Molecular signatures of three subpopulations of dermal fibroblasts and dermal equivalents containing one of these subpopulations.

Abstract

Identification of dermal fibroblast subpopulations. [Solution] The object of the present invention is a method for identifying dermal fibroblasts in vitro as papillary fibroblasts, reticular fibroblasts or dermal-subcutaneous junction fibroblasts (FJDH), the method comprising a step of measuring the levels of expression products of UCP2 and FGF9, and optionally at least one gene selected from the group consisting of the COL11A1 gene and the ACAN gene, and the level of expression product of the KLF9 gene.

Description

Technical Field

[0001] The present invention relates to the identification of subpopulations of dermal fibroblasts.

Background Art

[0002] The skin is composed of two related compartments, namely the epidermis and the dermis.

[0003] The epidermis is mainly composed of three cell types, namely keratinocytes, which are themselves the majority of the epidermal cells, melanocytes, and Langerhans cells. These cells form a keratinized epidermis that differentiates into a stratified layer in which there is a layer of dead cells that forms the stratum corneum on top.

[0004] The dermis provides a solid support for the epidermis. The dermis also supplies nutrients to the epidermis. The dermis is mainly composed of fibroblasts and the extracellular matrix.

[0005] The dermis more precisely contains two quite distinct layers: the papillary superficial layer (300 - 400 μm, in contact with the epidermis) and the underlying reticular layer (extending to the subcutaneous tissue). The connective columns of the dermis can also extend into the subcutaneous tissue.

[0006] The papillary dermis is characterized by a relatively thin extracellular matrix with a high density of cells, while the reticular dermis has a high-density network of matrix fibers and a low density of cells. The components of the matrix are also different in the two layers.

[0007] Furthermore, when culturing the fibroblasts of these two distinct layers, which are respectively called papillary fibroblasts and reticular fibroblasts, they have quite different morphological characteristics. For example, reticular fibroblasts have an extended and more square appearance, while papillary fibroblasts generally have a thin spindle-shaped form. Furthermore, differences in proliferation, culture matrix production, response to growth factors, and production of growth factors are observed between these two cell subpopulations.

[0008] Therefore, in normal skin, the dermis is composed of at least two subpopulations of fibroblasts, which inevitably produces essential consequences for the skin itself.

[0009] In the field of skin equivalents (or in vitro reconstructed skin), it is essential to reproduce the characteristics and properties of the various components of normal skin as accurately as possible in order to reflect the response of normal skin as faithfully as possible.

[0010] We have examined the differences in characteristics between subpopulations of dermal fibroblasts, and it is important that in vitro skin equivalents include subpopulations that have been clearly identified with respect to dermal fibroblasts using biomarkers.

[0011] This invention satisfies this need.

[0012] Janson et al. (2012), Journal of Investigative Dermatology 132:2565-2572, conducted transcriptome studies to identify the molecular signatures of the papillary and reticular phenotypes. They identified the MGP protein, which is exclusively expressed in reticular dermis, while the PDPN and NTN1 genes show generally higher expression in papillary fibroblasts.

[0013] Nauroy et al. (2017) in the Journal of Investigative Dermatology identified certain markers that are differentially expressed in reticular fibroblasts but not in papillary fibroblasts, using only skin samples from young donors. For example, COL11A1, MGP, FGF18, COMP, and ACAN genes were identified as being overexpressed in reticular fibroblasts.

[0014] However, these papers only consider papillary fibroblasts and reticular fibroblasts. However, we have demonstrated that another subpopulation of fibroblasts, called dermal-subcutaneous junction fibroblasts, can be separated by connective trabeculae released into the subcutaneous tissue by the dermis.

[0015] The inventors also identified molecular signatures, including a small number of biomarkers, which allowed them to identify and distinguish three subpopulations of dermal fibroblasts: papillary fibroblasts, reticular fibroblasts, and dermal-subcutaneous junction fibroblasts.

[0016] Apart from the first isolation of subpopulations of dermal-subcutaneous junction fibroblasts, the inventors have demonstrated that papillary, reticular, and dermal-subcutaneous junction fibroblasts can be identified by measuring the expression levels of the UCP2 gene, the FGF9 gene, and at least one gene optionally selected from the COL11A1 gene and the ACAN gene, as well as optionally selected from the KLF9 gene.

[0017] In fact, the expression level of the UCP2 gene was significantly higher in papillary fibroblasts than in reticular fibroblasts and dermal-subcutaneous junction fibroblasts, while the expression levels of the COL11A1, ACAN, and FGF9 genes were significantly higher in reticular fibroblasts and dermal-subcutaneous junction fibroblasts than in papillary fibroblasts. Finally, the expression level of the KLF9 gene was significantly higher in dermal-subcutaneous junction fibroblasts than in reticular fibroblasts. [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] EP789074 [Patent Document 2] EP285471 [Patent Document 3] EP285474 [Patent Document 4] EP502172 [Patent Document 5] EP418035 [Patent Document 6] WO91 / 16010 [Patent Document 7] EP197090 [License 8] EP20753 [License 9] FR2665175 [License 10] FR2689904 [Non-licensed literature]

[0019] [Non-licensed Document 1] Jansonら(2012) Journal of Investigative Dermatology 132:2565~2572 pages [Non-licensed Document 2] Nauroyら(2017) Journal of Investigative Dermatology [Non-licensed Document 3] Pecqueurら(1999) Biochemical and Biophysical Research Communications 255:40~46 pages [Non-licensed Document 4] Doegeら(1991) J. Biol. Chem. 15:894~902 pages [Non-licensed Document 5] Miyamotoら(1993) Molecular and Cellular Biology 13:4251~4259 pages [Non-licensed Document 6] Yoshioka (1990) J. Biol. Chem. 15:6423~6426 pages [Non-licensed Document 7] Sporlら(2012) Proc. Natl. Acad. Science USA 109:10903~10908 pages [Non-licensed Document 8] Regnier, Frontier of Matrix Biology, Vol. 9, pages 4~35 (Karger, Basel, 1981) [Non-licensed Document 9] Olsson et al. (1994) Acta. Derm. Venereol. 74:226-268 [Non-Patent Document 10] Asselineau et al. (1985) Exp. Cell. Res. 159:536-539 [Non-Patent Document 11] Asselineau et al. (1987), Models in dermato., col III, edited by Lowe & Mailbach, pp. 1-7. [Non-Patent Document 12] Asselineau et al. (1984) Br J Dermatol. 111 Suppl 27:219~22 [Non-Patent Document 13] Rheinwald and Green (1975) Cell 6:317~330 [Overview of the Initiative] [Means for solving the problem]

[0020] Therefore, the present invention provides an in vitro method for identifying dermal fibroblasts as papillary fibroblasts, reticular fibroblasts, or dermal-subcutaneous junction fibroblasts (FJDH), a) A step of preparing a biological sample containing at least one dermal fibroblast, b) A step of measuring the level of expression products of at least one gene selected from the group consisting of the UCP2 gene, the FGF9 gene, and optionally the COL11A1 gene and the ACAN gene, and optionally the level of expression products of the KLF9 gene, c) A step of identifying dermal fibroblasts as papillary fibroblasts, reticular fibroblasts, or dermal-subcutaneous junction fibroblasts (FJDH) based on the levels measured in step b), and Regarding methods including

[0021] Another object of the present invention is the use of expression products of at least one gene selected from the group consisting of the UCP2 gene and the FGF9 gene, and optionally the COL11A1 gene and the ACAN gene, in combination with optionally the expression product of the KLF9 gene, as a marker for in vitro identification of dermal fibroblasts as papillary fibroblasts, reticular fibroblasts, or dermal-subcutaneous junction fibroblasts (FJDH).

[0022] Another object of the present invention relates to an in vitro dermal equivalent comprising a subpopulation of dermal-subcutaneous junction fibroblasts (FJDH).

[0023] The present invention also relates to an in vitro skin equivalent containing a dermal equivalent according to the present invention.

[0024] The present invention also provides a kit for identifying dermal fibroblasts as papillary fibroblasts, reticular fibroblasts, or dermal-subcutaneous junction fibroblasts (FJDH), - At least one measuring means selected from the group consisting of means for measuring the level of UCP2 gene expression product, means for measuring the level of FGF9 gene expression product, means for measuring the level of COL11A1 gene expression product, and means for measuring the level of ACAN gene expression product. and - At least one means of measuring the level of KLF9 gene expression product Regarding the kit that includes this.

[0025] Another object of the present invention is a DNA microarray for identifying dermal fibroblasts as papillary fibroblasts, reticular fibroblasts, or dermal-subcutaneous junction fibroblasts (FJDH), - At least one probe selected from the group consisting of a probe for detecting UCP2 gene expression products, a probe for detecting FGF9 gene expression products, a probe for detecting COL11A1 gene expression products, and a probe for detecting ACAN gene expression products. and - At least one probe to detect KLF9 gene expression products Regarding DNA microarrays, including those mentioned. [Modes for carrying out the invention]

[0026] fibroblasts In this specification, "dermal fibroblasts" refers to fibroblasts derived from the dermis.

[0027] In this specification, “papillary fibroblasts” refers to fibroblasts of the papillary dermis, which is characterized by a relatively thin extracellular matrix and high density of cells. In culture, papillary fibroblasts typically have a thin, spindle-shaped morphology.

[0028] In this specification, “reticular fibroblasts” refers to fibroblasts of the reticular dermis, which is characterized by a relatively dense grid of matrix fibers and low-density cells. In culture, reticular fibroblasts typically have an expanded and more square appearance.

[0029] In this specification, “dermal-subcutaneous junction fibroblasts” or “FJDH fibroblasts” refers to fibroblasts from a zone located at the same level as the connective trabeculae released into the subcutaneous tissue by the dermis. FJDH fibroblasts in culture typically exhibit highly heterogeneous morphology. Thus, a wide variety of shapes are observed within the cell carpet, ranging from extremely small tricuspid cells to extremely large multipolar cells with fairly prominent intracellular trabecular networks (visible under a light microscope).

[0030] Identification method Step (a) in the identification method according to the present invention includes the step of preparing a biological sample containing at least one type of dermal fibroblast, as defined in the "fibroblast" section above.

[0031] Specifically, the biological sample may be an in vitro culture of dermal fibroblasts or a mixture of dermal fibroblasts, a sample derived from a skin biopsy, or a sample derived from an in vitro dermal equivalent or skin equivalent.

[0032] Specifically, the sample may be derived from a human skin biopsy performed on a young subject, such as one between 15 and 40 years of age, preferably between 17 and 31 years of age.

[0033] Step b) of the identification method according to the present invention includes measuring the level of the expression product of at least one gene selected from the group consisting of the UCP2 gene, the FGF9 gene, and optionally the COL11A1 and ACAN genes, and optionally the level of the expression product of the KLF9 gene.

[0034] In this specification, the “UCP2 gene” refers to the coding gene for “mitochondrial uncoupling protein 2.” The UCP2 gene is also known as the SLC25A8 gene, and the UCP2 protein is also known as UCPH or “Solute Carrier Family 25 Member 8.” It belongs to the family of mitochondrial anionic support proteins (MACPs) and regulates reactive oxygen species originating from mitochondria. This is typically described by Pecqueur et al. (1999) Biochemical and Biophysical Research Communications 255: pp. 40-46. The human UCP2 protein sequence is typically referred to by UniProt number P55851.

[0035] In this specification, the “ACAN gene” refers to the coding gene for “agrecan core protein.” The ACAN gene is also known as the AGC1 gene, CSPG1 gene, and MSK16 gene, and the ACAN protein is also known as “agrecan,” “cartilage-specific proteoglycan core protein,” or CSPCP, or “chondroitin sulfate proteoglycan core protein 1,” or “chondroitin sulfate proteoglycan 1.” It forms part of the extracellular matrix of cartilage tissue. It is a proteoglycan. It is typically described in Doege et al. (1991) J. Biol. Chem. 15:894-902. The human ACAN protein sequence is typically referred to as UniProt number P16112.

[0036] In this specification, “FGF9 gene” means the gene encoding fibroblast growth factor 9. The FGF9 protein is also called “glial activator,” GAF, “heparin-binding growth factor 9,” ​​or HBGF-9. It has a growth-stimulating effect on glial cells in culture. This is typically described by Miyamoto et al. (1993) Molecular and Cellular Biology 13: pp. 4251-4259. The human FGF9 protein sequence is typically referred to by UniProt number P31371.

[0037] In this specification, the “COL11A1 gene” refers to the gene encoding the α1(XI) chain of collagen. The COL11A1 gene is also called the COLL6 gene. This chain is one of two alpha chains of type XI collagen, a minor fibrous collagen. This is typically described in Yoshioka et al. (1990) J. Biol. Chem. 15:6423–6426. The human COL11A1 protein sequence is typically referred to by UniProt number P12107.

[0038] In this specification, “KLF9 gene” refers to the gene encoding Krueppel-like factor 9. The KLF9 gene is also called the BTEB gene or BTEB1 gene, and the KLF9 protein is also called the BTEB1 transcription factor, GC-box-binding protein 1, basic transcription element-binding protein 1, or BTE protein 1 ("BTE-binding protein 1"). It forms part of the family of type Sp1 C2H2 zinc finger transcription factors. This is typically described in Sporl et al. (2012) Proc. Natl. Acad. Science USA 109: pp. 10903-10908. The human KLF9 protein sequence is typically referred to as UniProt number Q13886.

[0039] In the context of this invention, the UniProt reference cited above was available on July 31, 2017.

[0040] In a particular embodiment, step (b) includes measuring the levels of expression products of at least two genes selected from the group consisting of the UCP2 gene and the FGF9 gene, and optionally the level of expression product of the KLF9 gene, preferably the levels of expression products of at least one gene selected from the group consisting of the UCP2 gene and the FGF9 gene, and optionally the level of expression product of at least one gene selected from the group consisting of the ACAN gene and the COL11A1 gene, and optionally the level of expression product of the KLF9 gene, preferably the levels of expression products of at least three genes selected from the group consisting of at least UCP2, ACAN, FGF9 and COL11A1 genes, and optionally the level of expression product of the KLF9 gene, and more preferably the levels of expression products of at least four genes selected from the group consisting of the UCP2, ACAN, FGF9 and COL11A1 genes, and optionally the level of expression product of the KLF9 gene.

[0041] In one particular embodiment, step (b) includes measuring the level of expression products of at least one gene, particularly at least two genes, selected from the group consisting of the UCP2 gene and the FGF9 gene. In another particular embodiment, step (b) includes measuring the level of expression products of at least one gene, selected from the group consisting of the UCP2 gene and the FGF9 gene, and at least one gene, selected from the group consisting of the COL11A1 gene and the ACAN gene. In yet another particular embodiment, step (b) includes measuring the level of expression products of at least three genes, particularly at least four genes, selected from the group consisting of the UCP2, ACAN, FGF9, and COL11A1 genes.

[0042] In another specific embodiment, step (b) measures the level of expression products of at least one gene, particularly at least two, three, or four genes, selected from the group consisting of the UCP2, ACAN, FGF9, and COL11A1 genes, and This includes measuring the level of the KLF9 gene expression product.

[0043] In this specification, the term “expression product of the X gene” means the mRNA encoded by the X gene or the protein encoded by the X gene. Therefore, the level of the expression product of the X gene can be measured by quantifying the mRNA or the corresponding protein. In a particular embodiment, the expression product of the X gene is the mRNA encoded by the X gene.

[0044] Preferably, the level of the expression product corresponds to the concentration or amount of the expression product.

[0045] The level of the expression product of the X gene can be measured in step (b) by any technique known to those skilled in the art. Specifically, if the expression product is a protein, the level of the expression product can be measured by an immunoassay such as an ELISA assay, immunofluorescence assay (IFA), radioimmunoassay (RIA), competitive binding test, or Western blot test. If the expression product is mRNA, the level of the expression product can be measured by sequencing, such as RT-PCR, qRT-PCR, or ddPCR (droplet digital PCR), for example, by NGS (next-generation sequencing) type sequencing or ddSEQ® single-cell isolator type sequencing.

[0046] It is said that the level of X gene expression product in a test sample increases when the ratio [X gene expression level in the test sample / control level of X gene] is 2 or higher.

[0047] It is said that the level of X gene expression product in a test sample decreases when the ratio [control level of X gene / level of X gene expression in the test sample] is 2 or higher.

[0048] If the ratio [control level of the X gene / expression level of the X gene in the test sample] is 2 or greater, the (-) symbol is placed before the obtained value.

[0049] This ratio has traditionally been called the "magnification change."

[0050] Step c) of the identification method according to the present invention includes identifying dermal fibroblasts as papillary fibroblasts, reticular fibroblasts, or dermal-subcutaneous junction fibroblasts (FJDH) based on the levels measured in step (b).

[0051] In a particular embodiment of the present invention, step (c) of the identification method according to the present invention includes a comparison of the level measured in step b) with one or more control levels.

[0052] In this specification, “control level” means a reference value that preferably corresponds to the level of the expression product of the gene in dermal fibroblasts known to be papillary, reticular, or FJDH fibroblasts, particularly those derived from the same donor.

[0053] In this specification, “dermal fibroblasts known to be papillary, reticular, or FJDH fibroblasts” means dermal fibroblasts whose type (papillary, reticular, or FJDH) has been previously determined in consideration of their morphology, source, or detected biomarkers.

[0054] Dermal fibroblasts, known to be papillary dermal fibroblasts, can be isolated from non-degreased human skin, particularly from tissue collected at a thickness of 300 μm, and then deepidermized after 16 hours of dispase treatment (Roche - 2.4 U / mL) at 4°C.

[0055] Dermal fibroblasts known to be reticular dermal fibroblasts can be isolated from non-dehydrated human skin derived from tissue removed from their dermal-subcutaneous junction; the tissue is then harvested at a thickness of 700 μm. Only the lower part of the tissue is retained.

[0056] Dermal fibroblasts known to be FJDH dermal fibroblasts can be harvested from connective trabeculae located at the dermal-subcutaneous junction. These can be harvested using a clamp and scissors.

[0057] In one particular embodiment, the level of the expression product of at least one gene selected from the group consisting of the UCP2 gene and the FGF9 gene is preferably measured in step b). Dermal fibroblasts are (i) If the level of the UCP2 gene expression product is higher than the control level, and / or (ii) When the level of FGF9 gene expression product is lower than the control level These were identified as papillary fibroblasts. The control levels in (i) and (ii) are preferably the levels of expression products of the UCP2 gene and the FGF9 gene, respectively, in dermal fibroblasts known to be reticular fibroblasts or FJDH fibroblasts.

[0058] In another specific embodiment, the levels of expression products of at least one gene selected from the group consisting of the UCP2 gene and the FGF9 gene, and the levels of expression products of at least one gene selected from the group consisting of the COL11A1 gene and the ACAN gene are preferably measured in step b). Dermal fibroblasts are 1)(i) If the level of the UCP2 gene expression product is higher than the control level, and / or (ii) If the level of the FGF9 gene expression product is lower than the control level, and 2)(iii) If the expression level of the COL11A1 gene expression product is lower than the control level, and / or (iv) If the level of ACAN gene expression product is lower than the control level These were identified as papillary fibroblasts. The control levels in (i), (ii), (iii), and (iv) are preferably the levels of expression products of the UCP2, FGF9, COL11A1, and ACAN genes in dermal fibroblasts known to be reticular fibroblasts or FJDH fibroblasts.

[0059] In another specific embodiment, the levels of the expression product of at least one gene selected from the group consisting of the UCP2, ACAN, FGF9, and COL11A1 genes, as well as the level of the expression product of the KLF9 gene, are preferably measured in step b). Dermal fibroblasts are 1)(i) If the level of the UCP2 gene expression product is lower than the control level, (ii) If the level of the ACAN gene expression product is higher than the control level, (iii) If the level of the FGF9 gene expression product is higher than the control level, and / or (iv) If the level of the COL11A1 gene expression product is higher than the control level, and 2) When the level of KLF9 gene expression product is lower than the control level. They were identified as reticular fibroblasts. The control levels in 1(i), 1(ii), 1(iii), and 1(iv) are preferably the levels of expression products of the UCP2, ACAN, FGF9, and COL11A1 genes, respectively, in dermal fibroblasts known to be papillary fibroblasts, and the control level in 2) is preferably the level of expression product of the KLF9 gene, in dermal fibroblasts known to be FJDH fibroblasts.

[0060] In another specific embodiment, the levels of the expression product of at least one gene selected from the group consisting of the UCP2, ACAN, FGF9, and COL11A1 genes, as well as the level of the expression product of the KLF9 gene, are preferably measured in step b). Dermal fibroblasts are 1)(i) If the level of the UCP2 gene expression product is lower than the control level, (ii) If the level of the ACAN gene expression product is higher than the control level, (iii) If the level of the FGF9 gene expression product is higher than the control level, and / or (iv) If the level of the COL11A1 gene expression product is higher than the control level, and 2) When the level of KLF9 gene expression product is higher than the control level. In some cases, these are identified as dermal-subcutaneous junction fibroblasts. The control levels in 1(i), 1(ii), 1(iii), and 1(iv) are preferably the levels of expression products of the UCP2, ACAN, FGF9, and COL11A1 genes, respectively, in dermal fibroblasts known to be papillary fibroblasts, and the control level in 2) is preferably the level of expression product of the KLF9 gene, in dermal fibroblasts known to be reticular fibroblasts.

[0061] In this specification, “higher level” means a level that is statistically significantly higher than the control level. Preferably, the higher level is at least 1.5 times, at least 2 times, at least 2.06 times, at least 2.5 times, at least 3 times, at least 3.2 times, at least 3.5 times, at least 4 times, at least 4.2 times, at least 4.28 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 5.8 times, at least 5.83 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 28 times, at least 28.5 times, or at least 28.6 times higher than the control level as defined above.

[0062] In this specification, “lower level” means a level that is statistically significantly lower than the control. Preferably, the reduced level is less than or equal to 1 / 1.5, 1 / 2, 1 / 2.06, 1 / 2.5, 1 / 3, 1 / 3.2, 1 / 3.5, 1 / 4, 1 / 4.2, 1 / 4.28, 1 / 4.5, 1 / 5, 1 / 5.5, 1 / 5.8, 1 / 5.83, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 28, 1 / 28.5, or 1 / 28.6 of the control level as defined above.

[0063] Preferably, dermal fibroblasts are (i) If the level of UCP2 gene expression product is at least 5 times higher than the control level defined above, particularly at least 5.5 times, at least 5.8 times, or at least 5.83 times higher, with reference to the level of UCP2 gene expression product in dermal fibroblasts, which are known to be reticular fibroblasts, and / or (ii) The level of FGF9 gene expression product is less than or equal to one-quarter of the control level defined above, particularly when referring to the level of FGF9 gene expression product in dermal fibroblasts known to be reticular fibroblasts, and especially less than or equal to one-four-and-a-half or one-four-and-a-half or one-four-and-a-half. These cells are identified as papillary fibroblasts.

[0064] Furthermore, preferably, dermal fibroblasts are 1)(i) If the level of UCP2 gene expression product is at least 5 times higher than the control level defined above, particularly at least 5.5 times, at least 5.8 times, or at least 5.83 times higher, with reference to the level of UCP2 gene expression product in dermal fibroblasts known to be reticular fibroblasts, and / or (ii) If the level of FGF9 gene expression product is less than or equal to one-quarter of the control level defined above, particularly with reference to the level of FGF9 gene expression product in dermal fibroblasts known to be reticular fibroblasts, and especially less than or equal to one-four-and-a-half or one-four-and-a-half or one-four-and-a-half, and 2)(iii) If the level of COL11A1 gene expression product is less than or equal to 1 / 25 of the control level defined above, particularly with reference to the level of COL11A1 gene expression product in dermal fibroblasts known to be reticular fibroblasts, and / or if it is less than or equal to 1 / 28, 1 / 28.5, or 1 / 28.6 (iv) If the level of ACAN gene expression product is less than or equal to one-third of the control level defined above, and especially less than or equal to one-third-2 of the control level, with reference to the level of ACAN gene expression product in dermal fibroblasts known to be reticular fibroblasts. These cells are identified as papillary fibroblasts.

[0065] Preferably, dermal fibroblasts are 1)(i) If the level of UCP2 gene expression product is less than or equal to one-fifth of the control level defined above, particularly with reference to the level of UCP2 gene expression product in dermal fibroblasts known to be papillary fibroblasts, and especially less than or equal to 5.5, 1 / 5.8, or 1 / 5.83, (ii) If the level of ACAN gene expression product is at least 3 times higher, and especially at least 3.2 times higher, than the control level defined above, with reference to the level of ACAN gene expression product in dermal fibroblasts known to be papillary fibroblasts, (iii) If the level of FGF9 gene expression product is at least four times higher than the control level defined above, particularly at least 4.2 times or at least 4.28 times higher, with reference to the level of FGF9 gene expression product in dermal fibroblasts known to be papillary fibroblasts, and / or (iv) If the level of COL11A1 gene expression product is at least 25 times higher than the control level defined above, particularly at least 28 times, at least 28.5 times, or at least 28.6 times higher, with reference to the level of COL11A1 gene expression product in dermal fibroblasts known to be papillary fibroblasts. These cells are identified as reticular fibroblasts or FJDH fibroblasts.

[0066] Preferably, dermal fibroblasts are also included, 2) If the level of KLF9 gene expression product is at least twice as high, and especially at least 2.06 times higher, than the control level defined above, with reference to the level of KLF9 gene expression product in dermal fibroblasts, which are known to be reticular fibroblasts. These cells were identified as FJDH fibroblasts.

[0067] Preferably, dermal fibroblasts are also included, 2) If the level of KLF9 gene expression product is less than or equal to half of the control level defined above, and especially less than or equal to 1 / 2.06, with reference to the level of KLF9 gene expression product in dermal fibroblasts known to be FJDH fibroblasts. These cells are identified as reticular fibroblasts.

[0068] Kits and microarrays Another object of the present invention is a kit for identifying dermal fibroblasts as papillary fibroblasts, reticular fibroblasts, or dermal-subcutaneous junction fibroblasts (FJDH), - At least one measuring means selected from the group consisting of means for measuring the level of UCP2 gene expression product, means for measuring the level of FGF9 gene expression product, means for measuring the level of COL11A1 gene expression product, and means for measuring the level of ACAN gene expression product. - At least one means for measuring the level of the KLF9 gene expression product, and - One or more controls from which a control level can be obtained. This is a kit that includes [the following items].

[0069] Specifically, the kit may also include, as a separate component, an antibody that recognizes the protein encoded by the gene in the biological sample defined above. The kit may also include, as a separate component, primers and / or probes that specifically hybridize to the mRNA encoded by the gene.

[0070] The kit may also include further optional components for carrying out the identification method according to the present invention. Such optional components include, for example, a container, a mixer, a buffer, instructions for carrying out the method, a marker, or a support.

[0071] Another object of the present invention is a DNA microarray for identifying dermal fibroblasts as papillary fibroblasts, reticular fibroblasts, or dermal-subcutaneous junction fibroblasts (FJDH), - At least one probe selected from the group consisting of a probe that detects UCP2 gene expression products, a probe that detects FGF9 gene expression products, a probe that detects COL11A1 gene expression products, and a probe that detects ACAN gene expression products. and - At least one probe to detect the KLF9 gene expression product, This is a DNA microarray composed of [the specified components].

[0072] The DNA microarray according to the present invention does not include any probes for detecting the expression products of genes other than UCP2, FGF9, COL11A1, ACAN, and KLF9.

[0073] In this specification, “DNA microarray” refers to the orderly arrangement of at least two types of probes on a substrate. Preferably, the DNA microarray according to the present invention also includes a control probe or a standard probe.

[0074] In this specification, “probe” means an oligonucleotide or polynucleotide, RNA or DNA that is naturally occurring as a purified digest of restriction enzymes or is produced synthetically and can specifically hybridize with a polynucleotide having a sequence complementary to the probe sequence. The probe may be single-stranded or double-stranded. Preferably, the probe contains or consists of 10 to 100 nucleotides, preferably 15 to 50 nucleotides or 15 to 25 nucleotides.

[0075] Dermal equivalents and skin equivalents The inventors have for the first time identified a subpopulation of dermal fibroblasts: fibroblasts located at the dermal-subcutaneous junction.

[0076] Therefore, using these new fibroblasts in dermal equivalents allows for a more accurate imitation of normal dermis.

[0077] Therefore, another object of the present invention is an in vitro dermis comprising a subpopulation of dermal-subcutaneous junction fibroblasts (FJDH) as defined in the “fibroblasts” section above, 1)(i) The level of the UCP2 gene expression product is reduced compared to the control level. (ii) The level of ACAN gene expression product is increased compared to the control level. (iii) The level of the FGF9 gene expression product is increased compared to the control level, and / or (iv) The level of the COL11A1 gene expression product is increased compared to the control level. and 2) Dermis in which the level of KLF9 gene expression product is increased compared to the control level.

[0078] The dermal equivalent according to the present invention also includes subpopulations of papillary fibroblasts and / or reticular fibroblasts, as defined in the “fibroblast” section above.

[0079] The dermal equivalent according to the present invention also preferably includes collagen.

[0080] The dermal equivalent collagen according to the present invention may be any type of collagen and of any origin. Preferably, the collagen is selected from type I, type III, or type V fibrous collagen. Preferably, the collagen is type I. Preferably, the collagen is of animal origin, particularly bovine origin. Particularly preferably, the collagen is bovine type I collagen. Alternatively, the collagen may be a mixture of various types of collagen of various origins in any proportion.

[0081] The fibroblasts in the dermal equivalent according to the present invention are preferably human fibroblasts, but may be of any origin.

[0082] The fibroblasts present in the dermal equivalent according to the present invention are preferably derived from a fibroblast culture, and by performing the identification method according to the present invention using a portion of the culture, it becomes possible to identify the entire culture as a culture of papillary, reticular, or FJDH fibroblasts.

[0083] The dermal equivalent according to the present invention may also include any other components that may constitutively exist in the skin, such as endothelial cells, macrophages, monocytes, macrophage precursors, dendritic cell precursors, or nerve cells.

[0084] The present invention also provides a method for preparing a dermal equivalent as defined above, comprising an initial step of identifying dermal fibroblasts as FJDH fibroblasts, A) A process of preparing a homogeneous culture of dermal fibroblasts, B) A step of sampling a portion of the dermal fibroblast culture prepared in step A), C) A step of identifying a portion of the dermal fibroblast culture sampled in step B) as a culture of FJDH fibroblasts using the identification method defined in the "Identification Method" section above, D) A step to prepare a dermal equivalent using a portion of the dermal fibroblast culture that was not sampled in step B), and This also relates to methods that include this.

[0085] A dermal equivalent can be prepared in step D) using any technique well known to those skilled in the art.

[0086] Therefore, the preparation of the dermal equivalent in step D) may include the steps of preparing a cell suspension of collagen-containing lattices and FJDH fibroblasts, and optionally a cell suspension of papillary fibroblasts and / or reticular fibroblasts.

[0087] As mentioned above, the collagen used may be any type of collagen from any origin, either alone or in mixtures.

[0088] Preferably, the grid is 11 × 10 5 ~5×10 6 At a concentration of cells / ml, preferably 2 × 10 5 ~2×10 6 Contains fibroblasts at a concentration of cells / ml.

[0089] The grid can be prepared by any technique well known to those skilled in the art.

[0090] Specifically, a solution containing collagen and dermal fibroblasts can be prepared and deposited on a support.

[0091] Preferably, the solution is incubated for, for example, 10 to 30 minutes, and then kept in incubation to allow lattice contraction so that the collagen can gel.

[0092] Therefore, preferably, the grid is kept for a further 1 to 7 days, and more preferably 3 or 4 days, during incubation.

[0093] Since the dermal content affects the epidermal compartment, the dermal equivalent according to the present invention can act as a support for the formation of a skin equivalent.

[0094] Therefore, the present invention also relates to in vitro skin equivalents, including the dermal equivalents defined above.

[0095] The skin equivalent according to the present invention includes an epidermal equivalent containing at least keratinocytes on the upper surface of the dermal equivalent.

[0096] Keratinocytes can be obtained from any source, but human keratinocytes are preferred. These can be prepared by any method well known to those skilled in the art. Therefore, keratinocytes can be prepared by culturing isolated epidermis derived from a normal skin sample, or by culturing keratinocytes obtained from hair follicle sheaths.

[0097] Preferably, the keratinocytes are normal human skin keratinocytes.

[0098] More preferably, keratinocytes are prepared from isolated human epidermis obtained from normal skin samples collected by the method described in Regnier et al., Frontier of Matrix Biology, Vol. 9, pp. 4-35 (Karger, Basel, 1981).

[0099] The epidermal equivalent may include Langerhans cells and / or Langerhans cell precursors and / or other cell types such as melanocytes.

[0100] Furthermore, the epidermal equivalent may advantageously include melanocytes and / or Langerhans cells and / or Langerhans cell precursors.

[0101] Melanocytes can be isolated from any organ containing them, such as normal skin or hair follicles. Preferably, melanocytes are isolated from normal skin. Any method well known to those skilled in the art can be used, such as the method described by Olsson et al. (1994) Acta. Derm. Venereol. 74: pp. 226-268.

[0102] Langerhans cells and / or Langerhans cell precursors may be as described in European Patent Application EP789074.

[0103] The present invention also relates to a method for preparing a skin equivalent as defined above, comprising a step of preparing a dermal equivalent using the method for preparing a dermal equivalent as defined above.

[0104] Preferably, the method for preparing a skin equivalent includes a step of preparing a dermal equivalent, followed by a step of reconstructing an epidermal equivalent containing at least keratinocytes on the dermal equivalent.

[0105] This epidermal reconstruction process can be carried out by any technique well known to those skilled in the art, such as the techniques described in the patent applications EP285471, EP285474, EP789074, EP502172, EP418035, WO91 / 16010, EP197090, EP20753, FR2665175 and FR2689904, or the techniques described in Asselineau et al. (1985) Exp. Cell. Res. 159: pp. 536-539, Asselineau et al. (1987), Models in dermato., col III, edited by Lowe & Mailbach, pp. 1-7, or Asselineau et al. (1984) Br J Dermatol. 111 Suppl 27: pp. 219-22.

[0106] Prior to this reconstitution step, it may be advantageous to perform an adhesion step in a culture dish of the prepared dermal equivalent using, for example, an adhesion solution consisting of MEM 1.76× medium, FCS, NaOH 0.1N, and MEM 25mM Hepes 10% FCS.

[0107] Preferably, the reconstruction step is carried out by seeding keratinocytes onto the dermal equivalent in a seeding loop.

[0108] After seeding keratinocytes onto the dermal equivalent, the culture can be advantageously maintained in a nutrient medium, such as the medium described in Rheinwald and Green (1975) Cell 6: pp. 317-330, or any medium that enables keratinocyte proliferation.

[0109] Preferably, the skin equivalent is maintained at the gas / liquid interface by depositing it, for example, onto a metal mesh, following an incubation period of 3 to 15 days, and more preferably 7 to 9 days. The liquid then preferably consists of the same nutrient medium as before.

[0110] Next, preferably, incubation is continued until a skin equivalent exhibiting skin characteristics is obtained, that is, until a skin equivalent covered by an epidermal equivalent exhibiting four standard types of cell layers, namely the basal layer, the upper layer of basal cells, the granular layer, and the stratum corneum.

[0111] In this way, incubation is preferably continued for a period of 5 to 30 days, and more preferably 7 to 10 days.

[0112] use The present invention also relates to the use of dermal equivalents as defined in the "Dermal Equivalents and Skin Equivalents" section above, or the use of skin equivalents as defined in the "Dermal Equivalents and Skin Equivalents" section above, for studying skin function.

[0113] The present invention also relates to a method for screening compounds that exhibit cosmetic activity after topical application to the skin, particularly in the field of anti-aging, such as the treatment of wrinkles and lines, and / or in the field of inflammation, and / or in the field of pigmentation, such as the treatment of pigmented spots, wherein the screening method includes the application of candidate compounds to a dermal equivalent or a skin equivalent according to the present invention.

[0114] Throughout this application, unless otherwise specified, the terms “comprising a” or “including a” mean “including at least one” or “including at least one,” in other words, “including one or more” or “including one or more.”

[0115] Throughout this specification, unless otherwise specified, the terms “between x and y” or “the range from x to y” refer to an inclusive range, i.e., the values ​​x and y are included within that range.

[0116] The present invention will be illustrated in more detail by the following examples. [Examples]

[0117] The following examples demonstrate the isolation of a new subpopulation of dermal-subcutaneous junction fibroblasts by the inventors, and the identification of the molecular signature of this subpopulation of fibroblasts present in the dermis.

[0118] Materials and methods Cell preparation Papillary fibroblasts (Fp), reticular fibroblasts (Fr), and dermal-subcutaneous junction fibroblasts (FJDH) were isolated from non-dehydrated human skin. These samples were collected after breast reduction surgery for aesthetic reasons. Six women were involved.

[0119] FJDH is isolated from connective trabeculae located at the dermal-subcutaneous junction. Connective trabeculae are sampled using clamps and scissors.

[0120] Fr isolation is performed on a portion of the tissue that has been removed as if its dermal-subcutaneous junction were intact. The tissue is then harvested at a thickness of 700 μm. Only the lower part of the tissue is retained.

[0121] Fp was isolated from tissue collected at a thickness of 300 μm, followed by dispase action (Roche - 2.4 U / mL) at 4°C for 16 hours, after which the tissue was dedermated.

[0122] After tearing, the dermal fragments are digested at 37°C with 0.2% (Gibco) collagenase under the action of type II collagenase.

[0123] Next, cells are amplified in MEM medium - 10% fetal bovine serum, supplemented with glutamine, sodium pyruvate, non-essential amino acids, penicillin, streptomycin, and fungizone, at 37°C and 5% CO2 in a humid atmosphere.

[0124] Transcriptome analysis and RT-qPCR After cell proliferation (the population doubles between 7 and 10 times), mRNA is extracted using a QIAgen column according to the supplier's instructions.

[0125] The sample was divided into two parts: one part was set aside for transcriptome analysis, and the other part was used for biomarker validation by PCR.

[0126] Transcriptome analysis was performed using an Affymetrix GeneChip HG-U133 Plus 2.0 type microarray. Probe sets that appeared to be differentially expressed showed a magnification change of >2 for an uncorrected p-value <0.05.

[0127] The primers used for validation by RT-qPCR are commercially available from Qiagen (QIAgen-Quantitech Primer Assay), as shown in Table 1 below.

[0128] [Table 1]

[0129] It is listed in [the document].

[0130] reconstructed skin Reconstructed skin was prepared using the protocol described in Asselineau et al. (1985) Exp. Cell. Res. 159: pp. 536-539.

[0131] In short, 10 originating from the dermal-subcutaneous tissue junction 6 5 × 10¹ fibroblasts (FJDH) were placed in a type I bovine collagen solution (Symatese). Four days after lattice organization and contraction, 5 × 10¹⁶ cells were placed. 4 Individual keratinocytes are seeded on the lattice surface. The cultures are maintained by immersion in MEM medium, 10% fetal bovine serum, EGF (10 ng / ml), hydrocortisone (0.4 μg / ml), and cholera toxin (0.1 nM) for one week. Complete stratification of the epidermis is obtained one week after emergence.

[0132] Throughout the entire reconstitution process, the culture is kept in an incubator saturated with 5% CO2 and humidity at 37°C.

[0133] result Therefore, we isolated and characterized three subpopulations of fibroblasts within the dermis: papillary fibroblasts (closer to the epidermis), reticular fibroblasts (embedded more deeply in the skin), and dermal-subcutaneous junction fibroblasts (a new subpopulation of cells separated from connective trabeculae released by the dermis into the subcutaneous tissue).

[0134] These three subpopulations of cells were isolated and cultured from breast reconstructive surgery samples taken from six individuals.

[0135] Transcriptome analysis was performed on these three subpopulations, and validation by RT-qPCR analysis demonstrated differential expression of five specific genes among these three subpopulations.

[0136] The results of the RT-qPCR analysis are shown in Table 2 below.

[0137] [Table 2]

[0138] Summarize it.

[0139] Therefore, we were able to demonstrate the following molecular signatures for three fibroblast subpopulations identified in the dermis:

[0140] [Table 3]

[0141] Next, reconstructed skin containing these three subpopulations was obtained as described above.

Claims

1. An in vitro dermal equivalent comprising a subpopulation of dermal-subcutaneous junction fibroblasts (FJDH), wherein in the dermal-subcutaneous junction fibroblasts (FJDH), 1)(i) The level of the UCP2 gene expression product is reduced compared to the control level. (ii) The level of ACAN gene expression product is increased compared to the control level. (iii) The level of the FGF9 gene expression product is increased compared to the control level, and / or (iv) The level of the COL11A1 gene expression product is increased compared to the control level. and 2) The level of KLF9 gene expression product is increased compared to the control level. The control levels in 1)(i), 1)(ii), 1)(iii), and 1(iv) are the levels of expression products of the UCP2, ACAN, FGF9, and COL11A1 genes, respectively, in dermal fibroblasts known to be papillary fibroblasts, and the control level in 2) is the level of expression product of the KLF9 gene, in dermal fibroblasts known to be reticular fibroblasts. Dermal equivalent.

2. The dermal equivalent according to claim 1, further comprising a subpopulation of papillary fibroblasts and / or a subpopulation of reticular fibroblasts.

3. The dermal equivalent according to claim 1 or 2, further comprising collagen.

4. An in vitro skin equivalent comprising a dermal equivalent according to any one of claims 1 to 3.

5. A method for preparing a dermal equivalent according to any one of claims 1 to 3, comprising an initial step of identifying dermal fibroblasts as FJDH fibroblasts, A) A process of preparing a homogeneous culture of dermal fibroblasts, B) A step of sampling a portion of the dermal fibroblast culture prepared in step A), C) The following steps a) A step of preparing a portion of the dermal fibroblast culture sampled in step B, b) A step of measuring the level of the expression product of at least one gene selected from the group consisting of the UCP2 gene and the FGF9 gene, as well as the level of the expression product of the KLF9 gene, and optionally the COL11A1 gene and the ACAN gene, and c) A step of identifying dermal fibroblasts as dermal-subcutaneous junction fibroblasts (FJDH) based on the levels measured in step b), The process involves identifying a portion of the dermal fibroblast culture sampled in step B) as a culture of FJDH fibroblasts using an in vitro identification method that includes the following: D) A step to prepare a dermal equivalent using a portion of the dermal fibroblast culture that was not sampled in step B), and A method that includes this.

Images