FR3043092A1 - MODULATION OF KLF FACTORS TO PROMOTE THE FUNCTIONALITY OF EPITHELIAL CELLS - Google Patents

Abstract

The present invention relates to the in vitro use of an inhibitor of the expression or activity of a Krüppel-like transcription factor (KLF) to increase proliferation and / or maintain and / or increase the immature nature of epithelial cells contained in a cell sample, or for enriching a cell sample with epithelial cells, as well as a cosmetic composition comprising said inhibitor and cosmetic and pharmaceutical uses of said inhibitor.

Description

GENERAL CONTEXT OF THE INVENTION

Systems, methods and methods for constructing or repairing epithelial tissues such as the epidermis have applications in areas as diverse as medical research and clinical development, tissue engineering and toxicology.

The pluristratified epithelial tissues (such as the epidermis, the cornea, the mucous tissues, etc.) share a number of common characteristics that impose constraints for the design of tests and tissues reconstructed in vitro. These characteristics are well exemplified in the case of the epidermis. The epidermis constitutes the most superficial structure of the skin and in particular ensures the barrier function. Mostly consisting of keratinocytes, it is renewed every 28 days on average. This tissue comprises 4 layers that correspond to the 4 stages of the differentiation program that keratinocytes undergo during their migration from the basal layer, the deepest, to the stratum corneum, the most superficial. The continuous physiological process of renewal of the different layers of keratinocytes is called keratinopoiesis. The basal layer of the epidermis, which has only one cell base, is the germinal compartment. It is at the level of this layer that the proliferation of keratinocytes takes place. Among the basal keratinocytes, there is a small proportion of cells called stem cells, which are believed to be responsible for long-term epidermal renewal. The immediate offspring of stem cells is called the progenitor population. These ensure the rapid renewal of the epidermis in the short term.

Like other pluristratified epithelial tissues, the epidermis thus consists of a heterogeneous set of cells with variable degrees of differentiation (or immaturity). It is generally accepted that basal keratinocytes represent approximately 10% of all epidermal keratinocytes, and the epidermal stem cell compartment only of the order of 0.1%.

Controlling the multiplication of the cells of the different human tissues without altering their characteristics and functionalities is a goal that brings applications, particularly in the areas of regeneration, tissue reconstruction and skin healing. However, this problem faces two main difficulties.

Firstly, the understanding of the molecular signals controlling the transmission of characters from a cell to its offspring, during successive mitoses, remains to this day very incomplete. This is particularly true for immature cells with high regenerative potential, whether of the 'precursor', 'progenitor' or 'stem cell' type. Secondly, unlike cells of transformed lines, a general property of primary cells (i.e., extracted from tissues and not transformed) is a progressive evolution towards a more differentiated then senescent phenotype, especially during a prolonged phase of proliferation.

When using the potential for organogenesis of normal epithelial cells by activating their proliferation, those skilled in the art are thus limited by the following points: 1) Quantitative limitation, inherent in the fact that the human cells epithelial unprocessed can not generally be obtained and produced in abundance, and 2) Qualitative limitation, due to the difficulty of preserving all the characters and potentialities of epithelial cells when a state of proliferation is induced. The importance of these difficulties is particularly exemplified in the case of cellular and tissue engineering methods implemented for the production of homologous cutaneous grafts intended for the treatment of burn victims, which imply a massive cell multiplication step in culture. Although methods for the production of functional skin grafts have been available for almost 30 years (Gallico et al., 1984, De Luca et al., 1989, Ronfard et al., 2000), the quality of engraftment remains at this variable day, in particular because of a lack of molecular targets to make these processes more reliable by a better preservation of the cellular potential (Barrandon et al., 2012). In this context, a particularly important objective is the maintenance of the immature character of precursor cells (progenitors and stem cells), which is a parameter intrinsically linked to their regenerative potential.

In this context, the objective of the inventors was to identify modulable molecular targets for directing a large quantity of normal epithelial cells having a given regenerative potential to an active proliferative state, while promoting the maintenance of their intrinsic characteristics ( regenerative potential, active proliferation, etc.) and the transmission of these to their offspring.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have succeeded in identifying such molecular targets. These are Krüppel-like transcription factors (or "Klf" or "KLF"). The present invention more particularly consists in applying, on epithelial cells of interest, a treatment making it possible to induce a decrease in the expression or activity of at least one of these factors (establishment of a cellular context "KLF repressed" otherwise known as "(KLF-rep)").

This treatment makes it possible to improve the function of the epithelial cells (hereinafter referred to as "gain of function"), especially in the cellular and tissue contexts listed below: 1) When the treatment is carried out on epithelial cells placed in two-dimensional culture in vitro, the effect obtained in response to the treatment is a gain in terms of proliferation and / or maintenance of the immature character of the precursor cells, which makes it possible to improve the cellular amplification, 2) When the context is that of a three-dimensional epithelium reconstructed in vitro, the effect obtained in response to treatment is a gain in terms of the richness of epithelial cells with a strong regenerative capacity of the tissue engineered product, 3) When cells involved in the constitution of a reconstructed epithelial tissue used as a graft, the gain obtained is then translated by an activation c ellular more effective during early stages of engraftment and healing in vivo.

In summary, the present invention provides a new molecular target (Klf transcription factors), which can be used to cause a controlled stimulation of epithelial cell proliferation without altering its characteristics and functionalities.

In a first aspect, the present invention thus aims at the in vitro use of an inhibitor of the expression or the activity of a Krüppel-like transcription factor (KLF) to increase the proliferation and / or or maintain and / or increase the immature character of epithelial cells contained in a cell sample, or to enrich a cell sample in epithelial cells.

In other words, the present application relates to a method for promoting the proliferation and / or maintaining and / or increasing the immature character of epithelial cells contained in a cell sample, or for enriching a cell sample in epithelial cells, said method comprising step of inhibiting the expression or activity of a Krüppel-like transcription factor (KLF). More specifically, this method comprises contacting a cell sample with an effective amount of said inhibitor. This method can be implemented in vitro or in vivo.

In the context of the present invention, the term "maintain the immature nature of the cells", limiting the loss or decrease and preferably maintain the degree or level of immaturity of the precursor cells, progenitors and strains.

"Krüppel-like" transcription factors are grouped together in a family (hereinafter referred to as "KLF family"). This family includes among others 17 members, numbered KLF1, KLF2, KLF3, (...) up to KLF17. Those skilled in the art can easily access the sequences of one or more of these factors from the data banks. Thus, by "Krüppel-like transcription factor" or "KLF family protein" is meant here a member of this family and, preferably, the KLF4 transcription factor. This transcription factor has, in humans, the protein sequence SEQ ID NO: 1 (accession number in the CCDS database of NCBI: CCDS6770, accession number in the UniProtKB / Swiss-Prot base: 043474.3).

The present invention therefore preferably relates to the use in vitro of an inhibitor of the expression or activity of a Krüppel-like transcription factor (KLF) to increase proliferation and / or maintain and / or to increase the immature nature of epithelial cells contained in a cell sample, or to enrich a cell sample with epithelial cells, characterized in that said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of SEQ ID NO: 1 sequence. In the KLF family, non-human homologues, in particular animal homologs of the transcription factors identified in humans, may also be included. By way of example, mention may be made of murine, canine, feline, equine, simian and other homologs.

The regulatory role of some of these KLF factors has been described in cellular and tissue models. Notably, transcription factors of the KLF family have been implicated in the control of pluripotent character of embryonic stem cell (ES) lines of murine and human origin (Jiang J. et al, 2008). However, the results of this study demonstrate that suppression of the expression of KLF factors in ES cells induces a loss of function, which is opposed to the function gain effect described in the present invention.

The link between KLF4 and the regulation of pluripotentity has been established in the context of reprogramming mature adult cells to a pluripotent stem cell state in an induced pluripotent stem cell model (iPS, Takahashi K. et al, 2007). In this study, the method for obtaining reprogramming of differentiated cells into pluripotent iPS (gain of function) stem cells involves the induction of KLF4 expression, not its inhibition. This can be explained by the fact that it is a particular model of stem cells, the properties of which are not necessarily extrapolable to native cells of adult tissues (especially with regard to molecular controls of proliferation) .

The function of the KLF4 factor in the biology of the normal epidermis has been described in several publications (Segre et al., 1999, Katz et al., 2002, Sen et al., 2012, Chew et al., 2013; Boxer et al, 2014). This transcription factor is identified as a regulator of terminal stages of keratinocyte differentiation, necessary for the establishment of the barrier function of the epidermis (Sen et al., 2012, Chew et al., 2013; et al, 2013, Boxer et al, 2014). KLF4 has also been described to intervene in the biology of hair follicle stem cells, with a possible role in controlling the ability of these cells to participate in skin regeneration processes (Li et al, 2012). In this study, the role of KLF4 was studied in a population of cells present in the hair follicle 'bulge' region in mice (which is very different from the biology of human interfollicular epidermis). In this model, it has been shown that an invalidation (total extinction) of the expression of KLF4 has the effect of causing a reduction of the follicular pool of stem cells associated with a delay in skin healing, which is exactly the opposite. of the present invention. On the other hand, another aspect of this study, carried out on keratinocytes of the transformed human carcinoma line A431, shows that decreasing the level of expression of KLF4 results in a reduction of the cell population exhibiting characteristics of stem cells. which is also an opposite effect to that described in the present invention.

In addition, expression of several members of the KLF transcription factor family, including KLF4, has been detected in corneal cells (especially keratinocytes). As for the epidermis, KLF4 is proposed as necessary for the establishment of the barrier function of the cornea (Swamynathan S. et al., 2011).

Finally, the involvement of KLF4 in the process of tumorigenesis is known, but the state of the art does not converge to a single conclusion on its exact role. Indeed, the Klf4 gene is identified either as an oncogene (Jiang W. et al, 2009) or as a tumor suppressor gene, depending on the cellular context considered. In the skin, the role of KLF4 in the formation of carcinomas is also controversial. An invalidation of the Klf4 gene has in particular been described as a tumorigenesis inducing parameter (Wei D. et al., 2005).

In conclusion, no study to date has demonstrated that it is possible to improve the regenerative potential and growth of epithelial cells by inhibiting the expression and / or activity of a KLF family transcription factor. as proposed by the present inventors.

According to the present invention, the reduction of the expression and / or the activity of KLF, and more particularly KLF4, within the epithelial cells makes it possible to promote the proliferation capacity of said cells and advantageously to maintain and / or increase the immature character of these cells.

In the context of the present invention, the epithelial cells of interest belong to the epithelial, preferably mammalian, and even more preferably, human lineages. It may be in particular human keratinocytes of the skin (interfollicular epidermis, hair follicle, sebaceous or sweat glands), cornea, or even the mucous membranes (in particular, the buccal, tracheal, vaginal, urethral, etc.).

Furthermore, the "cell sample" used in the invention consists of any biological sample containing or likely to contain epithelial cells as defined above. These epithelial cells may correspond to different states of differentiation or immaturity (precursors, progenitors, stem cells), as well as to different degrees of enrichment or purification (clonal progenitor progeny, meroclones, or clonal progeny of stem cells or holoclones).

More specifically, the sample used in the present invention may be a total population of unfractionated keratinocytes, a population or basal fraction of keratinocytes (consisting of a mixture of precursor keratinocytes, progenitors, and strains) or enriched subpopulations. stem cells or progenitors based on phenotypic criteria.

Preferably, the cell sample of the invention is derived from a biological sample extracted from its natural context, and obtained from a healthy or sick subject (for example from a biopsy). Preferably, this sample is extracted from cutaneous tissue, cornea or mucous membranes.

Alternatively, said cell sample may be tissue maintained in vivo in its natural context, on a subject (injured skin, aged skin, graft, etc.).

Preferably, said subject is an animal. Even more preferably, it is a mammal. Most preferably, it is a human. The cell sample of the invention is a sample of epithelial tissue. In particular, the tissue sample may be selected from the epithelia, for example the interfollicular epidermis of adult or neonatal human skin, the cornea, the mucous membranes, the hair follicles. Samples of diseased epithelial tissues are for example obtained by biopsy of patients suffering from a genetic disease (such as xeroderma pigmentosum, epidermolysis bullosa, etc.), or by cicatricial skin biopsy (particularly in burn victims). The diseased epithelial tissues may also be tumor tissues (carcinomas, etc.). The biological sample may also comprise cells derived from the epithelial (in particular keratinopoietic) differentiation of pluripotent stem cells chosen from embryonic, fetal and induced pluripotent stem cells. Examples of cells having an epithelial potential derived from fetal stem cells are: cells of the ectodermal embryonic leaflet, cells of the epithelial tissues, keratinocytes, etc. Cells with epithelial potential from so-called induced pluripotent stem cells (IPS) are generated by reprogramming cells from adult tissue that may be differentiated cells.

By "Krüppel-like" transcription factor (KLF) expression or activity inhibitor is meant herein any compound capable of interfering with the expression of said factor, or, once that it is expressed in protein form, with its transcriptional activity on DNA. This inhibitor is hereinafter referred to as "the inhibitor of the invention".

In a first embodiment, the inhibitor of the invention decreases or blocks the expression of the gene coding for the transcription factor KLF of interest. By "expression" of a gene is meant any process by which the information contained in the nucleotide sequence of a gene is converted into a functional protein, for example, all aspects of the transcription or translation of that gene. gene. For example, the term "inhibiting expression" may include inhibiting transcription, translation, or post-transcriptional or post-translational modifications of that gene. Preferably, in this case, said inhibitor is a chemical molecule or an inhibitory nucleic acid, such as an antisense oligonucleotide (for example a small interfering RNA (siRNA) or a microRNA (miRNA)), or a ribozyme which is specific for the polynucleotide encoding or regulating the expression of the KLF transcription factor of interest. An inhibitory nucleic acid can recognize either a sequence regulating the expression of this gene (for example its promoter), or the coding sequence of this gene. By "specific" of a polynucleotide coding for the - or regulating the expression of - the KLF transcription factor of interest, it is meant that the agent preferentially inhibits the expression of this transcription factor, with respect to the expression other genes. An agent which is specific for a particular sequence may bind preferentially to this sequence under conventional conditions of high stringency. The inhibitor of the invention "decreases the expression" of a given gene when, in its presence, the expression of said gene is at least 10%, at least 15%, at least 20%, at least 25%, preferably at least 30%, more preferably at least 40%, more preferably at least 50% than that measured in its absence. Moreover, the inhibitor of the invention "blocks the expression" of a given gene when, in its presence, the expression of said gene is at least 10%, at least 15%, at least 20% less than at least 25%, preferably at least 30%, more preferably at least 40%, more preferably at least 50% than that measured in its absence. The expression of a gene can be measured by any molecular biochemistry technique conventionally used by those skilled in the art, both at the transcriptional level (measurement of the formation of the mRNA from the DNA encoding said gene) and translational (measuring the formation of the protein encoded by said gene). Techniques such as northern blot, qRT-PCR, western-blot, or mass spectrometry can be used, for example. These techniques, well known to those skilled in the art, do not need to be detailed here.

In a preferred embodiment, the inhibitor of the invention is an antisense nucleic acid comprising a polynucleotide specific for a sequence coding for (or regulating the expression of a KLF transcription factor of interest. are, for example, available in publicly available databases, such as the NCBI-operated GenBank database, for example, the coding sequence for the transcription factor. KLF 4 is shown in SEQ ID NO: 1 (NCBI CCDS Access Number: CCDS6770; UniProtKB / Swiss-Prot Access Number: 043474.3).

On the basis of these sequences, one skilled in the art is able to design, manufacture and use appropriate antisense molecules without undue experimentation. The antisense nucleic acid of the invention may be, for example, an oligonucleotide or a nucleic acid comprising an antisense sequence which is operably linked to an expression control sequence, and which is expressed in a cell. The use of antisense nucleic acids to negatively regulate the expression of a particular protein in a cell is now well known (see for example Askari et al (1996) or Wagner, RW (1994)). ). An antisense nucleic acid molecule may comprise a nucleotide sequence that is complementary to the strand encoding another nucleic acid molecule (eg, an mRNA sequence), or a portion thereof, and therefore is capable of establishing hydrogen bonds with the strand encoding the other nucleic acid molecule. Alternatively, the antisense sequences may be complementary to a sequence present in the 5 'or 3' untranslated end of the mRNA or a region between a coding region and an untranslated region (e.g. at the junction of the 5 'untranslated region and the coding region). The antisense nucleic acid may be complementary to the sequence of a regulatory region of the gene, for example a transcription initiation or regulatory element sequence, or a splice site . Such antisense nucleic acid can be designed on the basis of the nucleotide sequence coding for the KLF protein or the sequences regulating its transcription or translation, and constructed according to the rules of Watson and Crick. To guide it in the construction of antisense molecules that are complementary to a region of a gene involved in transcription (thus blocking the transcription and / or production of isoforms, such as splice variants), the one skilled in the art can consult, for example, Lee et al. (1979); Cooney et al. (1988) and Dervan et al. (1991).

An antisense nucleic acid can exist in different forms. For example, it may be DNA, RNA or chimeric mixtures, derivatives or modified versions thereof, single-stranded or double-stranded. The nucleic acid can be modified at the nucleotide bases, sugar or phosphate port, using conventional procedures and modifications. Nucleotide base modifications include, for example, the methylated versions of purines or pyrimidines. Modifications may include grafting other molecules such as peptides, or agents facilitating transport across the cell membrane (see, eg, Letsinger et al., 1989), cleavage agents (eg, Krol et al. 1988) or intercalating agents (eg Zon et al., 1988).

The antisense nucleic acids of the invention can be constructed using chemical synthesis methods known in the art, for example using natural nucleotides or modified nucleotides to increase the biological stability of the molecules or to increase the stability physical duplex formed between the antisense and targeted nucleic acids. Such nucleotides are, for example, phosphorothioate and substituted acridine derivatives. To inhibit the expression of a KLF factor of interest in cells, these antisense nucleic acids may be added to the culture medium. In a preferred embodiment, the synthetic oligonucleotides are added at a final concentration of from about 10nM to about 1000nM, preferably from about 20nM to about 750nM, more preferably from about 30nM to about 500nM, more preferably from about 40nM to about 350 nM, more preferably between about 50 nM and about 200 nM (e.g., about 200 μg oligonucleotide / ml).

Alternatively, the antisense nucleic acids of the invention may be produced biologically using an expression vector in which a nucleic acid has been subcloned in an antisense orientation (i.e. the nucleic acid transcribed from the inserted nucleic acid will have an antisense orientation with respect to the target nucleotide sequence). Expression control sequences (e.g., regulatory sequences operably linked to the nucleic acid inserted into the vector) may be selected to allow the expression of the antisense molecule specifically in epithelial cells. 'interest. For example, promoters and / or activators or other regulatory sequences may be chosen to induce constitutive and specific expression of the antisense nucleic acid in these cells. It is also possible to use an inducible regulatory sequence, such as the Tet system (for example, as described in Gossen et al (1992)). The antisense expression vector may be in the form of, for example, a virus, plasmid, or attenuated recombinant phage. The expression vector can be introduced into the cells of interest using standard techniques well known in the art.

In a preferred embodiment, the antisense nucleic acid of the invention is a small interfering RNA ("siRNA"). A skilled worker is able to design, manufacture and use a variety of appropriate siRNAs without undue experimentation. Typical examples of making and using siRNA against KLF5, although for a purpose other than that of the present invention, are described in US Patent Application 2009/0011003. Other antisense nucleic acids can be used in the present invention, such as, for example, double-stranded RNAs ("dsRNA"), microRNAs ("miRNA"), short-hairpin RNAs ("shRNAs"). . These nucleic acids can be designed to target any region - either coding or non-coding of a gene. On the other hand, these molecules can be used to modify the structure of chromatin (epigenetic modification) and thus the expression of genes (see, for example, Allshire et al., 2002). The nucleic acids of the invention should not be limited to molecules containing natural RNA. They may also include chemically modified nucleotides. Methods of making antisense nucleic acids (eg, siRNA) are conventional for those skilled in the art. Methods to optimize the efficiency of siRNAs are well known to those skilled in the art (see Vickers et al (2003) and Yang et al (2003)). The antisense nucleic acid of the invention is of a length such that it is effective for the inhibition of the gene of interest. In general, it will be preferred to use an antisense nucleic acid of between about 6 and about 50 nucleotides in length (e.g., between about 10 and 30 nucleotides, or at least about 12, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides). The length of an efficient siRNA is generally between about 19 bp and about 29 bp in length (e.g., about 19, 21, 23, 25, 27 or 29 base pairs), but shorter or longer sequences are nevertheless preferred. acceptable.

In another embodiment, the inhibitor of the invention is a ribozyme. Ribozymes are catalytic RNA molecules having ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as mRNA, for which they have a complementary region. A ribozyme having specificity for an mRNA of interest may be designed depending on the targeted nucleotide sequence, for example the corresponding cDNA.

In general, it is preferable that the inhibitory nucleic acid or ribozyme comprises a strand that is 100% complementary to the gene sequence it is intended to inhibit. However, a 100% sequence identity between the nucleic acid and the target gene is not required to obtain effective antisense. Thus, sequence variations (due to genetic mutation, strain polymorphism, or natural evolutionary divergence) can be tolerated. Alternatively, nucleic acid sequences including, for example, small insertions, deletions, and point mutations relative to the target sequence may be effective for the inhibition of target gene expression. The degree of sequence identity can be optimized by the alignment algorithms known in the art, for example using the global algorithm described by Needleman-Wunsch (1970) or the local algorithm described by Smith-Waterman (1981) . Given the nature of the two compared sequences (one being likely to be identical to one fragment of the other), it is preferred in the present invention that the antisense nucleic acid and the target gene portion target are identical, by local alignment, to more than 90% (more precisely more than 95%, 98% or 99%).

In one embodiment, an inhibitory nucleic acid of the invention hybridizes to the sequence it is intended to inhibit, under conditions of high stringency. For example, siRNA may be functionally defined as a nucleotide sequence that is capable of hybridizing with a portion of the transcript of a target gene under high stringency conditions (eg, 400 mM NaCl, 40 mM pH PIPES). 6.4, 1 mM EDTA, 70 ° C for 12-16 hours hybridization, or equivalent conditions), followed generally by washing.

To conclude, the Krüppel-like transcription factor (KLF) expression inhibitor of the invention can be an antisense nucleic acid (for example a small interfering RNA, a double-stranded RNA). strand, a micro-RNA, or a short hairpin RNA) or a ribozyme.

A number of these inhibitors have been proposed to date. For example, antisense micro-RNAs (see US 2009/0099123), decoy oligonucleotides binding to the KLF4 transcription factor (see 2008/0300209), agents blocking the interaction between KLF and the p53 promoter. (see US 2009/0098054), or antisense RNA anti-KLF5 (see US 2009/0011003).

In a particular embodiment, said inhibitor is a short hairpin RNA (shRNA) specific for the mRNA of a KLF transcription factor. Said shRNA is, for example, a short hairpin RNA (shRNA) specific for KLF4 mRNA. More particularly, this shRNA has SEQ ID NO: 2 (see examples below).

In another preferred embodiment, the inhibitor of the invention is a chemical molecule that inhibits (or represses) the transcription of a gene coding for a Krüppel-like transcription factor (KLF).

In the context of the present invention, a molecule "inhibits" or "represses" the transcription of a given gene when, in its presence, the transcription of said gene is at least 10%, at least 15%, less than 20%, at least 25%, preferably at least 30%, more preferably at least 40%, more preferably at least 50% than that measured in its absence. The transcription of a gene can be measured by any molecular biochemistry technique conventionally used by those skilled in the art for this purpose, such as northern blot or qRT-PCR. These techniques, well known to those skilled in the art, do not need to be detailed here.

For example, said chemical molecule may be a molecule capable of suppressing the activity of a promoter regulating the transcription of a protein of the KLF family. In particular, said chemical molecule may be capable of repressing the activity of a promoter regulating the transcription of the human KLF4 protein.

Said chemical molecule may also be Kenpaullone (Yu et al., 2011, see examples below). This molecule, also known under the CAS number 142273-20-9, is marketed by numerous distributors (Sigma Aldrich, Tocris Bioscience, Santa Cruz Biotech, etc.). It has the formula:

In a second embodiment, the inhibitor of the invention decreases or blocks the activity of the KLF protein of interest (such an inhibitor is also called "antagonist"). It is then, for example, a protein closely related to the KLF protein of interest, which is in an inactive form and thus prevents the normal activity of this protein, in particular the transcriptional activity of the target KLF transcription factor on a of these target genes (such a protein is sometimes called "dominant negative" of this protein). Fragments of these "dominant negatives", blocking antibodies or antibody fragments having these properties, chemical molecules or aptamers specific for this protein are also aimed at insofar as these molecules are capable of preventing transcription. at least one target gene of a KLF transcription factor. The methods of designing, manufacturing and using such molecules are conventional and well known to those skilled in the art.

In the context of the present invention, a molecule "decreases" the activity of a KLF protein of interest when, in its presence, the transcription of at least one target gene of said KLF protein is less than 10 %, at least 15%, at least 20%, at least 25%, preferably at least 30%, more preferably at least 40%, more preferably at least 50% than that measured in its absence. Moreover, a molecule blocks the activity of a KLF protein of interest when, in its presence, the transcription of at least one target gene of said KLF protein is less than 10%, at least 15% less, at least 20%, at least 25%, preferably at least 30%, more preferably at least 40%, more preferably at least 50% than that measured in its absence. The transcription of said target gene can be measured by any molecular biochemistry technique conventionally used by those skilled in the art for this purpose, such as northern blot or qRT-PCR. These techniques, well known to those skilled in the art, do not need to be detailed here. A known direct target gene of KLF4 is for example p21.

In a preferred embodiment, said inhibitor is an antibody that has been generated against a protein of the KLF family or against a peptide fragment thereof. As used herein, the term "antibody" is used in its broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, and fragments thereof. antibodies (eg Fab, F (ab '), F (ab') 2, Fv). These antibodies may have been designed to block the activity of a protein of the KLF family. We will then speak here of "blocking" antibodies, that is to say that it can inhibit the function of a protein of the KLF family. Such antibodies can be produced by conventional technologies.

In another preferred embodiment, said inhibitor is a chemical molecule that binds to and occupies the active site of a KLF family protein, thereby blocking the normal biological activity of that protein. Examples of molecules include, for example, peptides or peptide-like molecules, or organic compounds (including synthetic compounds and compounds of natural origin).

Once the inhibitor of the invention is brought into contact with the cell sample, it enters the epithelial cells of interest either naturally (in the case of chemical molecules or small antisense nucleic acids), or by transfection techniques conventionally used (for example, by electroporation, precipitation with calcium phosphate, or using nanocapsules or liposomes).

According to a particular embodiment of the invention, a gene therapy is implemented so that said inhibitor is expressed in the cells of interest. In this case, the cells of the sample are transfected with a nucleic acid which codes for the inhibitor (s) which it is desired to introduce into these cells, under conditions such as the inhibitor (s) ( s) are synthesized in vivo by the cells into which the nucleic acid has been transferred. Such nucleic acid may in particular be in the form of a DNA vector, for example a plasmid vector or a viral vector. One or more vectors may be administered, each vector may carry one or more sequences coding for at least one inhibitor according to the invention (for example, for an anti-KLF4 siRNA or shRNA, as in the examples below) . The expression of the inhibitor in cellulo may be transient or stable.

Said viral vector may be chosen from an adenovirus, a retrovirus, a lentivirus, an adeno-associated virus (AAV), a herpes virus, a cytomegalovirus (CMV), a vaccinia virus, etc. Advantageously, said viral vector is a defective virus. The term "defective virus" refers to a virus unable to replicate in a target cell. Generally, the genome of the defective viruses is devoid of the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated, rendered non-functional or substituted by other sequences and in particular by the nucleic acid which encodes the peptide of interest. Lentiviral vectors have for example been described in Firat et al. (2002).

In a particular embodiment, the inhibitor of the invention is a shRNA that inhibits the expression of a protein of the KLF family carried by a lentiviral vector allowing its stable expression.

In an even more particular embodiment, the inhibitor of the invention is a shRNA inhibiting the expression of the KLF4 protein carried by a lentiviral vector allowing its stable expression (see Example 1 below).

COSMETIC AND PHARMACEUTICAL COMPOSITIONS OF THE INVENTION

In a preferred embodiment, the inhibitor of the invention is contacted with the cell sample in vitro (ie, apart from any animal body, in particular human). As indicated above, this bringing into contact makes it possible to improve the proliferation and the richness of the epithelial cells contained in this sample, and / or to maintain and / or increase the immature character, and / or to activate them. The cell sample, once treated in vitro, may be used, for example, in methods for screening active principles (for testing drugs or cosmetics on bi- or tri-dimensional cultures in vitro), to achieve tissue engineering or build functional epithelial cell banks. Alternatively, the sample thus treated (in vitro) can be grafted to an animal, as a graft. In the latter case, the treatment of the invention makes it possible to improve the regenerative potential of the graft and the engraftment in vivo.

In a non-exhaustive manner, the inhibitors of the invention thus find utility in the following areas: • In cellular and tissue engineering: - for the optimization of epithelial cell amplification processes, - for the improvement of the quality of epithelial cell libraries, and / or - for the improvement of in vitro models of reconstructed three-dimensional epithelia. • To promote care, healing, regeneration: - to promote healing and epithelial regeneration (cutaneous lesions, ocular), and / or - to improve and / or correct the condition of the skin of a subject, in particularly limit the aging of the skin (anti-aging strategies), improve the firmness, elasticity, structure, grain or complexion of the skin of said subject. • For cell therapy, transplantation, gene therapy: - for the optimization of ex vivo expansion protocols of epithelial cells for clinical purposes, - for the improvement of the regenerative potential of reconstructed epithelial tissue grafts, and / or for the improvement of the growth potential of epithelial cell samples intended to be genetically modified (in particular in the context of gene therapy approaches).

By "improving and / or correcting the state of the skin of a subject" is meant limiting, and / or preventing, and / or correcting the aging of the skin of one and / or the appearance of the signs, visible or no, skin aging subject and / or to slow down or mitigate the effects, promote the attenuation or resorption of wrinkles, improve and / or correct the firmness, elasticity, structure, grain or complexion of the skin of a subject. The expression "skin aging" is considered here in its broadest sense. It is associated with at least one condition selected from the disintegration of collagen fiber bundle structure, wrinkle formation, loss of skin elasticity, change of skin texture and reduction of skin texture. difference between a groove and an elevation of the surface of the skin.

In a particular embodiment, the inhibitor of the invention is contacted with a skin sample in vivo (ie, directly on an animal body). Such treatment is intended to promote healing and / or epithelial regeneration on cutaneous or ocular lesions, or to improve the skin condition of a subject. In this embodiment, the inhibitor of the invention is advantageously incorporated in a cosmetic or pharmaceutical composition.

A particular object of the invention is a cosmetic composition comprising at least one inhibitor as defined in the present invention, and optionally one or more cosmetically acceptable excipient (s).

Preferably, the cosmetic composition according to the invention is characterized in that said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of sequence SEQ ID NO: 1.

Another particular object of the invention is a pharmaceutical composition comprising at least one inhibitor as defined in the present invention, and optionally one or more pharmaceutically acceptable excipient (s) for use in the treatment of epithelial tissue lesions. a subject, preferably cutaneous or ocular lesions, in particular to promote healing and / or epithelial regeneration.

Preferably, the pharmaceutical composition according to the invention is characterized in that said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of sequence SEQ ID NO: 1.

In the present description, the term "pharmaceutically" or "cosmetically" acceptable excipients is understood to denote a compound or a combination of compounds used in a pharmaceutical or cosmetic composition, not causing side reactions and which allows, for example, the facilitation of administration of the active compound (s), increasing its life and / or efficiency in the body, increasing its solubility in solution or even improving its conservation. These excipients are well known and will be adapted by those skilled in the art depending on the nature and mode of administration of the selected inhibitor (s).

Preferably, the cosmetic or pharmaceutical composition of the invention is administered to the subject subcutaneously, or topically. More preferably, this composition is administered repeatedly, spread over time.

In another aspect, the present invention is directed to the use of the cosmetic composition of the invention for improving the skin condition of a subject. In this aspect, the composition of the invention is intended to improve the aesthetic appearance of the subjects treated.

In this aspect, topical administration is preferred. The cosmetic composition is then in particular in the form of aqueous or oily solutions or dispersions of the lotion or serum type, emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersion of an aqueous phase in a silicone phase (E / Si), a fatty phase in an aqueous phase (O / W: oil-in-water emulsion) or conversely (W / H: water-in-oil emulsion), or suspensions or emulsions of soft consistency of the cream or gel type aqueous or anhydrous, or microcapsules or microparticles, or vesicular dispersions of ionic and / or nonionic type or foams. The cosmetic composition of the invention is preferably in the form of a cream, an ointment, a balm, a milk, an oil, etc. also containing, if necessary, a number of additives: buffering agents, preservatives, thickeners, stabilizers, antioxidants, pH adjusting agents, etc. In general, any pharmaceutical or cosmetic composition of the invention may be applied to the skin.

The compositions are prepared according to the usual methods. The amounts of the various constituents of the compositions according to the invention are those conventionally used in the fields under consideration.

The cosmetic compositions comprise creams for cleaning, protecting or caring for the face, for the hands, for the feet, or for the body (for example, day creams, night creams, make-up removal creams, creams for complexion, sunscreen creams), fluid foundations, make-up removing milks, body care or protection milks, sunscreen lotions, lotions, gels or mousses for skin care, such as cleaning lotions , sunscreen lotions, artificial tanning lotions, bath compositions, deodorant compositions comprising a bactericidal agent, aftershave gels or lotions, depilatory creams.

The cosmetic compositions according to the invention may also consist of powdery or solid powdery preparations, for example in the form of a stick, a pressed powder, soaps or cleaning loaves. It can still be in the form of patches, pencils, brushes and applicators allowing a localized application on the spots of the face or hands. It can be used as a care product or as a makeup product.

In known manner, the cosmetic composition of the invention may also contain additives and / or adjuvants customary in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, buffering agents, preservatives, thickeners , stabilizers, antioxidants, solvents, fragrances, fillers, filters, pigments, odor absorbers, pH adjusting agents and dyestuffs. The amounts of these various adjuvants are those conventionally used in the fields under consideration, and for example vary from about 0.01% to 10% of the total weight of the composition. These adjuvants, depending on their nature, can be introduced into the fatty phase, into the aqueous phase and / or into the lipid spherules.

The subject of the present invention is also the use of at least one inhibitor according to the invention in a cosmetic composition for limiting or preventing the appearance of signs, visible or not, of cutaneous aging or for slowing down or attenuating the effects thereof, in particular to control skin remodeling, restructure the epidermis, firm the skin, and / or prevent or promote the attenuation or resorption of wrinkles.

Another subject of the invention relates to the cosmetic use of an inhibitor according to the invention, preferably to limit or prevent the appearance of signs, visible or not, of skin aging or to slow down or attenuate the effects thereof, particularly to control skin remodeling, restructure the epidermis, firm the skin, and / or prevent or promote the attenuation or resorption of wrinkles.

In particular, the cosmetic use according to the invention aims to reduce wrinkles and fine lines, especially those appearing on the face, neck, décolleté or hands.

The present invention also relates to a cosmetic treatment method for reducing or preventing the appearance of signs, visible or not, of intrinsic and / or extrinsic skin aging or to slow down or attenuate the effects thereof, in particular for controlling skin remodeling. , restructuring the epidermis, firming the skin, and / or preventing or promoting the attenuation or resorption of wrinkles and / or limiting the development of adipose tissue, consisting in applying to a skin area concerned body or face a composition cosmetic composition comprising at least one inhibitor according to the invention.

As indicated above, the present invention also relates to the pharmaceutical composition of the invention, for its use in the treatment of lesions of epithelial tissue, more particularly on cutaneous or ocular lesions of a subject, to promote healing and / or or epithelial regeneration.

In this aspect, topical or ocular administration, depending on the site of the lesion, will be preferred.

In the case where the administration is topical (skin lesion), the pharmaceutical composition of the invention may especially be in the form of aqueous or oily solutions or dispersions of the lotion or serum type, of consistency emulsions liquid or semi-liquid of the milk type, obtained by dispersion of an aqueous phase in a silicone phase (E / Si), a fatty phase in an aqueous phase (O / W: oil-in-water emulsion) or vice versa (W / O: water-in-oil emulsion), or suspensions or emulsions of soft consistency of the cream or gel type aqueous or anhydrous, or microcapsules or microparticles, or vesicular dispersions of ionic and / or nonionic type or of mosses. The cosmetic composition of the invention is preferably in the form of a cream, an ointment, a balm, a milk, an oil, etc. also containing, if necessary, a number of additives and / or adjuvant.

The pharmaceutical composition may be in any galenical form normally used for topical application to the skin. The pharmaceutical composition may in particular impregnate a patch or a dressing.

The compositions are prepared according to the usual methods. The amounts of the various constituents of the compositions according to the invention are those conventionally used in the fields under consideration. In known manner, the pharmaceutical composition of the invention may also contain additives and / or adjuvants customary in the pharmaceutical field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, buffering agents, preservatives, thickeners , stabilizers, antioxidants, solvents, fragrances, fillers, filters, pigments, odor absorbers, pH adjusting agents and dyestuffs. The amounts of these various adjuvants are those conventionally used in the fields under consideration, and for example vary from about 0.01% to 10% of the total weight of the composition.

In the case where the administration is ocular (ocular lesion), the pharmaceutical composition of the invention is then preferably in the form of an ophthalmic composition for local administration in the eye, for example as an eye drops , or an ophthalmic cream. The ophthalmic composition may be an aqueous solution comprising distilled water, a physiological saline solution, in which the peptides of the invention are dissolved. A number of additives may be included in the ophthalmic composition if necessary, for example buffer agents, isotonicity agents with tears, preservatives, thickeners, stabilizers, antioxidants, pH, chelating agents, etc. The bases for ophthalmic creams are, for example, petrolatum, jelene 50 or plastibase, macrogol, etc. Surfactants can be added to increase the hydrophilicity. Additives as described above, for example preservatives, may be added if necessary. In general, for local ophthalmic application, a satisfactory effect in the adult is obtained by the administration of a droplet in the eye of a preparation containing from 0.001 to 10%, preferably 0.01 to 1% by weight. of the compound of the invention or of a pharmaceutically acceptable salt thereof several times, preferably one to six times daily, and each time with preferably one to four droplets per eye, and in the case of use of an ophthalmic cream, a preparation containing 0.001 to 10%, preferably 0.01 to 1% w / v of the compound of the invention or a pharmaceutically acceptable salt thereof, with an application preferably one to six times a day in the eye.

The treatment of the cells of the sample with the inhibitor of the invention must be sufficient for the functions of the epithelial cells contained in the sample to be improved in a reasonable time. The exact dose of said inhibitor in the composition of the invention may vary depending on the desired effect, the type of inhibitor, the form of the composition, or the general condition of the subject to be treated.

In the context of the present invention, said subject is an animal, such as a rodent, a mammal, etc., preferably a mammal such as a primate, an equine, a canine, a felid, etc. Even more preferably, said subject is man.

METHOD OF SCREENING THE INVENTION

Another aspect of the invention relates to a screening method for identifying compounds capable of maintaining and / or improving the properties of epithelial cells. These properties are, for example, their proliferation, their immature nature or their degree of immaturity (ie of non-differentiation), or their regenerative potential.

By "maintaining and / or improving the proliferation of epithelial cells" is meant here a stabilization and / or an increase of at least 10%, at least 15%, at least 20%, at least 25%, preferably at least at least 30%, preferably at least 40%, more preferably at least 50% of the growth of epithelial cells contained in a cell sample. This increase in proliferation can be measured by any cell counting technique (by microscopy or flow cytometry for example). This improvement is found for example by comparing the amount of epithelial cells in the presence and absence of the candidate compound, after a given time.

By "maintenance and / or improvement of the immature character or the degree of immaturity (ie non-differentiation) of epithelial cells" is meant here a stabilization or an increase of at least 10%, at least 15%, at least 20%. %, at least 25%, preferably at least 30%, more preferably at least 40%, more preferably at least 50% of the degree of immaturity (ie non-differentiation) of stem cells, progenitors or precursors epithelial cells. Immature character is a necessary condition for the development of the stem potential, ie the potential to regenerate in the long term and to produce a functional skin graft. In other words, the more cells are immature, the greater the proportion of cells with regenerative potential in a given sample. By increasing the degree of immaturity is meant especially, but not exclusively, an increase as defined above the level of expression of immaturity markers. The degree of immaturity can be measured by studying the expression of phenotypic markers known to those skilled in the art, such as the integrin a6.

This screening method consists in fact of measuring whether candidate compounds are capable of reducing (or even blocking or suppressing) the expression or the activity of one or more "Krüppel-like" transcription factor (s) (KLF). ) in epithelial cells. The invention therefore aims in this aspect at a method for screening compounds capable of improving the properties of epithelial cells, said method comprising the step of measuring the capacity of each candidate compound to reduce the expression or activity of a or several Krüppel-like transcription factor (KLF) in epithelial cells.

In a preferred embodiment, said Krüppel-like transcription factor (KLF) is the KLF4 transcription factor. In a particularly preferred manner, said transcription factor KLF4 has the sequence SEQ ID NO: 1 (human KLF4).

Such a method is preferably implemented in vitro.

All types of compounds can be tested in such a screening method.

The epithelial cells used in this method may be of tissue origin (i.e., obtained by culturing primary cells derived from tissue) or transformed lines (e.g., keratinocytes of the transformed cell line). human carcinoma A431).

In this screening method, the candidate compounds are, for example, selected when the expression or activity of said member of the KLF family is "diminished" or "blocked" within the meaning of the present invention (as defined above for the inhibitor of the invention).

Methods for evaluating the expression and / or activity of members of the KLF family have also been described above.

CULTURE METHOD

The present invention also provides a method for culturing epithelial cells contained in a cell sample, or for enriching a cell sample with epithelial cells, comprising at least: - contacting an inhibitor of the expression or activity a Krüppel-like transcription factor (KLF) with said cell sample, so as to increase the proliferation and / or maintain and / or increase the immature character of epithelial cells contained in said cell sample.

Preferably, the method for culturing epithelial cells according to the invention is characterized in that said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of sequence SEQ ID NO: 1 .

The present invention also provides a method for increasing the proliferation and / or maintaining and / or increasing the immature character of epithelial cells contained in a cell sample, said method comprising a step of using the inhibitor or a composition according to the invention, or else a kit comprising such an inhibitor or such a composition, in cellular and tissue engineering (for example for the optimization of epithelial cell amplification processes, for the improvement of the quality of epithelial cell banks, and / or for the improvement of in vitro models of three-dimensional reconstructed epithelia), to promote care, healing, or epithelial regeneration (in cutaneous or ocular lesions), and / or to limit the aging of the skin (anti-aging strategies). age), or for cell therapy, transplantation, or gene therapy (eg for optimisatio n ex vivo clinical expansion of epithelial cell expansion protocols, for enhancing the regenerative potential of reconstructed epithelial tissue grafts, and / or for enhancing the growth potential of epithelial cell samples to be modified genetically (especially in the context of gene therapy approaches).

Preferably, in this method according to the invention, said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of sequence SEQ ID NO: 1.

The following figures illustrate, in connection with the examples below, embodiments of the present invention:

Figure 1 depicts the effect of treating keratinocytes of clonal origin with anti-Klf4 shRNAs at both the mRNA (qRT-PCR; A) and protein (western blot; B) levels.

Figure 2 depicts the proliferation of keratinocytes in response to treatment with anti-Klf4 shRNAs: short-term (7 days, A), medium-term (14 days, B), and long-term (13-week kinetic curve; .

FIG. 3 describes the cytological characteristics of keratinocytes whose Klf4 factor is repressed (more homogeneous, small cells, A), the amount of p63 mRNA expressed by these cells (B), or genomic alterations (duplications or deletions) present in these cells (C).

Figure 4 describes the clonogenic potential of keratinocytes whose Klf4 factor is repressed: clonal growth patterns (clonal microcultures, A), quantification of large clones (clonal microcultures, B), and conventional colony tests (low density cultures). C).

FIG. 5 describes the cell richness of a reconstructed skin graft in response to the Klf4 factor repression: increased graft thickness (measured on histological sections, A), increased number of cells within the grafts (measured by extraction then counting cells; B).

Figure 6 describes the clonogenic capacity of keratinocytes of the follicular epidermis in response to an addition of Kenpaullone (Ken) in the culture medium for 5 days: clonal growth profiles (clonal microcultures) enriched in large clones (A) , number of highly proliferative clones increased (B), number of clone exhibiting a very immature phenotype increased (high level of expression of IgG-a6) (C). Figure 6D shows examples of Itg-a6 phenotypic profiles (flow cytometry).

Figure 7. Impact of KLF4 repression on the sensitivity of keratinocytes to the differentiating effect of TGF-βΙ; (A) Study of the differentiating effect of TGF-βΙ in control cells and KLF4-rep exposed to TGF-βΙ (300 and 1000 μg / ml) for 7 days. A dose-dependent reduction of the fraction of immature keratinocytes (Itg-a6 +) is shown in typical alpha-6 integrin expression patterns (Itg-a6) obtained by flow cytometry (left panel), and on the histogram for independent experiments (n = 5, right pane); (B-C) Comparative transcriptional profiling and DNA methylation analysis at 5 weeks post-transduction confirm molecular linkages between KLF4 level, TGF-βΙ signaling, and immaturity of keratinocytes; (B) Examples of differentially expressed transcripts associated with stem cell properties and immaturity (left pane) or differentiation (right pane, microchips), and qRT-PCR validation; (C) Examples of genes displaying differential patterns of CpG methylation within the promoter region, in addition to transcriptional modulation. Data obtained with Illumina 450K chips are confirmed for specific genes by pyrosequencing (differential CpG methylation> 20% with 450K chips and> 10% with pyrosequencing); (D - E) Sensitivity of control cells and KLF4-rep to the differentiating effect of TGF-βΙ; (D) Western blot analysis of ΔΝρ63α in cells exposed to 150, 300, or 1000 μg / ml of TGF-βΙ for 7 days, with β-actin as a loading control: typical photographs of gels and histogram representing 5 independent experiences; (E) Cell surface expression of Ntg-a6 in cells exposed to 300 μg / ml TGF-βΙ for 7 days. The percentage of keratinocytes (Itg-a6 +) is determined by flow cytometry (14 control cultures, accumulated from 3 independent experiments); mean ± SEM of independent experiments, * significant at p <0.05, Student's t-test.

Figure 8. In vivo regeneration capacity of the epidermis: human epidermis xenograft produced by bioengineering in nude mice. (A) Typical histology of epithelial layers produced by bioengineering reconstructed on a fibrin gel, obtained using control cells and KLF4-rep. ; (B) Quantification of cellular content of epithelial layers: keratinocytes are extracted by enzymes from grafts (9.1 cm2) and counted (mean ± SEM, n = 3). (C - D) (left flaps) Histological visualization of representative graft sites, 4 weeks after transplant, with the area corresponding to the human reconstructed epidermis transplant, surrounded by the skin of the recipient mouse with the hair follicles (n > 10 transplants). (C - D) (right pane) Live imaging of xenograft sites based on the character (GFP +) of transduced human cells (n> 10 mice). (E) Evaluation of control cell capacity and KLF4-rep for a secondary xenograft. Human keratinocytes derived from primary grafts (4 weeks post-transplant) are sorted according to GFP expression, multiplied by 2 successive mass subcultures, and then used for secondary bioengineering and xenograft grafting. Samples that successfully pass these steps are classified as having the capability of a secondary xenograft. The histogram corresponds to the success rate of the secondary xenografts obtained with 22 and 16 primary transplants for the control cells and KLF4-rep, respectively. (F) Typical tissue sections showing the normal histological characteristics of secondary grafts produced with control cells and KLF4-rep (3 weeks post-transplant, n> 10 transplants); mean ± SEM, * significant to p <0.05, Bernouilli test.

Figure 9. Extinction of ΔΝρ63α expression following treatment with a high dose of TGF-βΙ. The effect of exposure for 7 days to 1000 μg / ml of TGF-βΙ on the expression of the ΔΝρ63α protein is analyzed by western blot in control keratinocytes and KLF4-rep. Untreated keratinocytes are used as positive control cells. Β-actin is detected in each sample. The data presented corresponds to a standard analysis of duplicate samples. ΔΝρ63α protein is not detected in any of the cells treated with 1000 μg / ml TGF-βΙ

Figure 10. Net boundary between the skin of the recipient mouse and human xenografts: Live imaging of the xenografted epidermis. (A-B) Live imaging of xenograft sites was performed based on the phenotype (GFP +) of control keratinocytes derived from transduced human holoclones (n> 10 mice; A), and KLF4-rep keratinocytes. holoclone derivatives (n> 10 mice, B).

Histograms showing fluorescence levels (arbitrary units, ua) detected within the human graft and within the surrounding mouse skin indicate a clear boundary between the skin of the recipient mouse and the xenografted human skin substitute.

Figure 11. Normal histology of the human epidermis after in vivo xenografting.

Histological tissue section of an epidermis produced with control keratinocytes and KLF4-rep after in vivo transplantation. (A - B) The histology of primary grafts of human skin produced by bioengineering is examined 4 weeks after xenografting on immunodeficient nude mice. Histological tissue sections are stained with hematoxylin-eosin-saffron (HES). The typical sections represented correspond to 4 replicates of grafts produced with control holoclonal keratinocytes and KLF4-rep, respectively (A) and (B).

Normal histological characteristics are observed for both control keratinocytes and KLF4-rep keratinocytes (n = 6 experiments, with a total of 30 grafted mice for both cell contexts).

Figure 12. Quantification of basal keratinocyte content in secondary xenografts. Histomorphometric analysis is conducted to quantify basal keratinocytes in secondary grafts produced with control keratinocytes and KLF4-rep, 3 weeks after xenograft. The number of basal keratinocytes per mm of epidermal cut is determined by counting in 10 grafts for each cellular context. The data presented correspond to the mean ± SEM. ** significant to p <0.01, Mann-Whitney U test.

Particular embodiments and advantages of the present invention are described in the examples below. These examples are provided for illustrative purposes only; they in no way limit the object or scope of the invention.

EXAMPLES A. Introduction

Despite its successful use in skin regeneration especially after massive burns, keratinocyte-based cell therapy strategies face a recurring problem of variability that makes the clinical outcome of extensive skin grafts uncertain. In particular, the production of large areas of skin produced by bioengineering requires a massive ex vivo cell expansion step, during which the maintenance of the immaturity of the stem and progenitor cells is a crucial criterion for preserving the regenerative potential of the grafted skin. At present, this highly important parameter remains incompletely controlled due to insufficient understanding of the cellular and molecular regulators involved in human keratinocyte stem cell transplantation capacity.

In order to overcome this need to preserve the immaturity of stem and progenitor cells, the inventors have studied the function of the KLF4 transcription factor in the biology of human keratinocyte precursors and the regeneration of the skin. In the examples described hereinafter, human holoclonal keratinocytes are used to study the functions of KLF4, as they are representative of the state of immature precursor cells with high growth potential and regenerative capacity. epidermis and thus have direct clinical relevance. A stable KLF4 lentivirus (KD for Knock-Down) approach has been developed as a proof of principle to study the properties of control keratinocytes and KLF4-rep. Using long-term cultures and clonal assays, the inventors show that the negative modulation of KLF4 increases the growth and clonogenic potential of precursor cells of human keratinocytes. In addition, KLF4-rep keratinocytes display improved graft capacity in a skin xenograft model in vivo, including in serial grafts. The decrease in KLF4 expression increases the immaturity of the keratinocyte precursors and the cell cycle, thus promoting self-renewal. This self-renewal is implemented by cross-antagonism against anti-proliferation and prodifferentiation effects mediated by the TGF-βΙ growth factor. In conclusion, the examples presented below show that KLF4 is a controller for the self-renewal of keratinocyte precursors.

The examples described below designate KLF4 as a major molecular target for enhancing the ex vivo expansion of fully functional human keratinocytes for epidermal regeneration. The present invention opens up direct prospects for the development of clinically relevant molecules that can modulate either KLF4 or its downstream pathway in keratinocytes to improve skin repair and skin regeneration. B. First part of the examples

Example No. 1 Improvement of the Growth of Cell Libraries Corresponding to Clonal Progeny of Keratinocyte Stem Cells 1.1. Materials and methods 1.1.1. Lentiviral vectors

The lentiviral constructs (Vectalys ®) used in the present example have the following general characteristics: - Skeleton of the vector derived from the HIV-1 virus. - Presence of a reporter gene (cDNA encoding GFP) allowing the selection of transduced cells. Expression of an anti-Klf4 shRNA under the control of the promoter EF1-a.

In the present example, the anti-Klf4 shRNA sequence selected to generate the KLF4-rep context is in the coding region of the mRNA. The negative control vector used in this example is distinguished from the active anti-Klf4 vector by the absence of shRNA. The strategy was further validated with a negative control vector encoding an action-free shRNA (data not shown). 1.1.2. Transduction of keratinocytes

The cultures, initially inoculated at a density of 1000 cells / cm 2, and brought to a confluence stage of 15-20%, are infected with a lentiviral suspension, the value of "multiplicity of infection" (MOI) being adjusted to 1 particle. active viral / cell. The cell cultures are incubated with the virus particles for 15 to 18 hours in the presence of a transduction promoting agent (hexadimethrin bromide (Sigma-Aldrich) at 8 μg / ml). During the keratinocyte transduction phase, the culture medium used has the particularity of comprising a fetal bovine serum (Hyclone) previously decomplemented by heating at 56 ° C. for 30 min. At the end of this step, the culture medium is renewed, and the cultures are continued until reaching 70-80% confluence, generally after 3-4 additional days of culture. The cells are then detached by trypsination, and cell sorting by flow cytometry (MoFlo, Beckman Coulter) is then performed, so as to purify the fraction transduced on the basis of the presence of the selection marker provided by the vector (in the case present, expression of the GFP fluorescent protein).

1.1.3. Q-RT-PCR

Four weeks after the lentiviral transduction step, the effect of anti-Klf4 shRNA on the expression level of Klf4 mRNA, as well as on the expression of p63 mRNA, was quantified by reverse transcription and Real-time PCR. Briefly, extractions of the total RNA from control cells or expressing the anti-Klf4 shRNA are carried out using the RNeasy kit (Qiagen). The RNA samples are then reverse transcribed into cDNA using the Superscript II Reverse Transcriptase kit (Life Technologies). The real-time PCR reactions are carried out according to a quantification principle using the SYBR® Green fluorescent molecule (Life Technologies) using specific primer pairs (Sigma-Aldrich) targeting the mRNA of different genes of interest, in particular Klf4, or 18S RNA, used for normalization (see Table 1 for the sequences of the different primers used). Each PCR amplification is performed in 7500 Fast Real-time PCR System (Life Technologies). The data are normalized with respect to the amplification of the cDNA corresponding to 18S RNA and analyzed in a comparative manner according to the AACt method (Livak and Schmittgen, 2001).

Table 1. Sequences of qRT-PCR primers

1.1.4. Western blot The effect of the anti-Klf4 shRNA on the level of expression of the KLF4 protein is studied by western-blot 4 weeks of culture after the lentiviral transduction step. This analysis is performed on nuclear extracts prepared according to Schreiber's method (Schreiber et al., 1989). Briefly, the cells are peeled off in the presence of protease inhibitors and phosphatases (Roche) and resuspended in a buffer solution containing HEPES, EGTA, KCI, DTT, NaF (Sigma -Aldrich) and protease inhibitors (Roche) and lysed on ice. After addition of NP-40 (Sigma-Aldrich), centrifugation is carried out, resulting in the separation of the cytoplasmic (supernatant) and nuclear (pellet) fractions. The pellet is then vortexed and centrifuged to obtain a nuclear protein extract, the concentration of which is determined using the Micro Protein Assay kit (BCA, Thermo Scientific). After denaturation at 70 ° C., the proteins are separated by electrophoresis (4-12% Novex Tris-Glycine precombined gels, Life Technologies) and then transferred to the PCDP membrane (Life Technologies). Detection of KLF4 protein and lamin (used as a standard for calibrating nuclear samples) is performed using specific primary antibodies: anti-KLF4 (sc-166100, Santa Cruz Biotechnology); anti-laminate A / C (JOL 2, Millipore). The signal is revealed and detected using an H RP conjugated secondary antibody (Pierce, Thermo Scientific), then by immersion in ECL (ECL detection kit, Life Technologies) and exposure of a hypersensitive film (Amersham Biosciences) . 1.1.5. Mass cultures

Proliferation is analyzed under previously described culture conditions: feeder layer of irradiated dermal fibroblasts and medium containing serum (Fortunel et al., 2010, 2011). Briefly, the cultures are made in flasks of 25 cm 2 cultures whose surface is covered with a type I collagen film (BioCoat, Becton-Dickinson), and in which irradiated human fibroblasts have been previously put in place (6000 cells / cm2). The keratinocytes whose proliferation capacity is evaluated are seeded at a density of 1000 cells / cm 2, and cultured in a medium based on a mixture consisting of DM EM and Ham F12 medium (3/1, Gibco), and supplemented. with 10% fetal bovine serum (Hyclone), 10 ng / ml EGF (Chemicon), 5 μg / ml transferrin (Sigma-Aldrich), 5 μg / ml insulin (Sigma-Aldrich), 0.4 μg / ml hydrocortisone (Sigma-Aldrich) Aldrich), 180 μΜ adenine (Sigma-Aldrich), 2 mM triiodothyronine (Sigma-Aldrich), 2 mM L-glutamine (Gibco), and 100 U / ml penicillin / streptomycin (Gibco). The cells are detached by trypsination (Gibco) after 1 week of culture, counted on Malassez cell, then reseeded in a similar way every week if it is to study the proliferation in the medium and long term. For long-term crops, the results are expressed as cumulative expansion curves (cumulative number of population doublings (DPs) during successive subcultures), using the formula DP = ( log N / N0) / log 2, where N0 represents the number of cells seeded, and N the number of cells obtained at the end of each culture. 1.1.6. Comparative Genomic Hybridization (CGH) The integrity of the genome in the context of repression of KLF4 expression is analyzed using pan genomic CGH chips (3x720K Whole-Genome Tiling v3.1 technology, according to the procedure developed by the supplier; Roche NimbleGen). 1.2. Results

The cellular material used consists of keratinocyte libraries of clonal origin produced from normal adult human skin samples, more precisely from interfollicular epidermis, and functionally characterized as corresponding to stem cell progeny. keratinocyte (also defined as 'holoclones', Fortunel et al., 2010, Patent FR2927633 / WO2009103731).

The embodiment used in the present case to generate a "KLF4-rep" holoclone context (ie, repressed for Klf4) is a stable transduction of an anti-Klf4 shRNA using a lentiviral vector.

On a molecular level, this implementation results in a reduction of Klf4 mRNA by a factor of 2.3 ± 0.7, a decrease equivalent to 57% ± 9% compared to control holoclones (p. <0.05; n = 3; Q-RT-PCR). Repression is also verified for the amount of nuclear KLF4 protein (western blot, Figure 1). The biological effect generated by the KLF4-rep cell context was first evaluated by short-term proliferation quantification. Cells corresponding to the KLF4-rep and control situations were placed in culture in exactly the same manner, in an mitogenic condition, and then comparative monitoring was performed over 2 weeks.

After 7 days of culture, the proliferation index obtained with the control cells is evaluated at 40 ± 2 while that of the KLF4-rep cells is 81 ± 7, which corresponds to a growth gain of one factor. 2.3. After 2 weeks, the proliferation indices are evaluated at 1.8x103 ± 0.3x103 (control cells) and 6x103 ± 1.2x103 (KLF4-rep cells), which brings the growth gain to a value of 3.3 ( p <0.05; n = 3; cf. Figures 2A and 2B). The effect of function gain generated by the KLF4-rep context was also followed in a more prolonged manner, in the context of long-term cultures maintained for 13 weeks. The gain persists for the duration of the observation, resulting in a cumulative growth increased by 40 population doublings in the KLF4-rep situation compared to the control configuration (13 weeks post-implementation of the KLF4-rep context; 2C).

At the cellular level, the KLF4-rep effect results in cytological characteristics indicative of a globally more immature (less differentiated) state of the cultures (small keratinocytes, high nucleo-cytoplasmic ratio, see Figure 3A). At a molecular level, the KLF4-rep effect results in an increased level of mRNA expression of the p63 immaturity marker, which reinforces the cytological observation (see Figure 3B). A key aspect is the question of the possible appearance of genomic alterations likely to lead to a transformation phenomenon, as a consequence of the establishment of the KLF4-rep cell context. Analysis of KLF4-rep cell DNA by the comparative genomic hybridization technique showed no detectable deletion or amplification (> 1 kb, Figure 3C). In addition, cells retained their potential to repair DNA damage after exposure to UVB (data not shown).

Example No. 2 Improving the Clonogenic Efficiency of Cell Libraries Corresponding to the Progeny of Keratinocyte Stem Cells 2.1. Material and methods 2.1.1. Lentiviral vectors and keratinocyte transduction

As in Example 1, the embodiment used to generate a KLF4-rep holoclone context is stable transduction of an anti-Klf4 shRNA using a lentiviral vector. 2.1.2. Clonal microcultures

The clonal microcultures test is performed in 96-well culture plates, the surface of which is covered with a type I collagen film (BioCoat, Becton-Dickinson). As in the case of the mass cultures used in the embodiment example 1, a feeder layer of irradiated fibroblasts is placed the day before the seeding of the keratinocytes (density: 6000 fibroblasts / cm 2). The following day, the keratinocytes (suspensions samples) are automatically deposited by flow cytometry with a single cell by culture microwells (cytometer equipped with a cloning module, MoFlo equipped with CyClone module, Beckman Coulter). The cultures are maintained for 2 weeks during which the culture medium, of composition identical to that described for the mass cultures of the embodiment example No. 1, is renewed 3 times a week. Culture wells in which the development of a keratinocyte clone occurs are observed by observation under an inverted microscope (Axio Observer D1, Zeiss). Their size is determined individually at the end of the 2 weeks of culture, by detachment of the cells (trypsination, Gibco) and counting in Malassez cell. Each individual clone is classified according to its size, which makes it possible to obtain clonal growth profiles representative of the samples of keratinocytes analyzed. 2.1.3. Colony test

These tests are carried out in petri dishes of 100 mm diameter whose culture surface is covered with a type I collagen film (BioCoat, Becton-Dickinson). The culture conditions are identical to those used for the test of clonal microcultures: feeder layer of irradiated fibroblasts put in place the day before seeding keratinocytes (density: 6000 fibroblasts / cm 2) and culture medium of the same composition. The keratinocytes of the tested samples are inoculated at a density of 20 cells / cm 2, then the cultures are maintained for 2 weeks. At the end of the test, the cultures are fixed with 70% ethanol, then stained with eosin and RAL blue (RAL Reagents). 2.2. Results

The functional parameter of clonogenicity is an indicator used to qualify the growth and regeneration potential of cell samples, in particular that of clinical or industrial interest banks.

In the present case, the clonal microcultures system (Fortunel et al., 2010: Patent FR2927633 / WO2009103731) has been implemented on cells corresponding to KLF4-rep and control holoclones (cell material derived from normal adult human interfollicular epidermis). ), so as to perform a comparative analysis of these two cellular contexts.

A gain in function is observed in the KLF4-rep context, concerning both the frequency of clone formation and their size. The generated clonal growth profiles indicate that KLF4-rep cells produce a greater number of clones than the control cells (in this example, 317 versus 174), this being associated with larger clone sizes (maximum observed sizes: 300 000 cells / clone versus 164,000 cells / clone). When the analysis is focused on clones of size> 60,000 cells, the clonogenic efficiency of KLF4-rep holoclones is 4.8 times higher than that of control holoclones (22.5% ± 3% versus 4, 6% o ± 2% o; p <0.05; n = 3) (Figure 4A and 4B).

Concordant results are obtained under standard clonogenicity test conditions (colony test: low-density non-clonal cultures): total clonogenic efficiencies of 40% ± 0.3% (KLF4-rep) versus 24% ± 1% o (control; <0.05; n = 3; Figure 4C).

Example No. 3 Improvement of the keratinocyte richness of a cutaneous graft. 3.1. Material and methods 3.1.1. Lentiviral vectors and keratinocyte transduction

As in Examples No. 1 and No. 2, the embodiment used to generate a KLF4-rep holoclone context is stable transduction of an anti-Klf4 shRNA using a lentiviral vector. 3.1.2. Preparation of skin grafts

A dermis equivalent is first reconstituted, the principle being to produce a fibrin matrix in which non-irradiated fibroblasts are incorporated. This cellularized fibrin gel is obtained by mixing fibroblasts (in this case, human fibroblast library of dermal origin) with human plasma, in the presence of NaCl (Sigma-Aldrich), tranexamic acid (Exacyl®, Sanofi ), and CaCl2 (Sigma-Aldrich). The mixture, prepared on ice, is cast in a petri dish of 9.6 cm 2 (BD Falcon, Becton-Dickinson), then the whole is placed at 37 ° C for 30 min, which allows the polymerization of fibrin . The fibrin gels are then covered with culture medium for keratinocytes (composition described in the examples of embodiment No. 1 and No. 2). The following day, the keratinocyte samples are seeded on these dermal supports at a density of 2400 cells / cm 2. The organotypic cultures are maintained for 2 weeks, which results in the development of a pluristratified epithelium on the dermal supports. 3.1.3. Extraction of keratinocytes from cutaneous grafts.

The cell richness of the epithelial component of cutaneous grafts is determined by extraction and counting of keratinocytes at the end of the 2 weeks of organogenesis in culture. The reconstructed epithelium is detached from the dermal component by enzymatic digestion using a trypsin mixture (Gibco) dispase (Life Technologies), followed by mechanical separation. It is then dissociated by enzymatic digestion using trypsin-EDTA (Gibco). The keratinocyte suspension obtained is filtered (cell strainers 70 μm, BD Falcon, Becton-Dickinson), then these cells are counted in Malassez cell. 3.2. Results

The epidermal reconstruction potential of cell libraries derived from KLF4-rep holoclones and controls was analyzed in a three-dimensional (3D) cutaneous organogenesis model using as bio-support a dermal equivalent reconstituted from a fibrin gel. This model has the advantage of being strictly comparable to cutaneous grafts used clinically in the context of grafts performed on burn victims (Ronfard et al., 2000).

Histological sections performed 2 weeks after initiation of the process of obtaining 3D grafts make it possible to visualize a thickness and a higher keratinocyte richness when the epidermal reconstruction is carried out from KLF4-rep cells (FIGS. 5A and B). This gain in cells is confirmed by counting the number of cells contained within grafts derived from KLF4-rep cells and controls: 8x105 ± 0.2x105 (KLF4-rep) versus 4x105 ± 1x105 (control, number of keratinocytes / d an area of 9.6 cm2; <0.05; n = 3; Figure 5B).

Example No. 4 Improvement of the clonogenic capacity of cell banks produced from keratinocytes corresponding to the total basal fraction. 4.1. Material and methods 4.1.1. Cellular material

In the present example, the basal keratinocyte library used was generated after type I collagen adhesion enrichment, as previously described (Fortunel et al., 2011). Briefly, the total keratinocytes are extracted from a skin sample, and the cell suspension obtained is then deposited on a culture surface covered with a type I collagen film (BioCoat, Becton-Dickinson culture flasks). An incubation for 10 to 15 minutes at 37 ° C of keratinocytes placed on a collagen support makes it possible to define and separate 2 cell populations: 1) population having a rapid adhesion capacity (Adh +++), capable of attaching during this short period of time incubation; 2) population with reduced adhesiveness (Adh + / '), not attaching to the support during this short incubation. The Adh +++ population corresponds to basal keratinocytes (~ 10% of total keratinocytes). It is used for primary amplification, which makes it possible to generate banks that are stored frozen in liquid nitrogen. The Adh + / 'population, consisting of differentiated keratinocytes, is not conserved. 4.1.2. Kenpaullone treatment

The treatment with Kenpaullone is carried out under mass culture conditions, as described in Example No. 1 (culture in medium containing serum, on the "feeder" layer of irradiated fibroblasts). The cell sample from basal keratinocytes with rapid adhesion (Adh +++) is thawed and then seeded at a density of 1000 keratinocytes / cm 2. Kenpaullone (Sigma) is added or not to the culture medium (concentration of 5 μΜ) 48 hours after initiation of cultures, the treatment being maintained for 5 days (until crop stop). The cells which have been treated with Kenpaullone and which result from the control condition are then removed by trypsination (Gibco) and then used for comparative evaluation of their clonogenic potential, by the test of clonal microcultures. 4.1.3. Clonal microcultures

The clonal microcultures test was implemented to characterize the clonogenic potential of a keratinocyte library that had or had not received the Kenpaullone treatment, according to a methodology identical to that used in the context of Example No. 2. Clonal microcultures were tested in the absence of Kenpaullone, whether treated or untreated. 4.1.4. Immuno-phenotypic analyzes

The degree of immaturity of keratinocyte clones from or without Kenpaullone-treated libraries is assessed by analysis of integrin-α6 expression (Itg-a6; CD49f). After detachment of the cells by trypsination (Gibco), the cells suspended in PBS / BSA (Sigma) are incubated with rat γ-globulins (Jackson ImmunoResearch) and then labeled by incubation with a specific anti-CD49f antibody coupled to phycoerythrin. (PE, anti-CD49f-PE, GoH3 clone, BD Pharmingen). An antibody with the same characteristics but not immunoreactive is used as isotype control (IgG-ΡΕ, clone R35-95, BD Pharmingen). The signals are collected and analyzed by flow cytometry (MoFlo equipped with a 488 nm argon laser, Beckman Coulter). 4.2. Results

Cellular libraries produced from keratinocytes derived from the basal layer of the interfollicular epidermis comprise cells covering different immaturity statuses: keratinocyte stem cells, early and late keratinocyte progenitors. This cell material has been subjected to the action of a chemical compound, Kenpaullone (Ken), whose property is to suppress the activity of the Klf4 promoter (Yu et al., 2011).

Mass cultures seeded from basal keratinocyte libraries were treated or not for 5 days with 5 μl of Kenpaullone (5 μM Ken, molecule added or not in the culture medium). At the end of this treatment, the clonogenic keratinocyte content was evaluated in a comparative manner in cultures exposed to 5 μM Ken and untreated, using the 'clonal microcultures' method (Fortunel et al., 2010; FR2927633 / WO2009103731).

The clonal growth profiles obtained indicate that the cultures which received the 5 μM Ken treatment are characterized by an increased richness in clones having a high growth potential compared to the untreated control cultures (FIG. 6).

These results are consistent with those presented in Example No. 2. Indeed, a gain in clonogenic keratinocytes is obtained by two different approaches: repression of Klf4 mRNA using a specific shRNA and pharmacological inhibition of the Klf4 promoter. C. Second part of the examples 1. Materials and methods

The following materials and methods are complementary to some or all of the materials and methods presented in the first part of the examples (part A. above). 1.1. Lentiviral vectors and keratinocyte transduction

As in Examples No. 1, No. 2, and No. 3, the embodiment used to generate a KLF4-rep holoclone context is a stable transduction of an anti-Klf4 shRNA using a lentiviral vector. 1.2. TGF-β 1 exposure

Recombinant human TGF-βΙ (R & D Systems) is added to the culture medium at the specified concentrations. For short-term (24- or 48-hour) treatments, TGF-βΙ is added when the culture has reached about 30% confluency. For longer treatments with TGF-βΙ (7 days), seeding densities are adjusted to compensate for its antiproliferative effect and to obtain sufficient cells for phenotype analysis. Initial keratinocyte densities were increased to 2000, 4000, and 8000 cells / cm2 in cultures exposed to low (150 μg / ml), intermediate (300 and 450 μg / ml), and high (1000 μg / ml) concentrations. ml) of TGF-βΙ 1.3. DNA Microarrays for Transcriptome Analysis The total RNA extracted with the RNeasy Mini and Micro kits (Qiagen) is amplified according to the Illumina TotalPrep RNA Amplification Kit (Life Technologies) protocol. Briefly, 500 μg of total RNA is reverse transcribed using ArrayScript® reverse transcriptase and a T7 oligo (dT) primer. Double-stranded cDNA templates are then used for in vitro transcription to produce biotin-modified cRNA. After fragmentation, the cRNAs are hybridized and processed on a BeadChiP HumanHT-12 v4 chip (Illumina Inc), according to the manufacturer's instructions. The BeadChip expression chips are scanned with a Beadarray reader (Illumina Inc). Statistical analysis is performed using GeneSpring GX software (v 11.5, Agilent). The standardized data is then subjected to a one-way ANOVA test to identify the differentially expressed transcripts (ratio threshold of 1.5), and the gene lists are deposited in GEO (geo@ncbi.nlm.nih.gov, access number pending). The functional annotation analysis is performed on the gene lists using DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/) on the Gene Ontology and KEGG Pathway databases. 1.4. Human DNA Methylation Microchips The DNA is prepared using the DNeasy Blood and Tissue Kit (Qiagen), according to the manufacturer's instructions. DNA is quantified using the Quant-iT dsDNA Broad-Range Assay kit (Life Technologies). One μg of DNA is converted with bisulfite using the EpiTect Bisulfite Conversion kit (Qiagen). Infinium 450K Methylation BeadChips chips (Illumina Inc) are used for analysis of whole genome methylation and scanned using the Illumina iScan system (Illumina Inc). The data is extracted using the Genome Studio version 2011.1 software, methylation module version 1.9.0 (Illumina Inc.) without any standardization step. Quality control and data standardization are performed using a refined version of an internally developed pipeline based on quantile standardization of subsets (Touleimat & Tost, 2012). The lists of probes are obtained by non-parametric tests (Mann Whitney) with a nominal p-value <0.05 and a median DNA methylation difference of 10 or 20% between the two conditions. 1.5. pyrosequencing

The quantitative validation of the methylation of the bisulfite-treated DNA is performed by pyrosequencing, as described in Tost &amp; Gut, 2007. In short, it was designed tests covering ~ 200 bp around a selected list of CpG (CpG showing at least 5% methylation difference). One μg of DNA is converted with bisulfite using the EpiTect 96 Bisulfite kit (Qiagen). The reactions are performed using HotStar Taq polymerase (Qiagen). Ten μΙ of PCR product is made single stranded and 4 pmol of the respective sequencing primer is used for analysis. Quantitative analysis of DNA methylation is performed on a PSQ 96MD system with the PyroGold SQA Reagent kit (Qiagen) and the results are analyzed using PyroMark CpG software (v. 1.0.11.14, Qiagen, see table 2 for the sequences of the primers used).

Table 2. Sequence of Primers Used for Pyrosequencing (Pyroseq)

1.6. Xenografting of human skin substitutes The experimental procedures are approved by the Ethics Committee CEEA-51 of the Center for Exploration and Experimental Functional Research (CERFE, Genopole®, Evry, France). Experiments and accommodation are managed at CERFE under appropriate aseptic conditions. Immunodeficient nude mice (Harlan Laboratories) are used as recipients of xenograft of human skin substitutes. The mice are anesthetized by intraperitoneal injection of ketamine (Centravet) and xylasin (Centravet), and are maintained on a heated surface to avoid hypothermia. A full thickness disc of dorsal skin (~ 1 cm2) is removed. This piece of mouse skin is then devitalized by series of freezing in liquid nitrogen and defrosting, and maintained for use in the form of biological bandage. The wound bed is covered with an equivalent surface of human skin substitute produced by bio-engineer. The piece of devitalized mouse skin is then sutured to the edge of the mouse skin to cover and transiently protect the xenograft site. This biological bandage is removed 1 week later, under anesthesia with isoflurane (Axience, anesthesia unit of Minerva).

The mice are euthanized 3 or 4 weeks after the xenograft for graft quality analysis, using the cervical dislocation procedure under anesthesia. 1.7. Qualification of xenotransplantation

Histology: formalin-fixed tissue sections embedded in 5 μm thick paraffin are stained with hematoxylin-eosin-saffron (HES), and then converted to high resolution digital slides using the NanoZoomer system 2.0-HT (Hamamatsu) on the histology platform of INRA / CEA (UMR 1313 GABI, Jouy en Josas). In addition to examining the overall architecture of the grafted human epidermis, quantification of basal keratinocyte content is performed. For each xenograft sample, at least 3 sections are taken into consideration, in which 3 to 4 regions of interest (ROI) are defined (~ 500 μm of graft cut length / ROI). The number of basal keratinocytes / mm of epidermal length is determined, based on a total of 9-12 KI.

Live Imaging: Xenograft monitoring, based on the detection of GFP fluorescence in transduced human keratinocytes, is performed using the Cellvizio® 488 confocal fluorescence laser endomicroscopy system (Mauna Kea Technologies, Paris). , France), equipped with the S-1500 signal acquisition microprobe. The signal is processed and managed using IC Viewer software (Mauna Kea Technologies). Full-size sections of the xenograft sites are digitally reconstructed by arranging successive signal acquisition frames, using the software's Advanced Mosaicing ™ feature.

Serial transplants: The qualification of the regenerative potential of keratinocytes includes the evaluation of their secondary xenograft capacity. After dissection, the graft samples are cut into 2 x 2 mm pieces, and incubated in a solution containing (v / v) 3/4 grade II dispase at 2.4 U / ml (Roche) and 1 / 4 of 0.25% trypsin (Gibco), for 2 hours at 4 ° C, and then for 30 min at room temperature. The epidermal layers are mechanically separated, and incubated in 0.05% trypsin in EDTA (Gibco) for 20 minutes at 37 ° C. The epidermal pieces are then dissociated by light pipetting to obtain single cell suspensions. Human keratinocytes are purified by sorting in flow cytometry (MoFlo, Beckman-Coulter) according to their phenotype (GFP +). The keratinocytes are expanded via 2 successive subcultures in bulk, and then used for a secondary graft by bioengineering and xenograft, as detailed above. Samples that have passed these different stages are classified as having capacity for secondary grafts. 1.8. Statistics

The statistical significance of the observed differences is determined using Student's t-test (proliferation, cell cycle, expression of markers in cultured cells), Mann-Whitney U-test (counting of basal keratinocytes within xenografts), and test of Bernouilli (successful xenografts). 2. Results

EXAMPLE 5 Control of the immature nature of the precursor cells The effect of TGF-βΙ on the differentiation of holoclonal keratinocytes is studied. The expression of integrin alpha-6 (ltg-αθ), a cell surface marker associated with an immature state of keratinocytes, is followed by flow cytometry on keratinocytes exposed for 7 days at 300 or 1000 μg / ml of TGF-βΙ A dose dependent decrease in IgG-α6 expression is observed in response to treatment with TGF-βΙ (Figure 7A, left panel). While 88.9 ± 5.1% of untreated keratinocytes express IgG-α6, exposure to 300 or 1000 μg / ml of TGF-β decreases expression of IgG-a6 to 60.2 ± 9.4% and 51.3 ± 5.0%, respectively (p <0.01; n = 3; Figure 7A, right pane). These results show that treatment of holoclonal keratinocytes with TGF-βΙ activates their differentiation. Conversely, the comparative transcriptional profiling of control cells and KLF4-rep shows an increase in the level of transcripts associated with stem cell properties and immaturity, such as ALDH3B3, ALDH4A1, and KRT15 (Figure 7B, left pane). ) and a decrease in the rate of differentiation-associated transcripts, such as KRT1, INV, and DSG1 (Figure 7B, right pane) in KLF4-rep cells. Transcriptome analysis and DNA methylation data on the entire genome are used to study the effect of KLF4 repression on the balance between immaturity and differentiation. This analysis identifies, in KLF4-rep cells, the genes associated with the control of stem cell and immaturity properties, displaying both transcriptional activation and promoter hypomethylation, as well as the genes associated with differentiation displaying at the same time. both transcriptional repression and hypermethylation of detectable promoters (Table 3). Differential methylation in the promoter region of the relevant candidate genes, such as ALDH3B2 and DKK1, is confirmed by pyrosequencing (Figure 7C). These results show that the negative modulation of KLF4 increases the immaturity of holoclonal keratinocytes.

Table 3. Genes displaying both transcriptional regulation and methylation changes. Gene selection displaying both transcriptional regulation (transcriptome microchips) and methylation changes (450K methylation chips) in KLF4-rep holoclonal keratinocytes against controls. Sixteen genes are selected according to their link with the TGF-β / ΒΜΡ network, or with stem cell properties. Five genes from this list were analyzed by pyrosequencing, and a median DNA methylation difference of at least 5% between the two conditions was confirmed: AKAP12, BMP6, DKK1, SPON2, ALDH3B2.

To investigate the interaction between KLF4 and the TGF-βΙ pathway in the control of immaturity as opposed to differentiation, control cells and KLF4-rep are exposed to TGF-βΙ for 7 days, and expression markers associated with immaturity, ΔΝρ63α and ltg-αθ, are studied. The expression of ΔΝρ63α is analyzed by Western blot, following a treatment with 150, 300, 450, or 1000 μg / ml of TGF-βΙ In the control cells, the expression of ΔΝρ63α is considerably reduced after the treatment. with TGF-βΙ at 300 and 450 μg / ml, whereas expression of ΔΝρ63α persists at a higher level in KLF4-rep cells exposed to any of these doses of TGF-βΙ (p <0.05; n = 5; Figure 7D). ΔΝρ63α disappears after exposure to the highest TGF-βΙ test dose of 1000 μg / ml, both in control cells and KLF4-rep (Figure 9). Treatment with 300 μg / ml TGF-βΙ reduced the proportion of (Itg-a6 +) from 89 ± 5% to 60 ± 9% in the control cells, whereas this fraction was only reduced by 90 ± 5.5% at 77 ± 4% in KLF4-rep cells (p <0.05; n = 3; Figure 7E). Overall, these results indicate that a deficiency of KLF4 may antagonize the activating effect of differentiation of treatment with TGF-βΙ in keratinocyte precursor cells.

Example No. 6: Gain observed on the ability of cells to ensure an epidermal grafting process and in vivo regeneration The effect of the KLF4 repression on the three-dimensional regeneration capacity of the epidermis by holoclonal keratinocytes is studied. Epidermis substitutes are produced by bioengineering using control cells and KLF4-rep. These substitutes are then analyzed after xenografting in nude mice. In vitro reconstructed epithelia generated using KLF4-rep cells appear thicker than those produced using control cells, and have a higher cell content (Figure 8A). Indeed, the quantification of the total keratinocytes contained in the grafts has shown that the cell content of the epidermal layers KLF4-rep is 1.8 times greater than that of the control controls (p <0.05; n - 3; Figure 8B). A similar effective coverage of the graft site is obtained in vivo with the control cells and KLF4-rep (Figures 8, C and D, left pane). Monitoring of live imaging grafting based on the detection of GFP fluorescence in transduced control cells and KLF4-rep shows that coverage is due to human epidermal substitutes, as expected (Fig. 8). , C and D, right flaps, and Figure 10). In addition, histological examinations carried out 4 weeks after the grafting show a normal tissue structure of the regenerated epidermis and terminal differentiation of the cells, with normal development of the granular and corneal layers, indicating that the repression of KLF4 does not produce any anomaly whatsoever. in the regeneration of the epidermis (n> 10 grafts, Figure 11).

In order to further study the regenerative potential of grafts produced with control cells and KLF4-rep, mice are euthanized 4 weeks after transplantation, and primary grafts are dissected and processed to extract keratinocytes. The total human keratinocytes are then sorted by flow cytometry according to the expression of GFP, and used for a second cycle of bioengineering and xenograft grafting. Three weeks after the transplant, only 3 out of 22 control samples tested resulted in a secondary transplant (13.6%). In contrast, for KLF4-rep cells, 7 out of 16 (43.8%) samples give rise to grafts, indicating a beneficial effect of KLF4 repression for a secondary graft (p. <0.05; Figure 8E). The qualitative and quantitative evaluation of the secondary grafts shows that the grafts obtained with the control cells and KLF4-rep present a normal organization and differentiation of the epidermis (Figure 8F). Quantification of cells on tissue sections reveals an increase in the number of basal keratinocytes (KB) in grafts produced with keratinocytes corresponding to the KLF4-rep cell context (160.1 ± 35.1 KB / mm cut vs. 125.3 ± 10.5 KB / mm in control grafts; <0.01; n = 10; Figure 12).

Taken as a whole, these results show that the negative modulation of KLF4 improves the self-renewal and the long-term regeneration capacity of keratinocyte precursor cells, without inducing a disturbance in the three-dimensional organogenesis of the epidermis.

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Claims (15)

1. In vitro use of an inhibitor of KRF expression or activity to increase proliferation and / or maintain and / or enhance immature epithelial cells contained in a cell sample, or for enriching a cell sample in epithelial cells, wherein said inhibitor is selected from a chemical molecule such as a peptide, peptide-like molecules, an organic compound, Kenpaullone, or a antibody or antibody fragment, or a nucleic acid such as an antisense oligonucleotide, or an aptamer, or a ribozyme. 2. Use according to claim 1, characterized in that said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of sequence SEQ ID NO: 1. 3. Use according to claim 1 or 2, characterized in that said cell sample of the invention is derived from a biological sample obtained from a healthy or sick subject, preferably human. 4. Use according to any one of claims 1 to 3, characterized in that said inhibitor is a chemical molecule, a nucleic acid or a ribozyme which decreases or blocks the expression of the gene coding for a transcription factor of the type "Krüppel -like "(KLF). 5. Use according to claim 4, characterized in that said inhibitor is an antisense nucleic acid selected from small interfering RNAs ("siRNA"), double-stranded RNAs ("dsRNA"), micro-RNAs (" miRNA "), and short hairpin (" shRNA ") RNAs. 6. Use according to claim 4, characterized in that said inhibitor is a chemical molecule inhibiting the transcription of a gene coding for a Krüppel-like transcription factor (KLF), preferably KLF4. 7. Use according to claim 6, characterized in that said inhibitor is Kenpaullone (CAS number 142273-20-9). 8. Cosmetic composition comprising at least one inhibitor of the expression or the activity of a transcription factor of the "Krüppel-liké" type (KLF), and optionally one or more cosmetically acceptable excipient (s), wherein said inhibitor is selected from a chemical molecule such as a peptide, peptide-like molecules, an organic compound, Kenpaullone, or an antibody or antibody fragment, or a nucleic acid such as an antisense oligonucleotide , or an aptamer, or even a ribozyme. 9. Cosmetic composition according to claim 8, characterized in that said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of sequence SEQ ID NO: 1. 10. Use of the cosmetic composition according to claim 8 or 9 for improving the condition of the skin of a subject. 11. Pharmaceutical composition comprising at least one inhibitor of the expression or the activity of a Krüppel-like transcription factor (KLF), and optionally one or more pharmaceutically acceptable excipient (s), for use in treating epithelial tissue lesions of a subject, preferably skin or ocular lesions, particularly for promoting healing and / or epithelial regeneration, wherein said inhibitor is selected from a chemical molecule such as a peptide, peptide-type molecules, an organic compound, Kenpaullone, or an antibody or antibody fragment, or a nucleic acid such as an antisense oligonucleotide, or an aptamer, or a ribozyme. 12. Pharmaceutical composition according to claim 11 for use according to claim 11, characterized in that said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of sequence SEQ ID NO: 1. 13. In Vitro Screening Method for Compounds Capable of Improving Properties Its epithelial cells, said method comprising the step of measuring the ability of each candidate compound to decrease expression or activity of one or more factor (s) ) of Krüppel-like transcription (KLF) in epithelial cells. 14. A method for culturing in vitro epithelial cells contained in a cell sample, or for enriching a cell sample with epithelial cells, excluding a sample of human embryonic stem cells, comprising at least: an inhibitor of expression or activity of a Krüppel-like transcription factor (KLF) with said cell sample, so as to increase proliferation and / or maintain and / or increase immature character epithelial cells contained in said cell sample; wherein said inhibitor is selected from a chemical molecule such as a peptide, peptide-like molecules, an organic compound, Kenpaullone, or an antibody or antibody fragment, or a nucleic acid such as an antisense oligonucleotide , or an aptamer, or even a ribozyme. 15. The method of claim 13 or 14, characterized in that said Krüppel-like transcription factor (KLF) is the transcription factor KLF4, preferably of sequence SEQ ID NO: 1.