Use of lif for tissue and cellular engineering

The present invention for the technical field of cell culture methods skin stem and / or human epidermal progenitors and their uses in particular for the preparation of epithelia multistoried, in particular of equivalents epidermal and / or skin.

It particularly relates to the use of an effective amount of a compound selected among LIF, an analogue of LIF, a LIF mimetic, a product capable of stimulating the endogenous expression of LIF and mixtures thereof to (i) promote the proliferation <i>in vitro</i>a population of skin stem cells and / or epidermal progenitors Human maintaining them in an undifferentiated state and / or (ii) maintain and / or increase their ability to generate a multilayered epithelium, especially epidermis multilayered and / or all or some of the skin appendages.

It also relates to a method for obtaining a bank or a culture skin stem cells and / or progenitors in human epidermal the presence of LIF, to a method for obtaining reconstructed epidermis and / or skins reconstructed, production kits cell banks or reconstructed epidermis, and the use of LIF including the stem cell preparation or of reconstructed epidermis for the treatment of subjects with intact skin (Burn victims and patients suffering from genetic diseases affecting the skin).

For 'population of skin stem cells and / or epidermal progenitors undifferentiated human 'according to the invention is meant a population of stem cells adult somatic naturally present in the basal layer of the epidermis, which are capable of self-renewal and / or proliferate and generate an epithelium pluristratified, particularly a multilayered epidermis and / or all or part of the annexes skin (sebaceous glands, follicular the hair, nail ...). Eg, you can get this population of skin stem cells and / or epidermal progenitors undifferentiated human adult skin from levies and / or neonatal skin.

By 'ability of a cell to self-renew, is meant a cell capable of divide to produce two daughter cells of which at least one is identical to the parent cell. At the scale of a complex cell population, the notion of self-renewal involves maintaining a compartment of cells and phenotypic characteristics functional constant during successive cell divisions. according to the invention, it is the maintenance of a stem cell compartment of compound skin and / or undifferentiated epidermal progenitors, in particular characterized by a high proliferative potential and an ability to generate a multilayered epithelium in particularly a multilayered epidermis.

By 'ability of a cell to proliferate' means a cell capable of multiplying to give two daughter cells, without necessarily transmitting features and potential of the parent cell to at least two cells girls. Proliferation, which may or may not be associated with the phenomenon of self-renewal, is likely to lead to the progressive reduction or disappearance of the compartment cell of interest within the cell population multiplies. According to the invention, cell proliferation is likely to be accompanied by progressive reduction and / or disappearance of the stem cell compartment skin and / or epidermal progenitors, in particular characterized with strong potential for growth and ability to generate a multilayered epithelium, in particular a multilayered epidermis.

This population includes skin stem cells and / or epidermal progenitors. The stem cell compartment, located most upstream of the hierarchy of the fabric Skin is characterized by a high potential for self-renewal (capacity multiplying the most important long-term), as well as a multipotentiality (Including ability to produce a multilayered epithelium, especially keratinocytes and / or different cell types from skin appendages such as follicles hair, sebaceous glands, nails ...). The epidermal progenitors from said stem cells, have the potential multiplication which may also be important, but generally smaller than the stem cells and are characterized by an ability to generate a epithelium pluristratified, particularly a multilayered epidermis.

Cells embryonic stem (ES) have potential for expansion <i>at priori</i> unlimited (Amit <i>et al</i>., <i>dev Biol</i>. 227: 271-278, 2000; Odorico<i>et al</i>., <i>Stem Cells</i>. 19: 193-204, 2001). Indeed, they can be multiplied without any signs of aging maintaining an undifferentiated state.

WO 02/097068 in particular discloses a method for inducing the differentiation of murine embryonic stem cells in murine keratinocytes.

However, the potential for expansion of adult somatic stem cells is limited by mitotic clock (Vaziri <i>et al</i>., <i>USA Proc Natl Acad Sci.</i> 91: 9857-9860, 1994; Chang <i>et al</i>., <i>USA Proc Natl Acad Sci.</i> 92: 11190-11194, 1995). this ability Limited expansion especially concerning stem cells and progenitors epidermal (Ramirez <i>et al</i>., <i>Genes Dev</i>. 15: 398-403, 2001; C ounter<i>et al</i>., <i>Lancet.</i>361: 1345-1346, 2003; Fortunel<i>et al</i>., <i>J Cell Sci</i>. 116: 4043-4052, 2003).

After a limited number of divisions, stem and progenitor cells from adult tissues invariably oriented towards differentiation, show signs of senescence and die. These characteristics represent limiting factors for realization of cell banks and for cell and tissue engineering (eg preparing skin equivalents).

The Applicant had previously proposed in WO 03/038073 (The OREAUCNRS) a method of enriching a population of stem cells keratinocyte from a preparation of keratinocytes, by accession preparation on an extracellular matrix component.

Cell culture methods, particularly for multiplication and / or differentiation cells must take into account the specific characteristics of stem cells somatic adult and embryonic stem cells, which differ in particular by (i) their potential for expansion, recognized as unlimited for cells Embryonic stem and limited to adult stem cells; and (ii) the stage differentiation, more advanced for skin stem cells and / or progenitors epidermal (capable of generating skin tissue) compared to stem cells Embryonic say 'totipotent' (capable of generating all types of tissues and organs the individual).

There is therefore a need to find agents capable of promoting the proliferation of skin stem cells and / or progenitor cells in the human epidermal Now in an undifferentiated state and are able to maintain or organogenic increase their potential, that is to say their ability to generate a multilayered epithelium, in particular a multilayered epidermis and / or all or part of skin appendages (hair follicle, sebaceous glands, nails ...).

Now the Applicant has just discovered, unexpectedly, that the use of Leukemia Inhibitory Factor or leukemia inhibitory factor (LIF) is used:<sl><li>(I) promote the proliferation <i>in vitro</i> a population of stem cells skin and / or human epidermal progenitor maintaining them in an undifferentiated state;</li><li>(Ii) appears and even necessary for obtaining a reconstructed epidermis pluristratified quality, that is to say, with characteristics histological comparable to those of a native epidermis (with a layer cornea, a granular layer, the suprabasal layers and a layer basal). It has shown that a reconstructed epidermis in the absence of LIF showed structural defects compared to a reconstructed epidermis in the presence of LIF.</li></sl>

LIF is described in US Patent 6,261,548 as a factor capable of suppressing proliferation of myeloid leukemia cells such as murine M1 cells, and promote macrophage differentiation, suggesting its use as an agent nonproliferative therapy to suppress certain forms of myeloid leukemia and to change the function of macrophages involved in the response to infections. At the skin, LIF is a cytokine produced naturally by keratinocytes<i>in vivo</i> and <i>in vitro</i> (Paglia <i>et al</i>., <i>Br J Dermatol</i>. 134: 817-823, 1996), and we know it factor is particularly involved in the control of inflammatory processes associated with various skin disorders such as psoriasis (Bonifati <i>et al</i>., <i>Arch Dermatol Res.</i>290: 9-13, 1998; Szepietowski<i>et al</i>., <i>J Dermatol</i>. 28: 115-122, 2001), or some allergies (Szepietowski <i>et al</i>., <i>Contact Dermatitis.</i> 36: 21-25, 1997).

However, it is not described nor suggested the use of LIF, an analogue of LIF, a LIF mimetic, a product capable of stimulating the expression of LIF endogenous or mixtures thereof to promote the multiplication <i>in vitro</i> stem cells Somatic adults known to have limited expansion potential compared to embryonic stem cells, and a more advanced stage of differentiation than these last; <i>a fortiori</i> the use of LIF to promote multiplication <i>in vitro</i> cells skin stem and / or human epidermal progenitors, nor to improve skin organ culture model (reconstructed epidermis and reconstructed skin).

We understand the importance of this discovery to obtain bank or Cultures of skin stem cells and / or progenitors from human epidermal undifferentiated, and for preparing skin equivalents for treating subjects with damaged skin (burn, subjects with a genetic disease affecting the skin).

The skin constitutes a physical barrier between the body and its environment. She is consists of two tissues: the epidermis and the dermis.

The dermis provides the epidermis with a solid support. It is also its feeder component. It consists mainly of fibroblasts and an extracellular matrix itself in particular composed of collagen, elastin, fibronectin, and a said substance ground substance, these components being basically synthesized by fibroblasts. There is also leukocytes, mastocytes and the tissue macrophages. It also contains blood vessels and fibers nerve.

The epidermis is a multilayered epithelium peeling, 100 microns thick average and is conventionally divided into a basal layer containing cells skin stem, epidermal progenitors, as well as cells engaged in the process of maturation / differentiation, called spinous layer consisting of several layers of polyhedral cells arranged on the cells basal called granular layer of flattened cells containing cytoplasmic inclusions, keratohyalin grains, and finally a top layer called stratum corneum (or <i>stratum corneum</i>), Consisting of keratinocytes at stadium terminal differentiation, called corneocytes. These are cells keratinised mummified, anucleate derived keratinocytes. The stack of corneocytes constitutes the horny layer which provides inter alia the barrier function the epidermis.

Epidermal differentiation follows a continuous maturation and oriented in wherein the basal keratinocytes are transformed by migrating to result in the formation of corneocytes, completely keratinized dead cells. This differentiation is the result of perfectly coordinated phenomena which will result in the maintenance of constant thickness and thus ensure the homeostasis of the epidermis. This implies a precise control of a number of cells which enter the differentiation process and the number of cells which are shed. During the normal process of scaling, only the most superficial corneocytes become detached from the surface of the epidermis.

Due to its exposure, the skin can be subjected to different types of abuse environmental, the most serious may lead to a fatal outcome. of the patients with severe burns may have tissue destruction skin on more than 80% of the body surface, and can be saved only if we manages to produce an equivalent amount sufficient to allow the skin regeneration of the skin throughout the areas destroyed.

Mastering the expansion potential of skin stem cells and / or progenitors epidermal and development of skin organotypic cultures from amplified cells <i>ex vivo</i> are therefore major issue for production grafts of autologous skin tissue (Ronfard <i>et al</i>., <i>Burns</i>. 17: 181-184, 1991<i>Transplantation.</i> 70: 1588-1598, 2000) and / or heterologous (Hefton <i>et al</i>., <i>Lancet.</i> 2: 428-430, 1983; Braye<i>et al</i>., <i>Med Biol Eng Comput</i>. 38: 248-252, 2000), for the treatment of severe burns.

Products epidermal equivalents from cells, derived from explants of skin and amplified <i>ex vivo,</i> are also used for the production of autologous allowing the regeneration of the skin tissue after surgical excision of a <i>nevus</i>(Kumagai <i>et al</i>., <i>Ann Plast Surg.</i> 39: 483-488, 1997), or a tattoo (Kumagai <i>et al</i>.,<i>An Plast Surg.</i> 33: 385-391, 1994).

The skin may also be affected by various diseases of genetic origin, compromising its integrity. This is for example bulbous epidermolysis, characterized by a permanent lack of cohesion between the dermis and epidermis (Eady <i>and al</i>., <i>J Dermatol</i>. 28: 638-640, 2001); ichthyosis, associated with a thickening pathologic corneal layer of the epidermis (DiGiovanna &amp; Robinson-Bostom, <i>Am J Dermatol Clin</i>. 4: 81-95, 2003).

Other types of genetic diseases of the skin such as <i>xeroderma pigmentosum,</i> lead to an alteration of the ability of this tissue to respond to 'Attacks' from the environment. These diseases are characterized by a deficiency excision mechanisms for DNA damage induced by ultraviolet radiation (UV), with the clinical consequence of increasing the frequency of occurrence skin cancer (Sarasin, <i>Mutat Res.</i> 428: 5-10, 1999).

It is therefore of serious medical conditions for which no pharmacological treatment efficient conventional currently available. However, the origin monogenic identified some of these diseases offers the prospect of treatment by therapy Gene (Spirito <i>et al</i>., <i>J Gen Med.</i> 3: 21-31, 2001; Magnaldo &amp; Sarasin,<i>Mutat Res.</i> 509: 211-220, 2002).

Indeed, a restoration of defective function was obtained on keratinocytes from skin samples taken from sick patients, grown <i>ex vivo</i> and corrected by gene transfer with a retroviral vector, in the case of several genetic diseases of the skin. By this approach, he has been able to obtain a genetic correction of keratinocytes with lamellar ichthyosis (Choate<i>et al</i>., <i>Hum Gene Ther.</i> 7: 2247-2253, 1996), for X-linked ichthyosis (Freiberg<i>et al</i>., <i>Hum Mol Genet.</i> 6: 927-933, 1997), as well as for different groups of<i>xeroderma pigmentosum</i> (Tile <i>et al</i>., <i>Hum Gene Ther.</i> 6: 1307-1315, 1995; Arnaudeau-Begard <i>et al</i>., <i>Hum Gene Ther.</i> 14: 983-996, 2003).

We now understand the importance of having culture systems to 'manipulate' <i>ex vivo</i> skin stem cells and / or epidermal progenitors from healthy subjects and / or patients suffering from genetic diseases affecting the skin, as well as to obtain epidermal equivalents and / or as physiological skin as possible from these cells.

A first object of the invention is therefore the use of an effective amount of a compound selected from Leukemia Inhibitory Factor (LIF), a LIF analogue, a mimetic LIF, a product capable of stimulating the expression of endogenous LIF, and mixtures thereof to (I) promote the proliferation <i>in vitro</i> a population of skin stem cells and / or human epidermal progenitor maintaining them in an undifferentiated state and / or for (ii) maintain and / or increase their ability to generate a multilayered epithelium.

In particular, the invention concerns the use of an effective amount of a compound selected from LIF, an analogue of LIF, a LIF mimetic, a product capable of stimulating the expression of endogenous LIF and mixtures thereof in a method for obtaining banks or skin stem cell cultures and / or human epidermal progenitors.

It also relates to the use of an effective amount of a compound selected from LIF, LIF analogue, a LIF mimetic, a product capable of stimulating expression of the endogenous LIF and mixtures thereof in a method for obtaining multistrata epithelia, especially reconstructed epidermis and / or skin rebuilt.

We speak in the context of the epidermis equivalent invention or epidermis reconstructed, as well as skin equivalent or reconstructed skin.

The action of LIF, a LIF analogue or mimetic of LIF on the proliferation <i>in vitro</i> a population of skin stem cells and / or epidermal progenitors human undifferentiated and / or differentiation and / or development of an epidermis pluristratified may be direct or indirect.

It may be direct, through stimulation of the proliferation potential, expansion, and / or organogenesis of skin stem cells and / or epidermal progenitors. She may also be indirect, including through factors and / or transmitted signals by the reactive cells to LIF in response to this factor, and capable of stimulating a beneficially these same cells and / or neighboring cells. This is e.g. signals transmitted by direct contact between the cells and / or factors involved in regulatory pathways autocrine and / or paracrine. Control the expression of potential of skin stem cells and / or epidermal progenitors involves including such 'dialogue' between the dermal fibroblasts and stem and / or epidermal progenitors. Said cells are therefore more receptive to stimuli exogenous and / or a signal from the environment of the cells.

Epidermal homeostasis results including a finely regulated balance between mitogenic signals promoting cell division and antiproliferative signals. these signals result in particular of the action of factors naturally produced by keratinocytes and / or from other cell types present in their environment, including secreted by the dermal fibroblasts.

As examples of known stimuli and / or signals to be involved in epidermal homeostasis', there may be mentioned:<ul><li>growth factors such as mitogenic growth factors 'Epidermal growth factor' (EGF) and 'keratinocyte growth factor "(KGF) (Cook <i>et al</i>., <i>J Cell Physiol</i>. 146: 277-289, 1991; Andreadis<i>et al</i>., <i>FASEB J.</i> 15: 898-906, 2001; Gamady <i>et al</i>., <i>J Cell Biochem.</i> 89: 440-449, 2003), or the 'transforming growth factor-β1 '(TGF-β1), including multifunctional growth factor identified for its anti-proliferative effect on keratinocytes <i>in vivo</i> and <i>in vitro</i> (Glick<i>et al</i>., <i>USA Proc Natl Acad Sci.</i> 90: 6076-6080, 1993; Van Ruissen<i>et al</i>., <i>J Cell Sci.</i> 107: 2219-2228, 1994; Cui<i>et al</i>., <i>Genes Dev</i>. 9: 945-955, 1995). These factors are involved in autocrine and paracrine regulation loops highly interactive and interlinked, ensuring both control proliferation and of differentiation (Reiss &amp; Sartorelli, <i>Cancer Res.</i> 47: 6705-6709, 1987; Hertle<i>et al</i>., <i>J Invest Dermatol</i>. 104: 260-265, 1995; Edmonson and<i>al</i>., <i>J Cell Physiol</i>. 179: 201-207, 1999; Yamasaki<i>et al</i>., <i>J Invest Dermatol</i>. 120: 1030-1037, 2003; Pasonen-Seppanen<i>et al</i>., <i>J Invest Dermatol</i>. 120: 1038-1044, 2003), and allowing a precise control of the exchange and / or maturation of keratinocytes; and or</li><li>molecules that can supplement, modify, and / or interfere with the action of growth factors, such as, for example: vitamin D and its derivatives for their ability to increase the sensitivity of keratinocytes the mitogenic actions KGF (Gamady <i>et al</i>., <i>J Cell Biochem.</i> 89: 440-449, 2003); retinoic acid for its ability to modulate the proliferation and / or differentiation keratinocytes (Choi &amp; Fuchs, <i>Cell Regul</i>. 1: 791-809, 1990; Gibbs<i>et al</i>., <i>Arch Dermatol Res.</i> 288: 729-738, 1996; Chapellier<i>et al</i>., <i>EMBO J.</i> 21: 3402-3413, 2002), including a cell sensitization mechanism to Action EGF and TGF-β (Tong <i>et al</i>., <i>J Invest Dermatol</i>. 94: 126-131, 1990).</li></ul>

The 'LIF' used according to the invention can in particular be provided in the form of a LIF purified a recombinant LIF, a homogenate or cell extract containing LIF, a culture supernatant of cells containing LIF, or mixtures thereof. Preferably used a LIF of human origin.

Use may in particular:<ul><li>LIF purified from organs, tissues and / or cells expressing naturally LIF [eg pituitary gland cells (Ferrara <i>et al</i>., <i>proc Natl Acad Sci USA.</i> 89: 98-702 6, 1992), dermal fibroblasts (Lorenzo <i>et al</i>., <i>wink Immunol Immunopathol</i>. 70: 260-265, 1994), lung fibroblasts (Elias<i>et al</i>.,<i>Am J Physiol</i>. 266: L426-435, 1994), placenta and endometrium (Kojima<i>et al</i>., <i>Biol Reprod.</i> 50: 882-887, 1994), bone marrow stromal cells (Lorgeot <i>et al</i>.,<i>cytokine</i>. 9: 754-758, 1997), kidney cells (Morel<i>et al</i>., <i>Cytokine.</i> 12: 265-271, 2000), cardiomyocytes (Ancey <i>et al</i>., <i>Cytokine.</i> 18: 199-205, 2002)];</li><li>a recombinant LIF, as obtained by culture of prokaryotic microorganisms (Eg bacteria, <i>E. coli</i>) Or eukaryotic cells (eg yeast, <i>Pichia pastoris, Saccharomyces)</i> form or not a fusion protein (eg LIF recombinant human sold by Chemicon International Inc.);</li><li>a homogenate or an extract of cells expressing LIF, in particular a mash or feeder cell extract (eg murine fibroblasts 3T3) expressing LIF, or genetically modified cells expressing LIF, or even cells stimulated to express LIF;</li><li>a cell culture supernatant containing LIF, such as a supernatant culture of 3T3 cells expressing LIF;</li></ul>   or mixtures thereof.

As' product capable of stimulating the expression of endogenous LIF, mention may be made such as IL-1β, cytokine described for its ability to induce the synthesis and secretion of LIF by fibroblasts, epithelial cells and smooth muscle cells of the lung Human (Knight <i>et al</i>., <i>Am J Respir Cell Mol Biol</i>. 20: 934-841, 1999). We can also include TNF-α, as well as cyclic AMP analoque bromoadénosine 8-3 ': 5' monophosphate (8BrcAMP), studied for their ability to induce the promoter LIF in a murine cell line derived from bone marrow stroma (Gollner <i>et al</i>., <i>Cytokine.</i>11: 656-663, 1999). According to an alternative, you can use any active ingredient can suppress the contrary the antagonistic activity of signaling pathways of anti-differentiating effect of LIF, and especially that involving the Stat5 protein. These include oligonucleotide antisense and / or interference methods by small RNA (siRNA), or yet modulators of kinases and phosphatases involved in activities activation of molecules Stats.

For analogue LIF 'according to the invention especially means any LIF polypeptide modified or LIF polypeptide fragment having LIF activity on cells skin stem and / or epidermal progenitors, that is to say which is capable (i) promote the ability of skin stem cells and / or epidermal progenitors undifferentiated self-renewal and / or to proliferate and (ii) to maintain and / or increase their ability to generate a multilayered epidermis.

For the modified LIF polypeptide having LIF activity "especially means a LIF polypeptide having undergone one or more modifications, for example to increase stability. By "modification" means any substitution, deletion and / or insertion of a amino acid or a small number of amino acids, in particular by substitution acid natural amino by unnatural amino acids or pseudo amino acids to positions such as the changes do not significantly affect the activity biological LIF. The modified LIF polypeptide may be obtained from a peptide sequence of LIF human selected from the sequence Genbank AAA59217 (195 aa), the GenBank sequence AAA51699 (202 aa), and the homologous sequences. By "homologous sequence" is meant a sequence at least 70%, preferably at least 85% and even more preferably at least 95% of a peptide sequence defined in the same species or from a different species; we then speaks of orthologous peptide sequence. We can also get this LIF polypeptide modified from the sequences of the gene or cDNA of human LIF (GenBank M63420 J05436, J03261, X13967), of sequences of the gene or cDNA of murine LIF (GenBank M63419 J05435, X06381, X12810, S73374), according to conventional techniques for cloning and expression.

By 'LIF polypeptide fragment having LIF activity "especially means a fragment of a sequence selected from the sequence Genbank AAA59217 (195 aa), the AAA51699 GenBank sequence (202 aa) and homologous sequences. Said fragment will include a size sufficient to reconstitute the tertiary structure LIF having binding sites to LIF-R and gp130. This polypeptide fragment may also be obtained according to the techniques Conventional cloning and expression from the sequences of the gene or cDNA of human LIF (GenBank M63420, J05436, X13967, J03261), the gene sequences or of the cDNA of murine LIF (GenBank X06381, J05435 M63419, X12810, S73374), and especially from the coding sequences.

Such analogues of LIF suitable for the implementation of the invention may thus be selected according to the process comprising the steps of:<sl><li>a) providing a preparation of cultured keratinocytes or stem cells skin and / or epidermal progenitors (i) in the presence or (ii) in the absence of the test product;</li><li>b) studying microscopic cells grown according to (i) and (ii);</li><li>c) measuring the product's ability to promote the proliferation <i>in vitro</i> cells skin stem and / or epidermal progenitors in comparing the number of cell clones obtained in the undifferentiated state (i) presence or (ii) in the absence of the test product;</li><li>d) the product is selected for which we get a number of cell clones increased the undifferentiated state in the presence of said product relative to the number of cell clones in the undifferentiated state in the absence of said product;</li><li>e) t then este ability of the product to regenerate epidermis p luristratifié according to the following steps:<sl><li>at. were seeded on a dermis support a preparation of keratinocytes (I) in the presence or (ii) in the absence of said test product;</li><li>b. is observed microscopically epidermis reconstructed structure according to (i) and (Ii);</li><li>c. the product is selected for which an epidermal structure is obtained reconstructed enhanced in the presence of said product relative to the structure of the skin in the absence of said product.</li></sl></li></sl>

By mimetic LIF 'according to the invention especially means any receptor agonist LIF (LIF-R) or any active extract or fraction of cell extract capable of activating gp130 and / or signaling pathways Jak / Stat and Ras / Map kinases, in particular expression and / or activity of Stat3. Eg, you can use agonistic antibodies of LIF receptors, synthetic peptides capable of interacting with receptors of LIF and to activate or any active ingredient capable of inducing activation of signaling pathways involved in the response to LIF, in particular the expression and / or activity of Stat3, or the activation of Janus-associated tyrosine kinases (JAK).

In particular, use may be agents capable of inducing the formation of a heterodimer LIFR-gp130, such as for example the cytokine Oncostatin M (OSM), the ciliary neurotrophic factor (CNTF) and Cardiotrophin-1 (CT-1), which have a strong homology tertiary structure with LIF. Such mimetics LIF suitable for the implementation of the invention may be selected by standard tests of binding to LIF-R and activating gp130.

These analogues of LIF or LIF mimetics may be naturally occurring or synthetic. By "natural origin" means a compound in pure form or in solution at various concentrations, obtained by various extraction processes from a tissue (skin ...) natural, in particular human skin or from original extracts plant. By "synthetic origin" means a compound in pure form or in solution at various concentrations obtained chemically or by production in an organism after introduction into this organism of the elements required for this production.

It is known that the cellular response to LIF involves a low affinity receptor (190 kD glycoprotein, gp 190) and a high affinity receptor (glycoprotein 130 kD, gp 130) (Taupin <i>et al</i>., <i>J Biol Chem.</i> 276: 47975-47981, 2001), and downstream of these receptors, signaling pathways Jak / Stat and Ras / MAP kinase (Ernst <i>et al</i>., <i>J Biol Chem.</i> 274: 9729-9737, 1999; Burdon<i>et al</i>., <i>Trends Cell Biol</i>. 12: 432-438, 2002). The transduction protein Stat3 signal plays an important role in maintaining the undifferentiated state of murine ES cells in response to LIF (Niwa <i>et al</i>., <i>Genes Dev</i>. 12: 2048-2060, 1998; Matsuda<i>et al</i>., <i>EMBO J.</i> 18: 4261-4269, 1999), whereas the expression of the Stat5 protein is instead associated with a commitment to these cells towards differentiation (Nemetz <i>et al</i>., <i>Differentiation.</i> 62: 213-220, 1998).

According to an alternative, we can also link to a LIF, a LIF analogue, a LIF mimetic or a product capable of stimulating the expression of LIF, a receiver LIF (LIF-R) or a product capable of stimulating the expression of endogenous LIF-R.

The invention also provides a method for obtaining a bank or a culture skin stem cells and / or epidermal undifferentiated progenitors, comprising at least one multiplication step of a preparation of keratinocytes humans in a culture medium containing an effective amount of a compound selected among LIF, an analogue of LIF, a LIF mimetic, a product capable of stimulating expression of the endogenous LIF and mixtures thereof.

For 'cell culture' according to the invention, is meant a preparation cells derived from a native tissue, cultured <i>in vitro</i> and kept alive in artificial conditions <i>(Ex vivo, in vitro</i>).

For 'cell bank' of the invention, is meant a preparation cells treated to be retained for later use. Cells may for example be frozen and stored as aliquots. Cells may be derived from native tissues and be either directly stored, for example in frozen or amplified <i>in vitro</i> before being stored.

By "preparation of human keratinocytes' according to the invention, is meant a set of keratinocytes obtained from a skin sample and / or any Other possible sources of skin stem cells and / or progenitor cells capable generate the epidermis and / or all or part of skin appendages. As an alternative source of said stem and / or progenitor cells include in particular the hair follicle.

A preparation of keratinocytes can be obtained according to conventional methods cellular culture. In particular, it is possible, from a skin explant from a subject, proceed as following:<ul><li>the subcutaneous tissue is removed with a scalpel;</li><li>the skin sample is decontaminated by antibiotic treatment (eg gentamycin);</li><li>the dermis of the skin is removed by proteolytic processing (eg trypsin dispase) and then dissection;</li><li>then promotes dissociation of the cells in the presence of a solution of 0.05% trypsin and 0.02% EDTA; and the effect is neutralized the trypsin by adding a DMEM culture medium containing 10% serum;</li><li>the cell suspension was homogenized, which was then washed in medium culture keratinocytes (KGM, Bullet kit, Clonetics Corp).</li></ul>

The step of propagating the preparation of keratinocytes may be carried out in a culture medium containing LIF, LIF analogue, mimetic or a LIF product capable of stimulating the expression of endogenous LIF, suitable for multiplication said cells and maintaining them in an undifferentiated state. Eg, you can use a semi-defined culture medium (KGM Bullet Kit, Clonetics, Cambrex B io Science Inc.), in the absence of feeder fibroblasts, and low density (Eg seeding at 2400 cells / cm²). We can also use the medium described by Rheinwald and Green (<i>Cell</i>. 6: 317-330, 1975), in the presence of irradiated fibroblasts, and low density (eg seeding at 2400 cells / cm²).

More generally, any culture medium comprising LIF and for promoting the multiplication of skin stem cells and / or epidermal progenitors and / or to generate a stratified epidermis, will also be part of the invention.

In particular, such a culture medium may, among at least a factor of growth mitogenic for keratinocytes [eg epidermal growth factor (EGF) and / or keratinocyte growth factor (KGF)], insulin, hydrocortisone, and an antibiotic (eg gentamicin, amphotericin B). Advantageously, the medium may further comprise a pituitary extract, by example of bovine origin, epinephrine, transferrin and / or amino acids nonessential. The medium may or may not contain serum and possibly be added factor growth 'transforming growth factor-β "(TGF-β).

The effective concentration of LIF, LIF analogue, mimetic LIF, or product capable of stimulating the expression of endogenous LIF or their mixtures in the present culture medium of said cells may be between 0.01pg / ml and 1mg / ml culture medium, preferably between 0.1ng / ml and 100ng / ml of culture medium, and even more preferably between 100 pg / ml and 1ng / ml of culture medium.

If using a ground product or a cell extract containing LIF, the effective concentration said pulp or extract present in the culture medium of the multiplying step said cells may be between 0.01pg / ml and 10mg / ml of culture medium, preferably between 10 pg / ml and 100 ug / ml of culture medium, preferably between 100 ng / ml and 1 .mu.g / ml of culture medium.

In a preferred embodiment, the preparation of human keratinocytes is first enriched skin stem cells and / or epidermal progenitors, especially by fast adhesion to an extracellular matrix component and cell recovery has acceded (cells with high adhesion capacity, appointed Adh⁺⁺⁺). By 'component of extracellular matrices, one in particular refers to molecules such as collagens, laminin, fibronectin, proteoglycans. Use may in particular the pre-enrichment step described in the application WO03 / 038 073 (The OREAUCNRS). Preferably, the adhering step is performed on a plastic substrate on which the Type I collagen is adsorbed; and this step is performed at 37 ° C for a period about 10 minutes to about 20 minutes, especially 12 to 15 minutes. And the peeling step can be performed by gentle trypsinization, eg presence of trypsin 0.05% and EDTA 0.02% for a short duration, preferably less than 10 minutes, especially during a period of 1 to 2 minutes.

In a particular embodiment, the cell suspension was placed in culture flasks "Covered" with collagen type I (collagen I solution (Sigma Chemical Co Ltd, Irvine, UK) diluted 2-fold in PBS, deposited for 45 minutes in the flasks, then drying after removal of surplus), at a density of 200 000 cells / cm². After 12 minutes, the keratinocytes that have not adhered are removed by washing in PBS buffer. Such selected adherent cells are detached from the support by a gentle trypsinization (0.05% trypsin-EDTA 0.02% (Biological Industries, Kibbutz Beit Haemek, Israel) for 3 to 5 minutes at 37 ° C). After neutralization of the trypsin (DMEM + 10% FCS), the cells are recovered, washed, and then returned to suspended in KGM medium. The fraction of adherent cells selected by this method is about 10% of total epidermal keratinocytes.

The percentage of adherent cells is usually 5 to 20% of the cells contained in the preparation of keratinocytes, in particular 10% of the cells contained in the preparation of keratinocytes. Said population Adh⁺⁺⁺ thus selected has a frequency of progenitors undifferentiated epidermal and / or skin stem cells clonogenic about 10 times that of the population of more mature keratinocytes characterized by a lower adhesion capacity (Adh^{- / +}).

The enrichment process and selects a cell population adhering to high expansion potential compared to non-adherent cells, mainly made up of keratinocytes mature. Said population of adherent cells Adh⁺⁺⁺ includes skin stem cells and / or epidermal progenitors in particular characterized by a potential for long-term expansion, and potential to generate a multilayered epithelium, in particular a multilayered epidermis, and / or a skin equivalent, and / or all or part Skin Structure.

In a particular embodiment, an enriched preparation of keratinocytes and / or the population of skin stem cells and / or epidermal progenitors in LIF responsive cells, that is to say cells having on their surface an expression or the LIF receptor (LIF-R phenotype of cells⁺) And / or cells capable of multiply in an undifferentiated state in the presence of LIF, may also be realized. The selection of LIF-R cells⁺ reactive LIF could for example be realized by immuno-phenotypic marking and sorting of viable cells by flow cytometry, using immuno-magnetic beads, and / or by immuno-selection on a carrier or are adsorbed anti-LIF-R antibody.

Advantageously, the method according to the invention further comprises a cryo-preservation step said proliferated cells under conditions favoring their maintenance in a differentiated state. The cryopreservation step used in particular for obtaining banks cells, is to freeze the liquid nitrogen in cells after expansion in the presence LIF, to allow storage and / or preservation of said cells for a later use.

This multiplication process <i>in vitro</i> in the presence of LIF according to the invention thus makes obtaining from a bank or a culture of skin stem cells and / or epidermal progenitors.

This bank or culture of skin stem cells and / or progenitors epidermal undifferentiated is in particular characterized in that it is enriched in cells able to respond to the action of LIF and / or express on their surface LIF receptor (LIF-R), called LIF-R⁺. It can be in liquid form or preferably frozen.

All production kit from a bank or a culture of skin stem cells and / or Undifferentiated human epidermal progenitors comprising at least (i) a preparation of human keratinocytes and (ii) a cell culture medium Human keratinocyte addition of an effective amount of LIF, an analogue of LIF or LIF mimetic will also be part of the invention. In particular, said kit will include a medium in which the effective amount of LIF is between 0.01pg / ml and 1mg / ml of culture medium, preferably between 0.1 and 100ng / ml of culture medium. If using a ground product or a cell extract containing LIF, the effective concentration said pulp or extract present in the culture medium may be between 0.01pg / ml and 10mg / ml of culture medium, preferably between 10 pg / ml and 100 ug / ml of culture medium, preferably between 100 ng / ml and 1 .mu.g / ml of culture medium.

The invention also relates to an epidermal process for preparing reconstructed and / or reconstructed skin comprising:<sl><li>a) a step of preparing a carrier or of a dermis equivalent; and</li><li>b) a step of seeding a population of human k Keratinocytes on said support;</li></sl>an effective amount of a compound selected from LIF, an analogue of LIF, a LIF mimetic and a product capable of stimulating the expression of endogenous LIF being added to the medium at one of steps a) or b), or both.

According to an alternative, the epidermis reconstructed process for preparing and / or skins reconstructed comprises:<sl><li>a) a step of preparing a carrier or of a dermis equivalent; and</li><li>b) a step of seeding a population of skin stem cells and / or undifferentiated epidermal progenitor cells cultured in the presence of a compound selected from LIF, an analogue of LIF, a LIF mimetic, a product capable of stimulating the expression of endogenous LIF, and mixtures thereof, on said support.</li></sl>

In particular, said support or dermis equivalent is selected from lattices of collagen / fibroblast, a previously désépidermisé dermis, membranes artificial.

We can quote as equivalent dermis preparation example the protocols described in patent applications (EP-A-285471, EP-A-285474, EP-A-789 074, EP-A-502 172, EP-A-418 035, WO-A-9116010, EP-A-197090, EP-A-20753, FR-A-2665175, FR-A-2689904) or preferably the protocol described by Asselineau <i>et al</i>., 1987, (Models in Dermatology., vol.III, Ed. Lowe &amp; Maibach, 1-7).

One example is of epidermal equivalents preparation protocols and / or skin, those described in the patents or in the patent applications EP 285471, EP 285474, EP 418 035, WO-A-90 02796, WO-A-9116010, EP 197090, EP 20753, FR 2665175, FR 2689904. In very general terms, the reconstructed skin models consist of human keratinocytes deposited on a support, often a dermis equivalent, and cultured under conditions such that they enter into a differentiation program resulting in the formation of an epidermis equivalent. Can also include other cell types such as cells of Langerhans (EP0789074) or melanocytes to reconstruct an epidermis and / or Skin close to native tissues.

First of epidermal equivalents production kit and / or skin equivalents comprising at least an effective amount of a compound selected from LIF, an analogue of LIF, a LIF mimetic, a product capable of stimulating the expression of endogenous LIF and mixtures thereof, is also part of the invention. In particular, the kit may include (i) a support or dermal equivalent, (ii) preparation of keratinocytes and (iii) an effective amount of a compound selected from LIF, LIF analogue, a LIF mimetic, a product capable of stimulating expression of the endogenous LIF and mixtures thereof. LIF, LIF analogue, mimetic LIF, a product capable of stimulating the expression the endogenous LIF, and mixtures thereof may be provided alone or as a mixture in a culture medium, said culture medium being suitable for the propagation and / or keratinocyte differentiation. As described above, such a medium can in particular contain at least a factor of growth mitogenic for keratinocytes [eg epidermal growth factor (EGF) and / or keratinocyte growth factor (KGF)], insulin, hydrocortisone, and an antibiotic (eg gentamicin, amphotericin B). Advantageously, the medium may further comprise a pituitary extract, by example of bovine origin, epinephrine, transferrin and / or amino acids nonessential.

The medium may or may not contain serum and possibly be added factor growth 'transforming growth factor-β "(TGF-β).

Another aspect of the invention relates to multiple uses of LIF as defined according to the invention and skin stem cells and / or epidermal progenitors undifferentiated amplified in the presence of LIF.

In particular, the invention relates to the use of skin stem cells and / or undifferentiated epidermal progenitors amplified in the presence of a compound selected among LIF, an analogue of LIF, a LIF mimetic, a product capable of stimulating expression of the endogenous LIF and mixtures thereof in a screening method and / or asset evaluation capable of modulating the Multiplication and / or differentiation of skin stem cells and / or epidermal progenitors in mature keratinocytes, or in any other cell type used in the construction of skin appendages.

In particular, the screening method comprises:<ul><li>culturing a skin stem cells and / or progenitors epidermal undifferentiated amplified in the presence of a compound selected from LIF, LIF analogue, a LIF mimetic, a product capable of stimulating expression of the endogenous LIF and mixtures thereof, under conditions enabling multiplication and / or their differentiation into mature keratinocytes or in any another cell type used in the formation of skin appendages, and presence of an asset test;</li><li>comparing the proliferation and / or differentiation of cells in presence of assets with the proliferation and / or differentiation of cells the absence of said assets.</li></ul>

The invention also relates to the use of skin stem cells and / or undifferentiated epidermal progenitors amplified in the presence of a compound selected among LIF, an analogue of LIF, a LIF mimetic, a product capable of stimulating expression of the endogenous LIF and mixtures thereof in a screening method and / or asset evaluation may modulate the ability of skin stem cells and / or epidermal progenitors generating a multilayered epithelium, in particular an epidermis equivalent and / or a skin equivalent.

In particular, this method comprises:<ul><li>culturing a skin stem cells and / or progenitors epidermal undifferentiated amplified in the presence of a compound selected from LIF, LIF analogue, a LIF mimetic, a product capable of stimulating the expression of endogenous LIF, and mixtures thereof, in their conditions for generating a multilayered epidermis and / or all or part of Annexes skin in the presence of an asset test;</li><li>comparing the ability of cells to generate a multilayered epidermis and / or all or some of the skin appendages in the presence of the asset with the ability cells to generate a multilayered epidermis and / or all or part of Annexes skin in the absence of said assets.</li></ul>

Another aspect of the invention is the use of a compound selected from LIF, a LIF analogue, a LIF mimetic and a product capable of stimulating the expression of Endogenous LIF for the preparation of skin stem cells and / or progenitors undifferentiated epidermal or reconstructed epidermis and / or reconstructed skin for treating skin lesions.

In particular, the skin stem cells and / or epidermal progenitors undifferentiated or reconstructed and / or reconstructed skin epidermis are intended to regenerate <i>in vivo</i> healthy skin on damaged skin or broken skin areas.

The causes of skin lesions can be multiple:<ul><li>accidental causes, such as a skin that has been burned over a large surface of the body or face (eg burn);</li><li>surgical causes such a skin that has undergone excision for treatment of nevus (Kumagai <i>et al</i>., <i>Ann Plast Surg.</i> 39: 483-488, 1997), or a tattoo (Kumagai <i>et al</i>., <i>An Plast Surg.</i> 33: 385-391, 1994). ;</li><li>genetic causes, e.g. having a skin-related changes a genetic disease; mention may be made bulbous epidermolysis, characterized by a permanent defect of cohesion between the dermis and epidermis (Eady <i>et al</i>., <i>J Dermatol</i>. 28: 638-640, 2001); ichthyosis, associated with a pathological thickening of the horny layer of the epidermis, such as lamellar ichthyosis (Choate <i>et al</i>., <i>Hum Gene Ther.</i> 7: 2247-2253, 1996), ichthyosis X-linked (Freiberg <i>et al</i>., <i>Hum Mol Genet.</i> 6: 927-933, 1997) ; of the<i>Xeroderma pigmentosum,</i> which are characterized by a deficiency excision mechanisms for DNA damage induced by ultraviolet radiation (UV), with clinical results in increased frequency appearance of skin cancers (Sarasin, <i>Mutat Res.</i> 428: 5-10, 1999).</li></ul>

Treatment of damaged skin, where the skin is injured accidentally or following surgery, may consist of:<ul><li>preparing a population of skin stem cells and / or human epidermal progenitors from a skin explant healthy subject (heterologous cells) or healthy areas of the subject with damaged skin (autologous cells);</li><li>multiplying said population of skin stem cells and / or progéniteurs épidermiques humains indifférenciés en présence de LIF;</li><li>re-implanting said cells at the injured skin areas or in the form of skin stem cells and / or epidermal progenitors human undifferentiated capable of generating <i>in vivo</i> a native epidermis and / or all or part of skin appendages, either in the form of reconstructed epidermis and / or reconstructed skin. In particular, the cells can be injected and / or applied to the areas to be treated, while the reconstructed epidermis or skin rebuilt will be grafted on the areas to treat.</li></ul>

The use of skin stem cells and / or epidermal progenitors undifferentiated amplified in the presence of LIF, or reconstructed epidermis and / or skin reconstructed prepared in the presence of LIF according to the invention particular be for treating burns. The use of skin stem cells and / or epidermal progenitors undifferentiated amplified in the presence of LIF, or reconstructed epidermis and / or skin reconstructed prepared in the presence of LIF according to the invention particular be for the treatment of skin lesions associated with a skin excision, particularly surgical origin (eg nevus, tattoo).

The treatment of skin lesions associated with a genetic disease may notably consist in a first mode, in: <ul><li>preparing a population of skin stem cells, and / or epidermal progenitors from a skin explant topic with a genetic disease affecting the skin;</li><li>the treatment <i>ex vivo</i> said cells for the defective function associated restorer genetic disease, in particular by transferring the non-mutated gene with a retroviral vector;</li><li>the re-location of said cells treated at the damaged skin areas, either in the form of skin stem cells and / or progenitors epidermal undifferentiated capable of generating <i>in vivo</i> a native epidermis and / or all or part of skin appendages, either in the form of reconstructed epidermis and / or reconstructed skin. In particular, the cells can be injected and / or applied to the areas to be treated, while the reconstructed epidermis or skin rebuilt will be grafted on the areas to treat.</li></ul>

According to an alternative, the skin lesions treatment the disease-associated Genetics may consist of:<ul><li>preparing a population of skin stem cells and / or human epidermal progenitors from a skin explant healthy subject (heterologous cells);</li><li>multiplying said population of skin stem cells and / or progéniteurs épidermiques humains indifférenciés en présence de LIF;</li><li>implanting said cells at the injured skin areas or in the form of skin stem cells and / or epidermal progenitors human undifferentiated capable of generating <i>in vivo</i> a native epidermis and / or all or part of skin appendages, either in the form of reconstructed epidermis and / or skin rebuilt. In particular, the cells can be injected and / or applied the areas to be treated, while the reconstructed epidermis or skin rebuilt will be grafted on the areas to treat.</li></ul>

The use of skin stem cells and / or epidermal progenitors undifferentiated amplified in the presence of LIF, or reconstructed epidermis and / or reconstructed skin in the presence of LIF, will thus for treating lesions skin associated with genetic diseases such as for example epidermolysis bulbous <i>Xeroderma pigmentosum,</i> lamellar ichthyosis and associated ichthyosis chromosome X.

The use of skin stem cells and / or epidermal progenitors undifferentiated prepared from a skin explant subject with a genetic disease and cultured in the presence of LIF may also be used for models donning <i>in vitro</i> Study of the efficiency of gene transfer protocols. This will include using said cells to assess the frequency correction genetic (percentage of cells with an insertion of the transgene), and / or assess and quantify the functional efficiency correction (for example, study correction of the initially deficient function in cultures in monolayers and / or the epidermis and / or reconstructed skin).

The invention will now be illustrated by the following nonlimiting examples.

<b>FIGURES</b>

<sl><li>The <b>Figure 1</b> presents an analysis of the effect of LIF on the clonogenic growth of stem cells and / or epidermal progenitors. cell clones obtained from cells at passage 1 cells cultured for 8 days in the absence of exogenous LIF, then fixed and stained <b>(AT) ;</b> cell clones obtained from the passage keratinocytes 1 cultivated for 8 days in the presence of exogenous LIF to 1ng / ml and then fixed and stained <b>(B)</b>; semi-quantitative analysis of the density of the clones obtained in control conditions and in presence of exogenous LIF <b>(C).</b></li><li>Figure 2 shows histological analyzes a reconstructed epidermis obtained presence or absence of a ground material of 3T3 fibroblasts depleted or not LIF. Chopped off Histologic a reconstructed epidermis obtained in the presence of ground material of non depleted 3T3 LIF (positive control) <b>(AT) ;</b> Histological section of a reconstructed epidermis obtained absence of ground material 3T3 (negative control) <b>(B);</b> Histological section of an epidermis rebuilt ground product obtained in the presence of 3T3 depleted of LIF (condition Experimental) (<b>C</b>).</li></sl>

EXAMPLES

<b>Example 1: Growth of stem cells and / or epidermal progenitors undifferentiated optimized in the presence of LIF.</b>

Keratinocytes were isolated from an adult skin sample (plasty Breast).

After removal of the subcutaneous tissue using a scalpel, the skin sample is cut into fragments of about 5mm x 5mm, then decontaminated by treatment antibiotic [Gentamicin (Life Technologies), 3 successive baths of 10 minutes in DMEM culture medium (Life Technologies)]. To allow separation of the dermis the epidermis, the sample is then subjected to proteolytic processing [dispase (Boehringer, Roche Diagnostics) + trypsin (Gibco, Invitrogen) 1 night at 4 ° C]. The epidermis is then separated from the dermis by dissection. The epidermis fragments separated tissue dermal are placed in a solution of trypsin-EDTA 0.05% 0.02% (Gibco, Invitrogen) (15 minutes at 37 ° C). The preparation is stirred periodically to promote the dissociation of cells. The effect of the trypsin is then neutralized by adding a culture medium containing 10% serum (DMEM + 10% serum). After neutralization of the trypsin (DMEM + 10% serum), cell preparation is homogenized mechanically (Pipetting) and then filtered. The cell suspension was washed and resuspended in KGM (Clonetics) medium. Cells in suspension are counted microscope using a Malassez cell. The viability of the samples is estimated by the exclusion method Trypan blue (Life Technologies).

A stage of enrichment with stem cells and / or epidermal progenitors undifferentiated was carried out by a quick adhesion step on a collagen substrate Type I (selection of a population with high adhesion capacity, Adh⁺⁺⁺).

Stem cells and / or epidermal undifferentiated progenitors can in fact be more mature keratinocytes separated based on their rapid adhesion property. This step allows a pre-enrichment of the preparation of stem cells and / or epidermal progenitors indifférenci ed.

The enrichment method by adhesion to collagen is described in the application WO 03/038073 and scientific publication (Fortunel <i>et al</i>., <i>J Cell Sci,</i> 116: 4043-4052, 2003).

The cell suspension was placed in culture flasks "covered" with Type I collagen [the adsorption of collagen on the adhesion support is provided by depositing a liquid solution of collagen I (Sigma Chemical) diluted 2-fold in PBS for at least 45 minutes and then drying after removal of surplus], a density of 150 000 to 200 000 cells / cm². After 12 to 15 minutes, the k Keratinocytes not party are washed in PBS buffer. The adherent cells, called Adh⁺⁺⁺, Thus selected are detached from the support by mild trypsinization (0.05% trypsin-EDTA 0.02% (Gibco, Invitrogen) for 3 to 5 minutes at 37 ° C). After neutralization of trypsin (DMEM + 10% serum), the cells are recovered, washed, then resuspended in culture medium (in this case, KGM medium). The fraction consisting of adherent cells selected by this method, represents about 5 to 10% of total epidermal keratinocytes.

Cultures initiated from cells Adh⁺⁺⁺, Were performed in culture medium semidefinite (Bullet kit KGM, Clonetics, Cambrex Bio Science Inc.), in the absence of feeder fibroblasts, and low density (seeding at 2400 cells / cm²) to achieve quantifiable isolated cell clones. The first planting (1 pass) the cultures were divided into two batches: 1) identical condition to that described above; 2) addition of LIF at a concentration of 1ng / ml (recombinant human LIF, Chemicon International Inc.). After 8 days of culture, cultures were fixed (70% ethanol) and stained (Giemsa and eosin) to analyze the characteristics of cell clones 2 obtained in these culture conditions <b>(Figure 1).</b>

The comparative analysis of cultures grown in the absence or presence of LIF shows this factor allows to optimize the growth of stem cells and / or progenitors undifferentiated epidermal from a culture of cells Adh⁺⁺⁺.

Macroscopic observation indicates an increase in the number of cell clones dense coloring large <b>(Figure 1 A, B).</b> Microscopic observation indicates that these clones whose development is favored consist essentially of small cells with morphological features associated with the state undifferentiated stem cells and / or epidermal progenitors, criterion reflecting a "youth" most important cultures treated with LIF. The increase in number of dense clones larger in response to LIF was confirmed by analysis computerized image, as shown in the histogram presented <b>1C</b>.

The use of LIF in culture system thus conducive multiplication a population of epidermal stem cells and / or epidermal progenitors undifferentiated human.

<b>Example 2: Positive effect of LIF on the epidermal reconstruction.</b>

We observed that crushed 3T3 fibroblast promote the recovery from epidermal stem cells and / or epidermal progenitors of cultivated <i>in vitro</i> and frozen. The reconstructed epidermis is good: it has a cellular organization and stratification similar to those of a native epidermal <b>(Figure 2A</b>).

To test the hypothesis of an involvement of the LIF in this property, depletion LIF by immunoprecipitation was performed, then the activity of the ground product free of LIF was compared to that of non depleted mash.

Depletion of the ground preparation 3T3 LIF is carried out using a polyclonal antibody in rabbits (Ac anti-LIF, Santa Cruz, no. SC-20087). The ground is 3T3 incubated with the Ab anti-LIF excess at 4 ° C for several hours to ensure a LIF good fixation on Ac. The free Ac and related LIF is then 'trapped' by adding sepharose beads to which are adsorbed G proteins (G proteins have a high affinity for the Fc domains of Ab). Centrifugation allows separation beads on which the LIF is retained in the LIF-depleted ground material.

The ability of epidermal stem cells from a frozen bank (isolated from a mammary skin sample) to generate a reconstructed epidermis was evaluated in the presence of ground material of non depleted 3T3 fibroblasts LIF (positive control), in absence of ground material 3T3 (negative control) and in the presence of crushed 3T3-depleted LIF (experimental condition). Histological characteristics of epidermis reconstructed obtained in each condition were compared on fixed sections and colored (<b>Figure 2</b>).

While organotypic cultures supplemented with crushed 3T3 fibroblast (not depleted LIF) allow obtaining reconstructed epidermis of good quality (<b>2A</b>), That is to say a cellular organization and a near laminating those a native skin, crops grown in the absence of ground material will allow to obtain that the epidermis not possessing the histological characteristics required<b>(Figure 2B).</b>

The skins obtained in the absence of ground material from 3T3 including present if:<ul><li>Vacuoles and large intercellular spaces;</li><li>Bad stratification;</li><li>poor basal layer cells;</li><li>suprabasal cells very stretched;</li><li>Misdirection of basal cells (parallel to the matrix);</li><li>Little or no granular layer;</li><li>thin stratum corneum.</li></ul>

The cells in the experimental condition, cultured in the presence of crushed 3T3 depleted of LIF, behave in a manner similar to those grown in the absence of crushed <b>(Figure 2C),</b> indicating that depletion of LIF resulted in a loss of beneficial properties of the ground material on the epidermal reconstruction.

Thus, the use of LIF, besides its ability to promote the proliferation of said cells in an undifferentiated state as shown in Example 1, keeps the potential organogenic said cells, ie their ability to generate epidermis reconstructed quality, with characteristics similar to those of the epidermis native.

<b>Example 3 Preparation of an epidermis equivalent and / or skin.</b>

Unless otherwise indicated, all media and buffers used in the examples are described in Bell <i>et al</i>. 1979 <i>(Proc Natl Acad Sci USA.</i> 76: 1274-1278, 1979) Asselineau and Prunieras 1984 <i>(Br J Dermatol</i>. 111: 219-222, 1984) or Asselineau<i>et al</i>., 1987 (Models in dermatology., Vol.III, Ed. Lowe &amp; Maibach, 1-7, 1987).

The supports or dermal equivalents are prepared as described in Asselineau <i>and al</i>. 1985 and 1987 <i>(Exp. Cell. Res.</i> 159: 536-539, 1985; Models in Dermatology, vol 3 pp 1-7 1987), in the following proportions:<tables><table><tgroup cols="2"><tbody><row><entry align="left">MEM (1,76X)</entry><entry align="left">45%</entry></row><row><entry align="left">Fetal calf serum</entry><entry align="left">9% </entry></row><row><entry align="left">NaOH (0.1N):</entry><entry align="left">5%</entry></row><row><entry align="left">acetic acid (1/1000):</entry><entry align="left">4%</entry></row><row><entry align="left">collagen:</entry><entry align="left">26%</entry></row><row><entry align="left">fibroblasts:</entry><entry align="left">11%</entry></row></tbody></tgroup></table></tables>

The collagen used is type I collagen (commercial solution), but we can also use collagen type III or IV. It is extracted from rat tails or calf skin by acid hydrolysis and stored in acidic medium at + 4 ° C; it polymerizes naturally by heating to 37 ° C and by decreasing the acidity (increase in PH). Collagen is dialyzed beforehand against successive baths of water + acid acetic.

The protocol is as follows: in a sterile Falcon tube, brought on MEM 1.76 X in the presence of additives (1% Glutamine, Acids mines nonessential 1% Pyruvate sodium 1%, 1% Fungizone and penicillin / streptomycin 1%), fetal calf serum, NaOH 0.1N sodium hydroxide was a joust at the fibroblasts isolated from skin explants human at a concentration of 1.4x 10⁵ cells per 1 ml of culture medium. then added slowly, against the wall of the tube so as to observe the appearance of a white cloud, a volume / volume mixture of collagen in dilute acetic acid 1/1000. The assembly is then carefully mixed and distributed into the wells of a plate of culture of 12 wells (type Costar reference 3512) at mixing 0.5ml per cm². The culture plate is then placed in an incubator at 37 ° C with 5% CO₂.

The second step is to seed a preparation of keratinocytes the presence of LIF or skin stem cells and / or epidermal progenitors undifferentiated amplified in a medium containing LIF on said support. In a particular embodiment, the medium in which lies the dermis equivalent is supplemented with LIF, prior to the seeding stage. The culture may then be kept immersed in a nutrient medium which 3F can be for example the medium described by Rheinwald and Green (Cell. 6: 317-330, 1975) environment that allows the proliferation of keratinocytes. After an incubation time of 3 to 15 days, preferably from 7 to 9 days, the equivalent of skin is maintained at the air / liquid interface for example by deposition on a grid metallic. The liquid is then preferably made of the same nutrient medium that the previous.

The incubation is then continued until a skin equivalent having the characteristics of a skin, namely the medium on which there is an equivalent epidermis having the four types of conventional cell layers, namely the basal layer, suprabasal, granular and cornea. Thus, the incubation is continued for a period of between 5 and 30 days, preferably between 7 and 10 days.

The reconstructed skin model thus produced includes two entities, the media and epidermis equivalent, it is possible to physically separate from each other.