (Tumor: freshly isolated tumor cells) and after (2 weeks of culture (pO) and 12 weeks of culture (p5)) culture.
  • B Percentage of EpCAM + CD34 + a6 + cells from papilloma CSC cultures quantified by flow cytometry.
  • Figure 24 Morphology of papilloma CSCs cultured under 3C conditions
  • Example 1 Development of culture conditions for culturing HFSCs and experimental characterization of such cultivated HFCSs
  • Keratinocytes were isolated from C57BL/6 mice on postnatal day (P) 21 , unless stated otherwise. 7-9 weeks old BALB/c-nude mice (CAnN.Cg-Foxn nu/Crl; Charles River, Germany) were used as recipients in transplantation experiments. Primary dermal fibroblasts were isolated from C57BL/6 mice on P2. Animals were housed and maintained according to FELASA guidelines in the animal facility of the Max Planck Institute for Biology of Ageing, Cologne, Germany. All experiments were approved by local authorities. Reagents and antibodies
  • the following components were used to prepare the basal keratinocyte growth medium (KGM basal medium): MEM (Spinners modification, Sigma), 5 pg/mL Insulin (Sigma), 10 pg/mL Transferrin (Sigma), 10 ⁇ Phosphoethanolamine (Sigma), 10 ⁇ Ethanolamine (Sigma), 0.36 pg/mL Hydrocortisone (Calbiochem), 2 mM Glutamine (Gibco), 100 U/mL Penicillin and 100 pg/mL Streptomycin (Gibco), 8% chelated fetal calf serum (FCS) or fetal bovine serum (FBS) (Gibco).
  • MEM Spinners modification, Sigma
  • 5 pg/mL Insulin Sigma
  • 10 pg/mL Transferrin Sigma
  • 10 ⁇ Phosphoethanolamine Sigma
  • 10 ⁇ Ethanolamine Sigma
  • 0.36 pg/mL Hydrocortisone Calbiochem
  • the following components were used to prepare the FAD medium: 2 parts of DMEM (Gibco) and 1 part of Ham's F12 (Gibco), 2 mM Glutamine (Gibco), 100 U/mL Penicillin and 100 pg/mL Streptomycin (Gibco), 10% chelated FCS or FBS (Gibco), 50 pg/ml Vitamine C (Sodium L-ascorbate; Sigma), 10 ng/ml EGF (Sigma), 5 pg/ml Insulin (Sigma), 0.5 pg/ml Hydrocortisone (Calbiochem, 0.1 nM cholera toxin (Sigma), 180 pM Adenin (Sigma).
  • Y-27632 Growth factor reduced Matrigel (BD Biosciences), (R)-(+)-trans-4-(1-aminoethyl)-N-(4- Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (referred to as Y-27632; Sigma-Aldrich), human recombinant FGF-2, human recombinant FGF-7, human recombinant FGF-10 (all from Miltenyi Biotec), human recombinant FGF-18 (Peprotech) mouse recombinant VEGF-164, human recombinant VEGF-121 (all from Miltenyi Biotec), human recombinant EGF (LONG-EGF; Sigma), rat tail collagen type I (Millipore), human recombinant Laminin-332 and human recombinant Laminin-51 (Biolamina) were used for cell culture experiments.
  • Y-27632 Sigma-Aldrich
  • Collagenase (C0130, Sigma) was used for cell recovery from collagen type I gels. 7AAD (eBioscience) and Fixable Viability Dye eFluor405 (eBioscience) were used to discriminate dead cells for flow cytometry.
  • eFluor660-CD34 (clone RAM34, eBioscience), Pacific blue-aplha-6 Integrin (clone GoH3, eBioscience), FITC- CD133 (clone 13A4, eBioscience), PE-CD200 (clone OX90, eBioscience), PE-Cy7- EpCAM (clone G8.8, eBioscience), PE-CD140a (clone APA5, eBioscience); PE-CD45 (clone 30-F11 , eBioscience), PE-CD31 (clone MEC13.3, eBioscience), APC-Cy7-beta-1 Integrin (clone HMB1-1 , eBioscience), SOX9 (H-90, Santa Cruz Biotechnology), Keratin15 (LHK 5, NeoMarkers, Fremon, CA). Secondary antibodies for immunofluorescence anti-mouse AlexaFluor 488 and anti-rabbit AlexaFluor 568
  • Keratinocytes were isolated from back skin of mice by incubating skin pieces in 0.8% Trypsin (Gibco) for 50 min at 37°C. After separating the epidermis from the underlying dermis, cells were homogenized, filtered through 70 pm and 45 pm cell strainers (BD Biosciences) and pelleted at 900 rpm for 3 min. For 2D culture of epidermis-derived cells, tissue culture petri dished were coated with a mixture of collagen I (30 pg/ml) + fibronectin (10 pg/ml; both from Millipore) in MEM for 1h at 37°C prior to plating of cells.
  • collagen I (30 pg/ml) + fibronectin (10 pg/ml; both from Millipore
  • Epidermis-derived cells were suspended in KGM or in FAD medium, and seeded on the coated plastic dishes. Medium was exchanged the next day after initial seeding and thereafter every second day. Cultures were incubated at 37°C, 5% CO 2 for 12-14 days. For 3D culture of HFSCs, 8 x 10 4 cells were resuspended in 40 ⁇ ice-cold 1 :1 KGM:Matrigel mixture that was dispensed as a droplet in 24-well cell culture dishes. The suspension was allowed to solidify for 15 min after which it was overlaid with 500 ⁇ of stem cell media.
  • KGM basal medium KGM basal medium
  • medium Y KGM, 5 ⁇ Y27632, 10 ng/ml human recombinant EGF
  • medium Y-E KGM, 5 ⁇ Y27632
  • medium YV KGM, 5 ⁇ Y27632, 10 ng/ml human recombinant EGF, 20 ng/ml mouse recombinant VEGF
  • medium YF KGM, 5 ⁇ Y27632, 10 ng/ml human recombinant EGF, 20 ng/ml human recombinant FGF-2
  • 3C KGM, 5 pM Y27632, 10 ng/ml human recombinant EGF, 20 ng/ml mouse recombinant VEGF, 20 ng/ml human recombinant FGF-2
  • 3C-E KGM, 5 pM Y27632, 20 ng/ml mouse recombinant VEGF, 20 ng/ml
  • hair follicle stem cells were extracted from Matrigel by mechanical homogenization and incubation in 0.5% Trypsin (Gibco), 0.5 mM EDTA, PBS or Accutase (Gibco) for 10 min at 37°C. Cells were passaged every 10-14 days in a 1 :3 to 1 :5 ratio in fresh 1 :1 KGM (where necessary with the respective supplements):Matrigel.
  • Proliferative potential assay A colony-forming assay to determine proliferative potential of stem and progenitor cells was conducted as previously described (Jensen et al., 2010, Nat Protoc 5: 898-911). Briefly, 2000-4000 cells were plated on 6-well plates containing Mitomycin C-treated feeder cells (J2 fibroblasts). Cultures were incubated at 37°C, 5% C0 2 for 12-14 days during which the medium was replaced every 2 days. Experiments were terminated when colonies reached a sufficient size to be visually identified and quantified. Colonies were fixed with 4% PFA 10 min and then stained with 1% crystal violet. Colony number and area were determined using the ImageJ software. All conditions were performed in triplicates.
  • Paraffin-embedded tissue sections were deparaffinized with Xylol and rehydrated with consecutive washes of 100 % isopropanol, 95 %, 75 %, 50 % ethanol, and distilled water. Sections were stained for 1 min with Hematoxylin and counterstained for 10 sec with Eosin. Sections were then dehydrated in 50 %, 75 %, 95 % ethanol and isopropanol, cleared with Xylol, and mounted in Entellan (Merk).
  • Cultured hair follicle stem cells were rinsed once in PBS, followed by fixation in 2% PFA, PBS for 30 min at room temperature (RT). Fixed cells were rinsed three times with 100 mM glycine, PBS, then permeabilized and blocked for 2 h at 37°C in 0.3% Triton-X 100, 5% BSA, PBS. Cells were stained with unlabeled or fluorescent primary antibodies in 0.3% Triton-X 100, 1 % BSA, PBS overnight at RT. Secondary, fluorescent antibodies were used to detect primary antibody binding and nuclei were visualized with DAPI. Slides were mounted with Elvanol mounting medium (0.2 M Tris pH 6.5, 12% polyvinyl alcohol, 30% glycerol, 2.5% DABCO-anti fade reagent).
  • Fluorescent images were collected by laser scanning confocal microscopy (TCS SP5X; Leica) with x63 or x40 immersion objectives using LAS X software. All images were recorded sequentially and averaged at least twice. Image processing (linear brightness and contrast enhancement) was performed with Fiji Software version 2.0.0 or Adobe Photoshop CS5.
  • Murine keratinocytes were cultured employing standard culture conditions. Standard growth conditions are defined by the use of KGM or FAD medium, a widely used standard keratinocyte culture medium (Watt & Green, 1982, Nature 295:434-436), as growth media. In addition the cells are cultured in 2-dimensional (2D) culture conditions, which are achieved by coating a tissue culture petri dish with ECM components (such as Collagen I), allowing them to polymerize and subsequently plating the cells on top of the ECM surface.
  • 2D 2-dimensional
  • keratinocytes were embedded in Matrigel or Culturex (from now on referred to as 3D-Matrigel ECM), and cultured in KGM comprising EGF. Under these conditions no cell growth was observed. Yet, keratinocyte survival and/or growth/expansion could be established in the presence in 3D-Matrigel ECM by adding EGF and a ROCK inhibitor (e.g. Y27632) to the KGM culture medium (Fig. 2 and Fig. 3, see Y). As determined by flow cytometry analyses the population of CD34 + a6 + HFSCs was significantly increased under this culturing condition employing a medium comprising EGF and the ROCK inhibitor Y27632 (termed Y medium; see Fig. 3).
  • EGF EGF
  • ROCK inhibitor e.g. Y27632
  • CD34 + a6 + HFSC population within the keratinocyte population in 3D-Matrigel ECM could be achieved by adding FGF-2 and/or VEGF (both mitogenic growth factors) in addition to EGF and the ROCK inhibitor Y27632 to the KGM culture medium (Fig. 2 and Fig. 3).
  • FGF-2 and/or VEGF both mitogenic growth factors
  • culturing keratinocytes in 3D- Matrigel ECM and KGM medium comprising Y27632, EGF and FGF-2 (from here on termed YF culturing conditions; the respective medium is referred to as YF medium) or Y27632, EGF and VEGF (termed YV culturing conditions; the respective medium is referred to as YV medium) increased the relative contribution of the CD34 + a6 + HFSC population.
  • the YF or YV culturing conditions did not increase the absolute numbers of CD34 + a6 + HFSCs, suggesting that both conditions rather promote HFSCs survival than HFSCs growth/expansion.
  • 3C culturing conditions in 3D-Matrigel ECM and KGM medium comprising Y27632, EGF, a FGF (e.g. FGF-2) and VEGF (referred from here on as 3C culturing conditions; the respective medium is referred to as 3 C medium) had a significant positive effect not only on the survival but also on the growth/expansion of CD34 + a6 + HFSCs within the cultured keratinocyte population.
  • ⁇ 6-fold increase in the percentage and ⁇ 5-fold increase in absolute numbers was achieved by the 3C culturing conditions (Fig. 3).
  • EGF is a preferred component of the 3C medium that is employed under the 3C culturing conditions.
  • ROCK inhibitor in the present example Y27632
  • 3C-Y 3C medium without ROCK inhibitor
  • a ROCK inhibitor and specifically Y27632 is a preferred component of the 3C medium that is preferably used during the complete culturing time.
  • keratinocytes cultured in the 3C medium in 3D-Matrigel ECM also expressed the surface marker CD200 (Fig. 6) that marks stem/progenitor cells located in the bulge and hair germ compartments in human and mouse skin (Garza et al., 201 1 , J Clin Invest. 121 (2): 613-622). While 19.9 ⁇ 1.5% of freshly isolated keratinocytes from P21 mice expressed CD200, 60.4 ⁇ 12.1 % of the cells grown under 3C culturing conditions were CD200 + (3-fold increase). Similar to the fold enrichment of the CD34 + a6 + HFSCs in 3C cultures the absolute numbers of CD200 + cells were increased 5-fold (Fig. 6).
  • RNA sequencing analysis of keratinocytes cultured in the 3C medium in the presence of 3D-Matrigel ECM confirmed that the global gene expression profile of these cells more closely resembled purified HFSCs than the total epidermal cells (see Principle component analyses (PCA) shown in Fig. 11A and B and Heat map shown in Fig. 11C), supporting the fact that the 3C culturing conditions enrich for HFSCs.
  • PCA Principle component analyses
  • Fig. 11C Heat map shown in Fig. 11C
  • HFSCs cultured in 3D-ECM under 3C conditions were passaged every two weeks into fresh 3D-Matrigel ECM for a period of up to 32 weeks with no evident change in their potential to grow/expand and survive.
  • the percentage of CD34 + a6 + HFSCs within the cell mixture remained constant from the first passage onward (Fig. 12).
  • freeze-thaw experiments demonstrated that cultured HFSCs could be stored frozen and cultured again without evident loss of HFSCs or proliferative capacity (Fig. 13).
  • HFSCs cultured under 3C conditions retain their proliferative potential and multi potency
  • colony-forming assays that are the golden standard to quantitatively assess the proliferative potential of SCs (Jensen et al., 2010, Nat Protoc 5: 898-911 ) were performed.
  • HFSCs (originating from cultivation under 3C conditions) plated on feeders at clonal density gave rise to more colonies that were also larger in size compared to freshly isolated keratinocytes containing 5.6 ⁇ 1.2% HFSCs (Fig. 14).
  • Colonies derived from HFSCs that originated from 3C culturing conditions contained small, tightly packed, cobble stone-like colonies (Fig. 15) that are characteristic for holoclones observed in feeder-dependent 2D-cultures of HFSCs (Blanpain et al., 2004, Cell 118: 635-648; Greco et al., 2009, Cell Stem Cell 4: 155-169). This indicates that the 3C culture system enriches for HFSCs with high proliferative potential.
  • HFSC cultures can be derived both from bona fide HFSCs as well as from committed progeny
  • FACS-sorted HFSCs CD34 + a6 +
  • non-HFSCs non-HFSCs
  • Both cell populations were able to give rise to cultures containing CD34 + a6 + and CD34 " a6 + under 3C culture conditions (Fig. 17A).
  • HFSC were completely depleted from cultures by culturing total epidermal cells in 2D for 14 days (2D 14d) and subsequently sub-culturing (passaging) these cells (non-HFSCs; CD34 " a6 + ) in 3C conditions.
  • HFSCs CD34 + a6 +
  • Fig. 17B Fig. 17B
  • 86 cultures established an equilibrium of approximately 50:50 ratio of HFSCs and non- HFSCs (Fig. 18). This indicates that the 3C culturing conditions are able to induce generation of CD34 + a6 + cells from non-HFSC cells, and on the other hand, to promote differentiation of CD34 + a6 + HFSCs into non-HFSCs, establishing a balance between HFSCs and differentiated progeny in culture.
  • HFSCs were cultured under 3C conditions in the additional presence of the BMP inhibitors dorsomorphin or K02288. Both BMP inhibitors caused a decrease on the percentage of CD34 + a6 + HFSCs (Fig. 20). Accordingly, addition of BMP inhibitors counteracts the expansion and enrichment of CD34 + a6 + HFSCs observed under 3C conditions.
  • Example 2 In vitro culture conditions for expansion and maintenance of skin cancer stem cells
  • mice of an age between 4 and 40 weeks were used for experiments. Tumor cells were harvested from Tg(Krt14-rtTA) x Tg(tetO-KRas2) mice (Fisher et al, 2001 , Genes Dev 15: 3249-3262; Nguyen et al, 2006, Cell 127: 171 -183), where overexpression of mutant KRas was induced by feeding the mice with doxycycline-containing chow (1 g/kg), leading to formation of benign papillomas and squamous cell carcinomas.
  • Tumor cells were isolated by mincing tumor biopsies and incubating them in 0.25% collagenase (Sigma), 62.5 U/mL DNasel (Roche) in HBSS (Hank's Balanced Salt Solution, Gibco) for 60 min with gentle agitation at 37°C. Cell suspensions were filtered through 45 ⁇ ⁇ strainers and centrifuged at 300 g for 10 min. Single cell suspensions were used for HFSC cultures as describe above. The cells were cultured in identical conditions as described in Example 1 for HFSCs.
  • Example 1 Additional materials and methods are described in Example 1.
  • CSCs cancer stem cells
  • CSCs account for 20-30% of the total cells in benign papillomas in mouse models (Lapouge et al., 2012, EMBOJ 31 : 4563-4575).
  • FACS analyses indicated presence of 22 ⁇ 16.6 % Lin " EpCAM + CD34 + a6 + cells (hereafter CSCs) in the pool of tumor cells isolated from papillomas obtained from the Tg(Krt14-rtTA) x Tg(tetO-KRas2) mouse model (Fig. 21 ). Culturing these tumor cells in 2D with standard keratinocyte growth conditions completely depleted the EpCAM + CD34 + a6 + population.

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