FR3071511A1 - MOLECULAR SIGNATURES OF AGING DERMAL FIBROBLASTS AND EQUIVALENT DERME COMPRISING THESE FIBROBLASTS - Google Patents

Abstract

The present invention relates to the use of an expression product of at least one gene selected from the group consisting of ACAN, PSG1, and optionally UCP2, SFRP2, COL11A1 and KANK4 genes, as a marker for identifying a dermal fibroblast in vitro. as a young or old dermal fibroblast.

Description

Molecular signatures of aging of dermal fibroblasts and dermis equivalent comprising these aged fibroblasts

The present invention relates to the identification of aged dermal fibroblasts.

We have been trying to develop, for several years, reconstructed skin models which allow us to carry out the studies necessary for a better understanding of the role of the skin both in the mechanical and physiological fields.

Thus, models more or less close to human skin have been developed. Very generally, the reconstructed skin models described so far include human keratinocytes associated or not with other skin cells such as melanocytes and / or Langerhans cells, deposited on a support, often the equivalent of dermis, and cultivated under conditions such that they enter into a differentiation program leading to the formation of an epidermis equivalent.

Among the dermis equivalents described so far, there are in particular mixed collagen / fibroblast lattices, the fibroblasts used generally coming initially from any type of donor regardless of age.

The skin has important functions during life. In particular, it has an anatomical barrier role against pathogens and damage. As the skin ages, it becomes thinner and more easily damaged.

The aging process of the skin is governed by changes in the epidermal, dermo-epidermal and dermal compartments. Distributed sparingly, the dermal fibroblasts (papillary and reticular) are considered to be responsible for the synthesis of the three major groups of proteins of the dermal extracellular matrix and this assembly of extracellular matrix, via an aberrant remodeling, influences the changing function of the aged skin.

Aged skin is characterized by a flattening of the dermoepidermal junction, marked atrophy and loss of elasticity of the dermal connective tissue, associated with a reduction and disorganization of the main components of the extracellular matrix, such as collagen and others. elastic fibers, proteoglycans and glycosaminoglycans. These changes in the dermal extracellular matrix would be associated with changes in the properties of dermal fibroblasts.

In view of the importance of the role of the components of the dermis in skin aging, there is therefore a significant need for dermis equivalents in vitro reflecting as much as possible the characteristics of a dermis of aged skin.

Assuming that papillary dermal fibroblasts differentiate into dermal reticular fibroblasts during aging, the authors of Janson et al. (2013) Exp. Dermatol. 22: 48-53 sought to mimic this process of differentiation by the implementation of a long-term culture (method known as replicative senescence). Reconstructed skins were then produced using as fibroblasts papillary fibroblasts obtained before and after replicative senescence and reticular fibroblasts.

In Mancini et al. (2012) Ag / ng 4: 843-853, the authors used replicative senescence after prolonged culture to model the aging of dermal fibroblasts. They have shown a correlation between the entry into senescence of fibroblasts and the overexpression of 2 microRNAs: miR 152 and miR 181a, which could target integrin 152 and collagen type XVI.

It has thus been proposed in European application EP 2 703 498 to implement a method for screening compounds for anti-aging purposes comprising contacting candidate compounds with a sample of fibroblasts, measuring the expression or the activity of micro-RNA 152 and the selection of the candidate compound as an anti-aging compound when the inhibition measured is at least 20% compared to untreated fibroblasts.

However, all of these methods are based on a modeling of skin aging based on replicative senescence after prolonged culture of fibroblasts. However, the fidelity of such a modeling remains subject to discussion (Rubin (2002) Arch. Gerontol. Geriatr. 34: 275-286).

There is therefore still a significant need for dermis equivalents in vitro, comprising fibroblasts having the characteristics of aged fibroblasts in situ.

The present invention meets this need.

The inventors have in fact identified, from skin samples from specifically young or old donors, molecular signatures, involving a reduced number of biomarkers, making it possible to identify and distinguish aged fibroblasts from young fibroblasts, both among papillary fibroblasts than among reticular fibroblasts.

The present inventors have in fact shown that aged papillary fibroblasts and aged reticular fibroblasts can be identified by measuring the level of expression of at least one gene chosen from the ACAN, PSG1, and possibly UCP2, SFRP2, COL11A1 and KANK4 genes. .

Thus, the levels of expression products of the UCP2 and SFRP2 genes are significantly lower and the levels of the expression products of the PSG1 genes,

COL11A1, ACAN and KANK4 are significantly higher in papillary fibroblasts from elderly donors, while levels of the expression products of the ACAN and PSG1 genes are significantly higher in reticular fibroblasts from elderly donors.

The present invention thus relates to an in vitro method of identifying a dermal fibroblast as being a young or old dermal fibroblast, comprising the following steps:

a) providing a biological sample comprising at least one dermal fibroblast,

b) measuring the level of an expression product of at least one gene chosen from the group consisting of the ACAN, PSG1, and optionally UCP2, SFRP2, COL11A1 and KANK4 genes, and

c) on the basis of the level (s) measured in step b), identify the dermal fibroblast as being a young or old dermal fibroblast.

The present invention also relates to the use of an expression product of at least one gene chosen from the group consisting of the ACAN, PSG1, and optionally UCP2, SFRP2, COL11A1 and KANK4 genes, as a marker for identifying in vitro a fibroblast dermal as a young or old dermal fibroblast.

By specifically selecting these aged papillary and reticular fibroblasts identified on the basis of the above biomarkers, the inventors were also able to prepare an aged dermis equivalent in vitro using only papillary and reticular fibroblasts identified as being aged.

Another subject of the invention thus relates to an aged dermis equivalent in vitro comprising:

(i) a subpopulation of papillary fibroblasts in which

- the level of an expression product of the UCP2 gene is reduced compared to a control level,

the level of an expression product of the PSG1 gene is increased compared to a control level,

- the level of an expression product of the SFRP2 gene is reduced compared to a control level,

the level of an expression product of the COL11A1 gene is increased compared to a control level,

the level of an expression product of the ACAN gene is increased relative to a control level, and / or

- the level of an expression product of the KANK4 gene is increased compared to a control level, and / or (ii) a subpopulation of reticular fibroblasts in which

the level of an expression product of the ACAN gene is increased relative to a control level, and / or

- the level of a product of expression of the PSG1 gene is increased compared to a control level.

The present invention also relates to a skin equivalent in vitro comprising a dermis equivalent according to the invention.

Another subject of the invention relates to a kit for identifying a dermal fibroblast as being a young or old fibroblast, said kit comprising at least one measuring means chosen from the group consisting of a means for measuring the level of a product d of the ACAN gene, a means of measuring the level of an expression product of the PSG1 gene, and optionally a means of measuring the level of an expression product of the UCP2 gene, a means of measuring the level of an expression product of the SFRP2 gene, a means of measuring the level of an expression product of the COL11A1 gene and a means of measuring the level of an expression product of the KANK4 gene.

The present invention also relates to a DNA chip for identifying a dermal fibroblast as being a young or old fibroblast, said chip comprising at least one probe chosen from the group consisting of a probe detecting an expression product of the ACAN gene, a probe detecting an expression product of the PSG1 gene, and optionally a probe detecting an expression product of the UCP2 gene, a probe detecting an expression product of the SFRP2 gene, a probe detecting an expression product of the COL11A1 gene and a probe detecting an expression product of the KANK4 gene.

Detailed description of the invention

fibroblasts

By dermal fibroblast is meant here any fibroblast originating from the dermis.

By papillary fibroblast is meant here a fibroblast of the papillary dermis, the papillary dermis being characterized by a relatively fine extracellular matrix and a high cell density. Papillary fibroblasts in culture typically have a thin, spindle-shaped morphology.

By reticular fibroblast is meant here a reticular dermis fibroblast, the reticular dermis being characterized by a very dense network of matrix fibers and a low cell density. Cultured reticular fibroblasts typically have an extended and more square appearance.

By fibroblast of the same type, here is meant a fibroblast of the same part of the dermis (papillary or reticular) and having a similar cellular morphology (papillary or reticular).

The expression “aged dermal fibroblast” is understood here to mean a fibroblast having characteristics similar to a dermal fibroblast of the same type originating from a donor over 50 years of age, in particular over 55 years of age, over 60 years of age or over 65 years of age.

By young dermal fibroblast is meant here a fibroblast having characteristics similar to a dermal fibroblast of the same type coming from a donor less than 30 years old, in particular less than 28 years old, less than 25 years old or less than 22 years old.

Identification method

Step (a) of the identification method according to the invention comprises the supply of a biological sample comprising at least one dermal fibroblast, as defined in the section Fibroblasts above.

The biological sample can in particular be an in vitro culture of dermal fibroblasts or of a mixture of dermal fibroblasts, a sample coming from a skin biopsy, or a sample coming from a dermis or skin equivalent in vitro.

Step (b) of the identification method according to the invention comprising the measurement of the level of an expression product of at least one gene chosen from the group consisting of the ACAN, PSG1, and optionally UCP2, SFRP2 genes , COL11A1 and KANK4.

By ACAN gene is meant here the gene coding \ aggrecan core protein. The ACAN gene is also called AGC1, CSPG1 and MSK16 and the ACAN protein is also called aggrecan or cartilage-specific proteoglycan core protein or CSPCP or chondroitin sulfate proteoglycan core protein 1 ”or chondroitin sulfate proteoglycan 1”. It is part of the extracellular matrix in the cartilage tissue. It is a proteoglycan. It is typically described in Doege et al. (1991) J. Biol. Chem. 15: 894-902. The sequence of the human ACAN protein is typically referenced under the number UniProt P16112.

By PSG1 gene is meant here the gene coding for pregnancy-specific beta1 glycoprotein β1 (pregnancy-specific beta-1-glycoprotein /). The PSG1 gene is also called the B1G1 gene, PSBG1 gene or PSGGA gene and the PSG1 protein is also called the PS-beta-G-1 protein, PSBG-1, protein-specific glycoprotein 1 (pregnancyspecific glycoprotein 1), CD66 antigen-like. family member F ', 1/2 nonspecific antigen of fêtai liver (fêtai liver non-specific cross-reactive antigen 1/2'), FLNCA-1/2 protein, PSG95 protein, pregnancy-specific β1 C / D glycoprotein (pregnancyspecific beta-1 glycoprotein C / D), protein PS-beta-C / D or CD66f. It works mainly as an immunomodulator to protect the developing fetus. It is typically described in Watanabe et al. (1988) J. Biol. Chem 263: 2049-2054. The sequence of the human PSG1 protein is typically referenced under the number UniProt P11464.

By UCP2 gene is meant here the gene encoding the mitochondrial uncoupling protein 2 '. The UCP2 gene is also called the SLC25A8 gene and the UCP2 protein is also called the UCPH or solute carrier family 25 member 8 '. It belongs to the family of mitochondrial anionic support proteins (MACP) and controls the reactive oxygen species derived from mitochondria. It is typically described in Pecqueur et al. (1999) Biochemical and Biophysical Research Communications 255: 40-46. The sequence of the human UCP2 protein is typically referenced under the number UniProt P55851.

By SFRP2 gene is meant here the gene coding for the secreted frizzled-related protein 2 '. The SFRP2 gene is also called the FRP2 gene or SARP1 gene and the SFRP2 protein is also called the FRP-2, a protein linked to secreted apoptosis 1 (secreted apoptosis-relatedprotein 1) or the SARP-1 protein. It acts as a soluble modulator of the SFRP signaling pathway. It is typically described in Melkonyan et al. (1997) Proc. Natl. Acad. Science USA 94: 13636-13641. The sequence of the human SFRP2 protein is typically referenced under the number UniProt Q96HF1.

By COL11A1 gene is meant here the gene coding for the α1 (XI) chain of collagen. The COL11A1 gene is also called the COLL6 gene. This chain is one of the two alpha chains of type XI collagen, a minor fibrillar collagen. It is typically described in Yoshioka et al. (1990) J. Biol. Chem. 15: 6423-6426. The sequence of the human COL11A1 protein is typically referenced under the number UniProt P12107.

By KANK4 gene is meant here the gene encoding the KN motif and ankyrin repeat domain-containing protein 4 (KANK4). The KANK4 gene is also called the ANKRD38 gene and the KANK4 protein is also called the ankyrin repeat domain-containing protein 38 '. It would be involved in controlling the formation of the cytoskeleton by regulating the polymerization of actin. It is typically described in Zhu et al. (2008) Biochemical Biophysica Acta 1780: 128-133. The sequence of the human KANK4 protein is typically referenced under the number UniProt Q5T7N3.

In the context of the invention, the UniProt references cited above are those which were available on July 31, 2017.

In a particular embodiment, step (b) comprises measuring the level of an expression product of at least two genes chosen from the group consisting of the ACAN, PSG1, and possibly UCP2, SFRP2, COL11A1 and KANK4, preferably at least 3, 4, 5 or 6 genes selected from the group consisting of ACAN, PSG1, UCP2, SFRP2, COL11A1 and KANK4 genes.

In a particular embodiment, in particular when the dermal fibroblast is a reticular fibroblast, step (b) comprises measuring the level of an expression product of at least one gene chosen from the group consisting of ACAN genes and PSG1, preferably measuring the level of the expression products of the ACAN and PSG1 genes. In another particular embodiment, in particular when the dermal fibroblast is a papillary fibroblast, step (b) comprises measuring the level of an expression product of at least one gene, preferably at least two , in particular at least 3, 4 or 5 genes, chosen from the group consisting of the ACAN, PSG1, UCP2, SFRP2, COL11A1 and KANK4 genes, preferably the measurement of the level of the expression products of the ACAN, PSG1, UCP2 genes, SFRP2, COL11A1 and KANK4.

The expression product of gene X is understood here to mean the mRNA encoded by said gene X or the protein encoded by said gene X. The level of expression product of gene X can therefore be measured by quantifying the mRNA or the corresponding protein. In a particular embodiment, said X gene expression product is the mRNA encoded by said X gene.

Preferably, the level of the expression product corresponds to the concentration or the quantity of the expression product.

The level of the expression product of a gene X can be measured in step (b) by any technique well known to those skilled in the art. In particular, when the expression product is a protein, the level of the expression product can be measured by means of immunological assays such as ELISA assays, immunofluorescent assays (IFA), radioimmunoassays (RIA), competitive binding tests or Western Blots. When the expression product is an mRNA, the level of the expression product can be measured by RT-PCR, qRT-PCR, ddPCR (Droplet Digital PCR), by sequencing, for example by NGS type sequencing (Next generation sequencing). ) or by sequencing with ddSEQ ™ single cell isolator.

Step (c) of the identification method according to the invention comprises the identification, on the basis of the level (s) measured in step b), of the dermal fibroblast as being a dermal fibroblast young or old.

In a particular embodiment, step (c) of the identification method according to the invention comprises comparing the level (s) measured in step b) with one or more levels (x ) control.

By control level is meant here a reference value preferably corresponding to the level of said expression product of said gene in a dermal fibroblast of the same type (as defined in the section Fibroblasts above), known as being a young dermal fibroblast , in particular a dermal fibroblast of the same type originating from a subject of less than 30 years, in particular of 28 years, of less than 25 years or of less than 22 years. In particular, when the dermal fibroblast studied is a papillary fibroblast, the control level is preferably the level of said expression product of said gene in a papillary fibroblast known as being a young papillary fibroblast, in particular a papillary fibroblast from a subject under 30, especially 28, under 25 or under 22. When the dermal fibroblast studied is a reticular fibroblast, the control level is preferably the level of said expression product of said gene in a reticular fibroblast known as being a young reticular fibroblast, in particular a reticular fibroblast originating from a subject of less than 30 years, in particular 28 years, less than 25 years or less than 22 years.

In a particular embodiment, when the dermal fibroblast is a papillary fibroblast, the level of an expression product of at least one gene chosen from the group consisting of the genes UCP2, PSG1, SFRP2, COL11A1, ACAN and KANK4 is preferably measured in step b), and the papillary fibroblast is identified as being an aged papillary fibroblast when:

(i) the level of an expression product of the UCP2 gene is decreased compared to a control level, (ii) the level of an expression product of the PSG1 gene is increased compared to a control level, (iii ) the level of an expression product of the SFRP2 gene is decreased compared to a control level, (iv) the level of an expression product of the COL11A1 gene is increased compared to a control level, (v) the level of an expression product of the ACAN gene is increased relative to a control level, and / or (vi) the level of an expression product of the KANK4 gene is increased relative to a control level, the control levels of (i), (ii), (iii), (iv), (v) and (vi) preferably being respectively the level of the expression product of the genes UCP2, PSG1, SFRP2, COL11A1, ACAN and KANK4 in a papillary fibroblast known as a young papillary fibroblast.

In another particular embodiment, when the dermal fibroblast is a reticular fibroblast, the level of an expression product of at least one gene chosen from the group consisting of the ACAN and PSG1 genes is preferably measured in step b), and the reticular fibroblast is identified as an aged reticular fibroblast when:

(i) the level of an expression product of the ACAN gene is increased relative to a control level, and / or (ii) the level of an expression product of the PSG1 gene is increased relative to a control level , the control levels of (i) and (ii) preferably being respectively the level of the expression product of the ACAN and PSG1 genes in a reticular fibroblast known as being a young reticular fibroblast.

By increased level here is meant a statistically significantly increased level compared to the control level. Preferably, the increased level is increased at least 1.3 times, at least 1.38 times, at least 1.4 times, at least 1.5 times, at least 1, 59 times, at least 1.6 times, at least 1.7 times, at least 1.73 times, at least 2 times, at least 2.5 times, at least 2.9 times, at least 2.92 times, at least 5 times, at least 10 times, at least 10.9 times, at least 15 times, at least 20 times , at least 21 times, at least 21.5 times or at least 21.56 times compared to the control level as defined above.

Preferably, the papillary fibroblast is identified as being an aged papillary fibroblast when:

(ii) the level of a product of expression of the PSG1 gene is increased by at least 20 times, in particular by at least 21 times, by at least 21.5 times or by at least 21.56 times relative to a control level as defined above, (iv) the level of a product of expression of the COL11A1 gene is increased by at least 1.4 times, in particular by at least 1.5 times or at least 1.59 times compared to a control level as defined above, (v) the level of an expression product of the ACAN gene is increased by at least

1.3 times, in particular at least 1.38 times relative to a control level as defined above, and / or (vi) the level of an expression product of the KANK4 gene is increased by at least

1.6 times, in particular at least 1.7 times or at least 1.73 times compared to a control level as defined above.

Preferably, the reticular fibroblast is identified as an aged reticular fibroblast when:

(i) the level of an expression product of the ACAN gene is increased by at least 2.5 times, in particular by at least 2.9 times or by at least 2.92 times compared to a level control as defined above, and / or (ii) the level of a product of expression of the PSG1 gene is increased by at least 10 times, in particular at least 10.5 times or by at least 10, 9 times compared to a control level as defined above.

By reduced level is meant here a statistically significantly reduced level compared to the control. Preferably, the decreased level is decreased by at least 2 times, by at least 2.5 times, by at least 2.51 times, by at least 3 times, by at least 4 times, by at least 4.2 times or at least 4.23 times compared to the control level as defined above.

Preferably, the papillary fibroblast is identified as being an aged papillary fibroblast when:

(i) the level of an expression product of the UCP2 gene is decreased by at least 2 times, in particular by at least 2.5 times or by at least 2.51 times compared to a control level such as defined above, and / or (iii) the level of an expression product of the SFRP2 gene is decreased by at least 3 times, in particular by at least 4 times, by at least 4.2 times or at least 4.23 times compared to a control level as defined above.

The present invention also relates to the use of an expression product, as defined above, of at least one gene chosen from the group consisting of the ACAN, PSG1, and optionally UCP2, SFRP2, COL11A1 and KANK4, as a marker to identify in vitro a dermal fibroblast, as defined in the section Fibroblasts above as a young or old dermal fibroblast.

Dermis and skin equivalents

Thanks to the identification method described above, the inventors were able to prepare for the first time an aged dermis equivalent in vitro from exclusively aged dermal fibroblasts, having the same characteristics as the dermal fibroblasts obtained from aged donors .

The subject of the present invention is therefore an equivalent of an aged dermis in vitro comprising:

(i) a subpopulation of papillary fibroblasts, as defined in the section Fibroblasts above, in which

- the level of an expression product of the UCP2 gene is reduced compared to a control level,

the level of an expression product of the PSG1 gene is increased compared to a control level,

- the level of an expression product of the SFRP2 gene is reduced compared to a control level,

the level of an expression product of the COL11A1 gene is increased compared to a control level,

the level of an expression product of the ACAN gene is increased relative to a control level, and / or

- the level of an expression product of the KANK4 gene is increased compared to a control level, and / or (ii) a subpopulation of reticular fibroblasts, as defined in the section Fibroblasts above in which

the level of an expression product of the ACAN gene is increased relative to a control level, and / or

- the level of a product of expression of the PSG1 gene is increased compared to a control level.

The terms expression product, increased, decreased and control level here have the same definitions as those provided in the Identification method section above.

The dermis equivalent according to the invention preferably also comprises collagen.

The collagen present in the dermis equivalent according to the invention can be any type of collagen of any origin. Preferably, the collagen is chosen from type I, III or V fibrillar collagens. Preferably, the collagen is type I collagen. Preferably, the collagen is of animal origin, in particular of bovine origin. In a particularly preferred manner, the collagen is bovine collagen type I. Alternatively, the collagen can be a mixture of collagen of different types in any proportion and / or of different origins.

The fibroblasts present in the dermis equivalent according to the invention can come from any origin, but are preferably fibroblasts of human origin.

The fibroblasts present in the dermis equivalent according to the invention preferably come from a culture of fibroblasts, part of which was used to implement the identification method according to the invention, thus making it possible to identify the whole. culture as a culture of aged fibroblasts.

The dermis equivalent according to the invention may also contain any other component which may be constitutively present in the skin such as endothelial cells, macrophages, monocytes, macrophage precursors, dendritic cell precursors or nerve cells.

The present invention also relates to a process for preparing a dermis equivalent as defined above comprising an initial step of identifying dermal fibroblasts as being aged dermal fibroblasts, comprising the following steps:

A) the supply of a homogeneous culture of dermal fibroblasts,

B) the removal of part of the culture of dermal fibroblasts provided in step A),

C) the identification of the part of the dermal fibroblast culture taken in step B) as being a culture of aged dermal fibroblasts using the identification method as defined in the section Identification method above on it, and

D) the use of the part of the dermal fibroblast culture not taken in step B) to prepare a dermis equivalent.

The preparation of the dermis equivalent in step D) can be carried out by any technique well known to those skilled in the art.

The preparation of the dermis equivalent in step D) can thus comprise a step of preparing a lattice comprising collagen and a cell suspension of aged dermal fibroblasts, preferably of aged papillary and reticular dermal fibroblasts, in particular identified as aged in step C).

As indicated previously, the collagen used can be any type of collagen, of any origin, alone or as a mixture.

Preferably, the lattice comprises fibroblasts at a concentration of 1 x 10 ⁵ to 5χ 10 ⁶ cells / ml, preferably at a concentration of 2x 10 ⁵ to 2x 10 ⁶ cells / ml.

The lattice can be prepared by any technique well known to those skilled in the art.

In particular, a solution comprising collagen and aged dermal fibroblasts can be prepared and deposited on a support.

Preferably, the solution is incubated so as to allow the gelation of the collagen, for example for 10 to 30 min, then maintained in incubation to allow the contraction of the lattice.

Preferably, the lattice is thus kept to incubate for 1 to 7 days, more preferably for 3 or 4 days.

Insofar as the dermal content influences the epidermal compartment, the aged dermis equivalent according to the invention can serve as a support for the formation of an aged skin equivalent.

The present invention thus also relates to a skin equivalent in vitro comprising a dermis equivalent as defined above.

The skin equivalent according to the invention comprises, on the dermis equivalent, an epidermis equivalent comprising at least keratinocytes.

The keratinocytes can come from any origin but are preferably keratinocytes of human origin. They can be prepared by any method well known to those skilled in the art. Thus, the keratinocytes can be prepared by culture of dissociated epidermis originating from normal skin sampling or by culture of keratinocytes originating from the sheath of the hair follicle.

Preferably, the keratinocytes are keratinocytes of normal human skin.

More preferably, the keratinocytes are prepared from dissociated human epidermis obtained from normal human skin samples according to the method described in Régnier et al., Frontier of Matrix Biology, Vol. 9, 4-35 (Karger, Basel, 1981).

The epidermis equivalent can include any other cell type, such as Langerhans cells and / or Langerhans cell precursors and / or melanocytes.

Advantageously, the epidermis equivalent also comprises melanocytes, and / or Langerhans cells and / or Langerhans cell precursors.

Melanocytes can be isolated from any organ containing it such as normal skin or the hair follicle. Preferably, the melanocytes are isolated from normal skin. Any method for preparing melanocytes known to a person skilled in the art can be used, such as the method described in Olsson et al. (1994) Acta. Derm. Venereol. 74: 226-268.

Langerhans cells and / or Langerhans cell precursors can be as described in European patent application EP 789074.

The present invention also relates to a process for preparing a skin equivalent as defined above comprising a step for preparing a dermis equivalent by the process for preparing dermis equivalent as defined above.

Preferably, the process for preparing a skin equivalent comprises, after the step for preparing the dermis equivalent, a step for reconstituting, on the dermis equivalent, an epidermis equivalent comprising at least keratinocytes.

This step of reconstituting an epidermis equivalent can be carried out by any technique well known to those skilled in the art, such as the techniques described in patent applications EP 285471, EP285474, EP789074, EP502172, EP418035, W091 / 16010 , EP197090, EP20753, FR2665175, FR2689904 or that described in Asselineau et al. (1985) Exp. Cell. Res. 159: 536-539, in Asselineau et al. (1987), Models in dermato., Col III, Ed. Lowe & Mailbach, 1-7 or in Asselineau et al. (1984) Br J Dermatol. 111 Suppl 27: 219-22.

This reconstitution step can advantageously be preceded by a bonding step, in a culture dish, of the dermis equivalent prepared, for example with an adhesive solution comprising MEM 1.76X medium, SVF, NaOH 0 , 1 N and MEM 25 mM Hepes 10% SVF.

Preferably, the reconstitution step is carried out by seeding keratinocytes on the dermis equivalent, preferably in a seed ring.

Advantageously, after sowing the keratinocytes on the dermis equivalent, the culture can be kept immersed in a nutritive medium which can for example be the medium described by Rheinwald and Green (1975) Cell 6: 317-330, medium which allows proliferation keratinocytes.

After an incubation time of preferably 3 to 15 days, more preferably 7 to 9 days, the skin equivalent is preferably maintained at the air / liquid interface, for example by deposition on a metal grid. The liquid then preferably consists of the same nutritive medium as above.

The incubation then continues preferably until a skin equivalent having the characteristics of a skin is obtained, namely a dermis equivalent covered with an epidermis equivalent having the four types of cell layers. classical, namely the basal, suprabasal, granular and corneal layers.

Thus, the incubation preferably continues for a period of between 5 and 30 days, preferably between 7 and 10 days.

uses

The present invention also relates to the use of a dermis equivalent as defined in the section Dermis and skin equivalent above or of a skin equivalent as defined in the section Dermis and skin equivalent above, to study the aging of the skin.

The dermis equivalent and / or the skin equivalent according to the invention are in particular useful for studying the appearance of wrinkles, the appearance of pigment spots, or any other phenomenon associated with aging of the skin, in particular associated photoaging, more particularly associated with the effect of ultraviolet radiation on the skin, such as actinic keratoses.

The present invention also relates to a method for screening a compound exhibiting activity after topical application on aged skin, in particular in the anti-aging field such as for the treatment of wrinkles and fine lines and / or the treatment of spots pigments, said screening method comprising applying a candidate compound to the dermis equivalent according to the invention or the skin equivalent according to the invention.

Kit and chip

The present invention also relates to a kit for identifying a dermal fibroblast as being a young or old fibroblast, said kit comprising at least one measuring means chosen from the group consisting of means for measuring the level of a product of expression of the ACAN gene, a means of measuring the level of an expression product of the PSG1 gene, and optionally a means of measuring the level of an expression product of the UCP2 gene, a means of measuring the level of a expression product of the SFRP2 gene, means for measuring the level of an expression product of the COL11A1 gene and means for measuring the level of an expression product of the KANK4 gene.

The kit may in particular comprise, as separate components, antibodies recognizing the proteins encoded by said genes in a biological sample as defined above. The kit may comprise, as separate components, primers and / or probes which hybridize specifically to the mRNAs encoded by said genes.

The kit can also include optional additional components for implementing the identification method according to the invention. Such optional components are for example containers, mixers, buffers, instructions for implementing the method, markers or supports.

The kit according to the invention can also comprise a control or several controls from which the control level, as defined in the section Identification method ”above, can be obtained.

The present invention also relates to a DNA chip for identifying a dermal fibroblast as being a young or old fibroblast, said chip comprising at least one probe chosen from the group consisting of a probe detecting an expression product of the ACAN gene, a probe detecting an expression product of the PSG1 gene, and optionally a probe detecting an expression product of the UCP2 gene, a probe detecting an expression product of the SFRP2 gene, a probe detecting an expression product of the COL11A1 gene and a probe detecting an expression product of the KANK4 gene.

By DNA chip is meant here an ordered arrangement of at least two probes on a substrate. Preferably, the DNA chip according to the invention further comprises a control or standard probe.

By probe is meant here an oligonucleotide or polynucleotide, RNA or DNA, which exists naturally as in a digestion product purified from a restriction enzyme or which is produced synthetically, and which is capable of hybridizing specifically with a polynucleotide carrying a sequence complementary to that of the probe. The probe can be single strand or double strand. Preferably, the probe comprises or consists of 10 to 100 nucleotides, preferably 15 to 50 nucleotides or 15 to 25 nucleotides.

Throughout the above description, unless otherwise stated, the term between x and y or going from x to y corresponds to an inclusive range, that is to say that the values x and y are included in range.

The present invention will be illustrated in more detail by the example below.

Example

Example

The example below shows the identification by the inventors of molecular signatures making it possible to distinguish subpopulations of young and old papillary and reticular fibroblasts.

Material and methods

Cell preparation

The papillary (Fp) and reticular (Fr) fibroblasts were isolated from undegreased human skin. These samples are collected after breast reduction for cosmetic reasons. Six women were included: 3 young donors (22, 25 and 28) and 3 elderly donors (55, 61 and 65).

The isolation of the Fr is performed on the part of tissue thus depleted of its hypodermis. The tissue is then dermatomed up to 700 qm. Only the lower part of the fabric is kept.

The isolation of Fp is carried out on dermatomated tissue at 300 qm then de-epidermized after dispersing action (Roche - 2.4 Units / ml) for 16 h at 4 ° C.

After shredding, the dermis fragments are digested under the action of 0.2% type II collagenase (Gibco) at 37 ° C.

The cells are then amplified in MEM medium - 10% fetal calf serum supplemented with Glutamine, sodium pyruvate, nonessential amino acid, penicillin, streptomycin and fungizone, under a humid atmosphere, at 37 ° C and 5% CQ>.

Transcriptomic analysis and RT-qPCR

After expansion of the cells (between 7 and 10 population doublings), the mRNAs are extracted on a QIAgen column according to the instructions given by the supplier.

The samples were divided in 2: one part was reserved for transcriptomic analysis, the other part was used for the validation of biomarkers by PCR.

The transcriptomic study was carried out on an Affymetrix chip of the GeneChip HGU133 Plus 2.0 type. The probes sets considered as differentially expressed had to present a fold change> to 2 for an unadjusted p-value <to 0.05.

The primers used for RT-qPCR validations are commercially available from Qiagen (QIAgen - Quantitech Primer Assay) and are listed in Table 1 below.

Table 1: Primers used

Uncomfortable Reference QIAgen of the primer ACAN QT00001365 COL11A1 QT00088711 UCP2 QT00014140 PSG1 QT01006775 SFRP2 QT00073220 KANK4 QT00030905 GAPDH QT01192646

Reconstructed skins

The preparation of the reconstructed skins was carried out by following the protocol described in Asselineau et al. (1985) Exp. Cell. Res. 159: 536-539.

Briefly, 10 ⁶ fibroblasts identified as aged were included in a bovine collagen type I solution (Symatèse). After 4 days of organization and contraction of the lattice, 5.10 ⁴ keratinocytes are seeded on the surface of the lattice. The culture is maintained in immersion for 1 week in MEM medium, 10% fetal calf serum, EGF (1 Ong / ml), Hydrocortisone (0.4 g / ml) and Choiera toxin (0.1 nM). A complete stratification of the epidermis is obtained one week after being put into emersion.

Throughout this reconstruction process, the culture is carried out in a humidity-saturated incubator containing 5% CO ₂ at 37 ° C.

Results

Cells from subpopulations of papillary and reticular fibroblasts were isolated and cultured from breast plasties collected from 3 young individuals (under 28 years of age) and 3 histologically aged individuals (between 55 and 65 years of age). 4 sets of sub-populations were thus formed: a sub-population of papillary fibroblasts from young donors, a sub-population of papillary fibroblasts from elderly donors, a sub-population of reticular fibroblasts from young donors and a sub-population of fibroblasts reticular from elderly donors.

Transcriptomic analyzes of these 4 sets of sub-populations were performed. Differential expression of 6 specific genes has been demonstrated between the subpopulations of young and old papillary fibroblasts and differential expression of 2 specific genes has been demonstrated between the subpopulations of young and old reticular fibroblasts, with validation by analysis by RT-qPCR.

The results of the RT-qPCR analyzes are summarized in Table 2 below.

Table 2: Analysis by RT-qPCR of the 3 sub-populations of dermal fibroblasts

Fp young vs old (change in number of times) Conclusion RNA ACAN -1.38 Upwardly regulated in older FPs COL11A1 -1.59 KANK -1.73 PSG1 -21.56 SFRP2 4.23 Downgraded in older FPs UCP2 2.51

Fr young vs old (change in number of times) Conclusion RNA ACAN -2.92 Upwardly regulated in the elderly Fr PSG1 -10.9

Fp: papillary fibroblast, Fr: reticular fibroblast

The inventors were thus able to demonstrate the following molecular signatures for the subpopulations of young and old papillary fibroblasts and for the subpopulations of young and old reticular fibroblasts:

UCP2 PSG1 SFRP2 COL11A1 ACAN KANK4 Young papillary fibroblast strong Negative strong Negative Negative Negative fibroblast Low Positive Low Positive Positive Positive papillary = = = = = = age Decreases Increases Decreases Increases Increases Increases

Young reticular fibroblast - Negative Decreases - - Positive Decreases - Aged reticular fibroblast - Positive Increases - - Strong positive Increases -

Reconstructed skin comprising the sub-populations identified as elderly was then obtained as indicated above.

Claims (9)

1. In vitro method for identifying a dermal fibroblast as being a young or old dermal fibroblast, comprising the following steps: a) providing a biological sample comprising at least one dermal fibroblast, b) measuring the level of an expression product of at least one gene chosen from the group consisting of the ACAN, PSG1, and optionally UCP2, SFRP2, COL11A1 and KANK4 genes, and c) on the basis of the level (s) measured in step b), identify the dermal fibroblast as being a young or old dermal fibroblast. 2. Method according to claim 1, in which the dermal fibroblast is a papillary fibroblast and the level of an expression product of at least one gene chosen from the group consisting of the genes UCP2, PSG1, SFRP2, COL11A1, ACAN and KANK4 is measured in step b), and in which the papillary fibroblast is identified as being an aged papillary fibroblast when: (i) - the level of an expression product of the UCP2 gene is decreased compared to a control level, (ii) the level of an expression product of the PSG1 gene is increased compared to a control level, ( iii) the level of an expression product of the SFRP2 gene is decreased compared to a control level, (iv) the level of an expression product of the COL11A1 gene is increased compared to a control level, (v) the level of an expression product of the ACAN gene is increased relative to a control level, and / or (vi) the level of an expression product of the KANK4 gene is increased relative to a control level. 3. Method according to claim 1, in which the dermal fibroblast is a reticular fibroblast and the level of an expression product of at least one gene chosen from the group consisting of the ACAN and PSG1 genes is measured in step b ), and in which the reticular fibroblast is identified as an aged reticular fibroblast when: (i) the level of an expression product of the ACAN gene is increased relative to a control level, and / or (ii) the level of an expression product of the PSG1 gene is increased relative to a control level . 4. Method according to claim 2 or 3, in which the said level (s) of control is (are) the level (s) of the said product (s) corresponding expression (s) in a dermal fibroblast of the same type from a subject under 30 years of age. 5. Equivalent aged dermis in vitro comprising: (i) a subpopulation of papillary fibroblasts in which - the level of an expression product of the UCP2 gene is reduced compared to a control level, the level of an expression product of the PSG1 gene is increased compared to a control level, - the level of an expression product of the SFRP2 gene is reduced compared to a control level, the level of an expression product of the COL11A1 gene is increased compared to a control level, the level of an expression product of the ACAN gene is increased relative to a control level, and / or - the level of an expression product of the KANK4 gene is increased compared to a control level, and / or (ii) a subpopulation of reticular fibroblasts in which the level of an expression product of the ACAN gene is increased relative to a control level, and / or - the level of a product of expression of the PSG1 gene is increased compared to a control level. 6. Dermis equivalent according to claim 5, in which the said level (s) of control is (are) the level (s) of the said product (s) corresponding expression (s) in a dermal fibroblast of the same type from a subject under 30 years of age. 7. In vitro skin equivalent comprising a dermis equivalent according to claim 5 or 6. 8. Kit for identifying a dermal fibroblast as being a young or old fibroblast, said kit comprising at least one measuring means chosen from the group consisting of means for measuring the level of an expression product of the ACAN gene, a means for measuring the level of a product of expression of the PSG1 gene, and optionally a means of measuring the level of a product of expression of the UCP2 gene, a means of measuring the level of a product of expression of the gene SFRP2, a means of measuring the level of an expression product of the COL11A1 gene and a means of measuring the level of an expression product of the KANK4 gene. 9. DNA chip to identify a dermal fibroblast as being a young or old fibroblast, said chip comprising at least one probe chosen from the group consisting of a probe detecting an expression product of the ACAN gene, a probe detecting a product d expression of the PSG1 gene, and optionally a probe detecting an expression product of the UCP2 gene, a probe detecting an expression product of the SFRP2 gene, a probe detecting an expression product of the COL11A1 gene and a probe detecting a product d of the KANK4 gene.