CELL AND TISSUE MODELS COMPRISING FIBROBLASTS OF THE DERMO-HYPODERMIC JUNCTION AND APPLICATIONS THEREOF

Abstract

The present invention relates to an in vitroprocess for preparing cell and tissue models comprising at least one step of culturing fibroblasts of the dermo-hypodermic junction; the models obtained and also the implementation thereof in processes for screening active agents that promote the differentiation of fibroblasts of the dermo-hypodermic junction into adipocyte, osteoblast and/or chondroblast cells.

Claims

1. An in vitro process for preparing a cell model comprising at least one step of culturing fibroblasts of the dermo-hypodermic junction.

2. The process as claimed in claim 1, wherein the fibroblasts are cultured between 24 h and 72 h after reaching at least 80% confluence.

3. The process as claimed in claim 1, also comprising a step of centrifugation of the fibroblasts obtained at the end of said culture step.

4. An in vitro process for preparing a tissue model comprising at least one step of culturing fibroblasts of the dermo-hypodermic junction on collagen sponge.

5. The process as claimed in claim 4, wherein said fibroblasts are cultured for 10 to 20 days.

6. The in vitro process for preparing a cell model as claimed in claim 1 or an in vitro process for preparing a tissue model comprising at least one step of culturing fibroblasts of the dermo-hypodermic junction, also comprising a step of identification of a dermal fibroblast as a fibroblast of the dermo-hyperdermic junction, said identification step being prior to said culture step and comprising: a) providing a biological sample comprising at least one dermal fibroblast, b) measuring, in the biological sample provided in step a), the level of an expression product of at least one gene selected from the group consisting of the genes UCP2, ACAN, FGF9 and COL11A1, and the level of an expression product of the gene KLF9, c) identifying the dermal fibroblast of step a) as a fibroblast of the dermo-hypodermic junction when: 1) (i) the level of the expression product of the gene UCP2 is decreased relative to a control level, (ii) the level of the expression product of the gene ACAN is increased relative to a control level, (iii) the level of the expression product of the gene FGF9 is increased relative to a control level, and/or (iv) the level of the expression product of the gene COL11A1 is increased relative to a control level, and 2) the level of the expression product of the gene KLF9 is increased relative to a control level, wherein the control levels of 1(i), 1(ii), 1(iii) and 1(iv) being respectively the level of the expression product of the genes UCP2, ACAN, FGF9 and COL11A1 in a dermal fibroblast known to be a papillary fibroblast and the control level of 2) being the level of the expression product of the gene KLF9 in a dermal fibroblast known to be a reticular fibroblast.

7. An in vitro cell model obtainable according to the process as defined in claim 1.

8. An in vitro tissue model obtainable according to claim 4.

9. The cell model as claimed in claim 7 or tissue model obtainable by an in vitro process comprising at least one step of culturing fibroblasts of the dermo-hypodermic junction on collagen sponge, comprising fibroblasts of the dermo-hypodermic junction for which: 1) (i) the level of the expression product of the gene UCP2 is decreased relative to a control level, (ii) the level of the expression product of the gene ACAN is increased relative to a control level, (iii) the level of the expression product of the gene FGF9 is increased relative to a control level, and/or (iv) the level of the expression product of the gene COL11A1 is increased relative to a control level, and 2) the level of the expression product of the gene KLF9 is increased relative to a control level, wherein the control levels of 1(i), 1(ii), 1(iii) and 1(iv) being respectively the level of the expression product of the genes UCP2, ACAN, FGF9 and COL11A1 in a dermal fibroblast known to be a papillary fibroblast and the control level of 2) being the level of the expression product of the gene KLF9 in a dermal fibroblast known to be a reticular fibroblast.

10. The use of in vitro cell or tissue models as defined in claim 7 as tool for screening for active agents that promote the differentiation of fibroblasts of the dermo-hypodermic junction into adipocyte, osteoblast and/or chondroblast cells.

11. A process for screening active agents that promote the differentiation of fibroblasts of the dermo-hypodermic junction into adipocyte, osteoblast and/or chondroblast cells, comprising the following steps: i. providing a cell as defined in claim 7, ii. bringing said model into contact with at least one agent to be screened, iii. carrying out a qualitative and/or quantitative measurement of the expression of at least one marker of the adipocyte, osteoblast and/or chondrocyte cells or of their biological activity (activities), then iv. comparing the measurement carried out in step iii) with that obtained from a control.

12. A process for screening active agents that promote the differentiation of fibroblasts of the dermo-hypodermic junction into adipocyte, osteoblast and/or chondroblast cells, comprising the following steps: i. providing a as defined in claim 8, ii. bringing said model into contact with at least one agent to be screened, iii. carrying out a qualitative and/or quantitative measurement of the expression of at least one marker of the adipocyte, osteoblast and/or chondrocyte cells or of their biological activity (activities), then iv. comparing the measurement carried out in step iii) with that obtained from a control.

13. The process as claimed in claim 1, wherein the fibroblasts are cultured for 48 h after reaching at least 80% confluence.

14. The process as claimed in claim 2, also comprising a step of centrifugation of the fibroblasts obtained at the end of said culture step.

15. The in vitro process for preparing a cell model as claimed in claim 2, also comprising a step of identification of a dermal fibroblast as a fibroblast of the dermo-hyperdermic junction, said identification step being prior to said culture step and comprising: a) providing a biological sample comprising at least one dermal fibroblast, b) measuring, in the biological sample provided in step a), the level of an expression product of at least one gene selected from the group consisting of the genes UCP2, ACAN, FGF9 and COL11A1, and the level of an expression product of the gene KLF9, c) identifying the dermal fibroblast of step a) as a fibroblast of the dermo-hypodermic junction when: 1) (i) the level of the expression product of the gene UCP2 is decreased relative to a control level, (ii) the level of the expression product of the gene ACAN is increased relative to a control level, (iii) the level of the expression product of the gene FGF9 is increased relative to a control level, and/or (iv) the level of the expression product of the gene COL11A1 is increased relative to a control level, and 2) the level of the expression product of the gene KLF9 is increased relative to a control level, wherein the control levels of 1(i), 1(ii), 1(iii) and 1(iv) being respectively the level of the expression product of the genes UCP2, ACAN, FGF9 and COL11A1 in a dermal fibroblast known to be a papillary fibroblast and the control level of 2) being the level of the expression product of the gene KLF9 in a dermal fibroblast known to be a reticular fibroblast.

16. The in vitro process for preparing a cell model as claimed in claim 3, also comprising a step of identification of a dermal fibroblast as a fibroblast of the dermo-hyperdermic junction, said identification step being prior to said culture step and comprising: a) providing a biological sample comprising at least one dermal fibroblast, b) measuring, in the biological sample provided in step a), the level of an expression product of at least one gene selected from the group consisting of the genes UCP2, ACAN, FGF9 and COL11A1, and the level of an expression product of the gene KLF9, c) identifying the dermal fibroblast of step a) as a fibroblast of the dermo-hypodermic junction when: 1) (i) the level of the expression product of the gene UCP2 is decreased relative to a control level, (ii) the level of the expression product of the gene ACAN is increased relative to a control level, (iii) the level of the expression product of the gene FGF9 is increased relative to a control level, and/or (iv) the level of the expression product of the gene COL11A1 is increased relative to a control level, and 2) the level of the expression product of the gene KLF9 is increased relative to a control level, wherein the control levels of 1(i), 1(ii), 1(iii) and 1(iv) being respectively the level of the expression product of the genes UCP2, ACAN, FGF9 and COL11A1 in a dermal fibroblast known to be a papillary fibroblast and the control level of 2) being the level of the expression product of the gene KLF9 in a dermal fibroblast known to be a reticular fibroblast.

17. The in vitro process for preparing a tissue model as claimed in claim 4, also comprising a step of identification of a dermal fibroblast as a fibroblast of the dermo-hyperdermic junction, said identification step being prior to said culture step and comprising: a) providing a biological sample comprising at least one dermal fibroblast, b) measuring, in the biological sample provided in step a), the level of an expression product of at least one gene selected from the group consisting of the genes UCP2, ACAN, FGF9 and COL11A1, and the level of an expression product of the gene KLF9, c) identifying the dermal fibroblast of step a) as a fibroblast of the dermo-hypodermic junction when: 1) (i) the level of the expression product of the gene UCP2 is decreased relative to a control level, (ii) the level of the expression product of the gene ACAN is increased relative to a control level, (iii) the level of the expression product of the gene FGF9 is increased relative to a control level, and/or (iv) the level of the expression product of the gene COL11A1 is increased relative to a control level, and 2) the level of the expression product of the gene KLF9 is increased relative to a control level, wherein the control levels of 1(i), 1(ii), 1(iii) and 1(iv) being respectively the level of the expression product of the genes UCP2, ACAN, FGF9 and COL11A1 in a dermal fibroblast known to be a papillary fibroblast and the control level of 2) being the level of the expression product of the gene KLF9 in a dermal fibroblast known to be a reticular fibroblast.

18. The in vitro process for preparing a tissue model as claimed in claim 5, also comprising a step of identification of a dermal fibroblast as a fibroblast of the dermo-hyperdermic junction, said identification step being prior to said culture step and comprising: a) providing a biological sample comprising at least one dermal fibroblast, b) measuring, in the biological sample provided in step a), the level of an expression product of at least one gene selected from the group consisting of the genes UCP2, ACAN, FGF9 and COL11A1, and the level of an expression product of the gene KLF9, c) identifying the dermal fibroblast of step a) as a fibroblast of the dermo-hypodermic junction when: 1) (i) the level of the expression product of the gene UCP2 is decreased relative to a control level, (ii) the level of the expression product of the gene ACAN is increased relative to a control level, (iii) the level of the expression product of the gene FGF9 is increased relative to a control level, and/or (iv) the level of the expression product of the gene COL11A1 is increased relative to a control level, and 2) the level of the expression product of the gene KLF9 is increased relative to a control level, wherein the control levels of 1(i), 1(ii), 1(iii) and 1(iv) being respectively the level of the expression product of the genes UCP2, ACAN, FGF9 and COL11A1 in a dermal fibroblast known to be a papillary fibroblast and the control level of 2) being the level of the expression product of the gene KLF9 in a dermal fibroblast known to be a reticular fibroblast.

19. An in vitro cell model obtainable according to the process as defined in claim 2.

20. An in vitro tissue model obtainable according to claim 5.

Description

FIGURES

[0149] FIG. 1: View of the region from which the fibroblasts are taken, of the dermo-hypodermic junction.

[0150] FIG. 2: Demonstration of the differentiation of fibroblasts of the dermo-hypodermic junction into adipocyte cells in a cell model according to example 1.

[0151] FIG. 3: Demonstration of the differentiation of fibroblasts of the dermo-hypodermic junction into osteoblast cells in a cell model according to example 1.

[0152] FIG. 4: Demonstration of the differentiation of fibroblasts of the dermo-hypodermic junction into chondroblast cells in a spheroid cell model according to example 1.

[0153] FIG. 5: Demonstration of the differentiation of fibroblasts of the dermo-hypodermic junction into adipocyte cells in a tissue model according to example 2.

[0154] FIG. 6: Demonstration of the differentiation of fibroblasts Fp (outside the invention), Fr (outside the invention), and F-DHJ (according to the invention) into adipocyte cells in a cell model.

[0155] FIG. 7: Demonstration of the differentiation of fibroblasts Fp (outside the invention), Fr (outside the invention), and F-DHJ (according to the invention) into osteoblast cells in a cell model.

[0156] FIG. 8: Demonstration of the differentiation of fibroblasts Fp (outside the invention), Fr (outside the invention), and F-DHJ (according to the invention) into chondroblast cells in a cell model.

EXAMPLE 1: PROCESS FOR PREPARING A CELL MODEL OBTAINED FROM DHJ FIBROBLASTS AND VALIDATION OF ITS USE AS A TOOL FOR SCREENING ACTIVE AGENTS THAT PROMOTE THE DIFFERENTIATION OF DHJ FIBROBLASTS INTO ADIPOCYTE, OSTEOBLAST AND CHONDROBLAST CELLS

[0157] 1) Taking Fibroblasts from the DHJ

[0158] The fibroblasts of the dermo-hypodermic junction (F-DHJ) were isolated from non-defatted human skin. These samples are collected after breast reduction for esthetic reasons. The F-DHJs are isolated from the connective trabeculae present at the dermo-hypodermic junction. The latter are taken using tweezers and scissors. (see FIG. 1)

[0159] After comminution, the fragments of dermis are digested under the action of type II collagenase at 0.2% (Gibco) at 37° C.

[0160] The cells are then amplified in MEM medium—10% fetal calf serum supplemented with glutamine, sodium pyruvate, non-essential amino acids, penicillin, streptomycin and fungizone, under a moist atmosphere, at 37° C. and 5% CO.sub.2.

[0161] 2) Method for Identifying Fibroblasts of the DHJ (Verification of the Cellular Phenotype by RT-qPCR)

[0162] After amplification of the fibroblasts (between 7 and 10 population doublings), the mRNAs are extracted on QlAgen column according to the instructions given by the supplier.

[0163] The probes considered to be differentially expressed had to have a fold change of >2 for a p-value<0.05.

[0164] The molecular signature of the cells is verified according to the method below. Thus, the F-DHJs will exhibit relative levels of expression of ACAN, Col11a1, FGF9, UCP2 and KLF9 consistent with those summarized in the tables below.

TABLE-US-00001 TABLE 1 Fp vs FDHJ (change in number of times) RNA Conclusion ACAN −5.38 Upregulated in FDHJs COL11A1 −24.43 FGF9 −3.50 UCP2 4.36 Downregulated in FDHJs Fr vs FDHJ (change in number of times) RNA Conclusion KLF9 −2.06 Upregulated in FDHJs Fp: papillary fibroblast, Fr: reticular fibroblast, FDHJ: fibroblast of the dermo-hypodermic junction

TABLE-US-00002 UCP2 COL11A1 ACAN FGF9 KLF9 FDHJ Negative = Positive = Positive = Positive = Positive = fibroblast Decreased Increased Increased Increased Increased
Table of primers (QIAgen—quantitech primer assay) used for RT-qPCR validations

TABLE-US-00003 Name Ref ACAN QT00001365 Col XI QT00088711 FGF9 QT00000091 GAPDH QT01192646 UCP2 QT00014140

[0165] 3) Process for Preparing a Cell Model According to the Invention

[0166] a. 2D Cell Model

[0167] The fibroblasts of the dermo-hypodermic junction are seeded in a Petri dish at 1400 cells per cm.sup.2 in a medium of MEM culture medium—10% fetal calf serum supplemented with glutamine, sodium pyruvate, non-essential amino acids, penicillin, streptomycin and fungizone, under a moist atmosphere, at 37° C. and 5% CO.sub.2. The fibroblasts of the dermo-hypodermic junction are cultured for 48 hours after reaching confluence.

[0168] At the end of this process, a cell model according to the invention is thus obtained.

[0169] b. Cell Model in Spheroid Form

[0170] The fibroblasts of the dermo-hypodermic junction are seeded in a Petri dish at 1400 cells per cm.sup.2 in a medium of MEM culture medium—10% fetal calf serum supplemented with glutamine, sodium pyruvate, non-essential amino acids, penicillin, streptomycin and fungizone, under a moist atmosphere, at 37° C. and 5% CO.sub.2. The fibroblasts of the dermo-hypodermic junction are cultured for 48 hours after reaching confluence.

[0171] 48 hours after reaching confluence, spheroids are formed after centrifugation of 100 000 DHJ cells.

[0172] At the end of this process, a cell model in spheroid form according to the invention is thus obtained.

[0173] 4) Validation of the Use of the Cell Model According to the Invention as Tool for Screening Active Agents:

[0174] a. That Promote the Differentiation of DHJ Fibroblasts into Adipocyte

[0175] In order to validate the cell model produced in paragraph 3) a. for use as a tool for screening active agents that promote differentiation of F-DHJs into adipocytes, a positive control is used that consists of a mixture of active agents comprising indometacin, IBMX and dexamethasone, known for their pro-differentiating activities of fibroblasts into adipocytes (Peiffer et al., Leukemia 2007 April; 21 (4): 714-24).

[0176] The culture medium in point 3) a. is therefore substituted by a cocktail composed of: 60 μM indometacin/0.5 mM IBMX/10.sup.−6 M dexamethasone diluted in DMEM/20% fetal calf serum. The cells are kept in culture for 3 weeks in the presence of the differentiation induction cocktail. The media are renewed 3 times a week.

[0177] At the end of the 3 weeks of induction, the cell culture is stopped by fixation in paraformaldehyde at 4%. The detection of cells oriented towards an adipocyte differentiation profile is carried out under a microscope. Highly refractive spheres are observable inside the cells. Oil Red O staining, known to stain lipid droplets present inside the adipocytes red, can also be performed. (see FIG. 2)

[0178] Conclusion: differentiation of DHJ fibroblasts into adipocytes is observed; thus, this model can be used as a tool for screening active agents that promote the differentiation of DHJ fibroblasts into adipocytes.

[0179] b. That Promote the Differentiation of DHJ Fibroblasts into Osteoblasts

[0180] In order to validate the cell model produced in paragraph 3) a. for use as a tool for screening active agents that promote differentiation of F-DHJs into osteoblasts, a positive control is used that consists of a mixture of active agents comprising 2β-glycerophosphate, ascorbate 2-phosphate and dexamethasone, known for their pro-differentiating activities of fibroblasts into osteoblasts (Peiffer et al., Leukemia 2007 April; 21 (4): 714-24).

[0181] The culture medium in point 3) a. is therefore substituted by a cocktail composed of: 2 mM 2β-glycerophosphate, 0.15 mM ascorbate 2-phosphate, 10.sup.−7M dexamethazone and 10% fetal calf serum. The cells are kept in culture for 3 weeks in the presence of the differentiation induction cocktail. The media are renewed 3 times a week.

[0182] At the end of the 3 weeks of induction, the cell culture is stopped by fixation in paraformaldehyde at 4%. The detection of cells oriented towards an osteoblast differentiation profile is carried out by means of alizarin staining. This product stains calcified extracellular matrices red. (see FIG. 3)

[0183] Conclusion: differentiation of DHJ fibroblasts into osteoblasts is observed; thus, this model can be used as a tool for screening active agents that promote the differentiation of DHJ fibroblasts into osteoblasts.

[0184] c. That Promote the Differentiation of DHJ Fibroblasts into Chondroblasts

[0185] In order to validate the cell model produced in paragraph 3) b. for use as a tool for screening active agents that promote differentiation of F-DHJs into chondroblasts, a positive control is used that consists of a mixture of active agents comprising insulin, transferrin, sodium selenite, linoleic acid, oleic acid, bovine serum albumin, sodium pyruvate, ascorbate 2-phosphate, and dexamethazone, known for their pro-differentiating activities of fibroblasts into chondroblasts (Mesenchymal and hematopoietic stem cells form a unique bone marrow niche, Mendez-Ferrer S, Michurina T V, Ferraro F, Mazloom A R, Macarthur B D, Lira S A, Scadden D T, Ma'ayan A, Enikolopov G N, Frenette P S, Nature. 2010 Aug. 12; 466(7308):829-34. doi: 10.1038/nature09262).

[0186] 24 hours after forming the spheroids, the culture medium from point 3) b. is thus replaced by an induction cocktail composed of 0.5 μg/ml of insulin, 0.5 μg/ml of transferrin, 0.5 ng/ml of sodium selenite, 6.25 μg/ml of linoleic acid, 6.25 μg/ml of oleic acid, 1.25 mg/ml of bovine serum albumin, 1 mmol/l of sodium pyruvate, 0.17 mmol/l of ascorbate 2-phosphate, 0.1 μmol/l of dexamethazone, 0.35 mmol/l of proline and 0.01 μg/ml of TGF-β. The medium is renewed 3 times a week for 2 weeks.

[0187] The cultures are stopped by freezing the spheroids after inclusion in OCT. The spheroids are then cut with a microtome at 5 μm. The differentiation of F-DHJ into chondroblasts is demonstrated by staining with toluidine blue supplemented by staining with safranin O (see FIG. 4).

[0188] Conclusion: differentiation of DHJ fibroblasts into chondroblasts is observed; thus, this model can be used as a tool for screening active agents that promote the differentiation of DHJ fibroblasts into chondroblasts.

EXAMPLE 2: PROCESS FOR PREPARING A TISSUE MODEL ON COLLAGEN SPONGE FROM DHJ FIBROBLASTS AND VALIDATION OF ITS USE AS A TOOL FOR SCREENING ACTIVE AGENTS THAT PROMOTE THE DIFFERENTIATION OF DHJ FIBROBLASTS INTO ADIPOCYTE CELLS

[0189] 1) Taking Fibroblasts from the DHJ (According to Example 1)

[0190] 2) Method for Identifying Fibroblasts of the DHJ (Verification of the Cellular Phenotype by RT-qPCR) (According to Example 1)

[0191] 3) Process for Preparing a Tissue Model According to the Invention

[0192] The cells of the dermo-hypodermic junction are seeded at 250 000 cells per sponge (Symatese Biomaterials) in the MEM culture medium—10% fetal calf serum supplemented with glutamine, sodium pyruvate, non-essential amino acids, penicillin, streptomycin and fungizone, in a moist atmosphere, at 37° C. and 5% CO.sub.2. The cultures are maintained for 14 days.

[0193] At the end of this process, a tissue model according to the invention is thus obtained.

[0194] 4) Validation of the Use of the Tissue Model According to the Invention as Tool for Screening Active Agents:

[0195] In order to validate the tissue model produced in paragraph 3) for use as a tool for screening active agents that promote differentiation of F-DHJs into adipocytes, a positive control is used that consists of a mixture of active agents comprising indometacin, IBMX and dexamethasone, known for their pro-differentiating activities of fibroblasts into adipocytes (Peiffer et al., Leukemia 2007 April; 21 (4): 714-24).

[0196] The culture medium in point 3) is therefore substituted by a cocktail composed of: 60 μM indometacin/0.5 mM IBMX/10.sup.−6 M dexamethasone diluted in DMEM/20% fetal calf serum. The induction of differentiation is maintained for 3 weeks. The media are renewed 3 times a week.

[0197] At the end of the 3 weeks of induction, the cell culture is stopped by fixation in paraformaldehyde at 4%. The detection of cells oriented towards an adipocyte differentiation profile is carried out under a microscope after staining with Oil Red 0, known to stain lipid droplets present inside the adipocytes red. (see FIG. 5)

[0198] Conclusion: differentiation of DHJ fibroblasts into adipocytes is observed; thus, this model can be used as a tool for screening active agents that promote the differentiation of DHJ fibroblasts into adipocytes.

EXAMPLE 3: COMPARATIVE, OUTSIDE THE INVENTION: TISSUE MODEL ON COLLAGEN LATTICE OBTAINED FROM DHJ FIBROBLASTS

[0199] The lattices were prepared following the previously published protocol (Asselineau et al.—Exp Cell res, 1985). 10.sup.6 F-DHJ fibroblasts were included in a bovine type I collagen solution (Symatése Biomateriaux).

[0200] In order to study the sensitivity of the tissue model on lattice (outside of the invention) in the context of its implementation as a tool for screening active agents that promote the differentiation of DHJ fibroblasts into adipocytes, use is made, as for examples 1), 4) a. and 2), 4), of a positive control consisting of a mixture of indometacin, IBMX and dexamethasone, known for their pro-differentiating activities of fibroblasts into adipocytes, (Peiffer et al., Leukemia 2007 April; 21(4): 714-24).

[0201] Thus, after 4 days of organization and contraction of the lattice, the culture medium was substituted with an induction cocktail composed of: 60 μM indometacin/0.5 mM IBMX/10.sup.−6 M dexamethasone diluted in DMEM/20% fetal calf serum. The induction of differentiation is maintained for 3 weeks. The media are renewed 3 times a week.

[0202] At the end of the 3 weeks of induction, the cell culture is stopped by freezing the lattices after inclusion in OCT. The lattices are then cut with a microtome at 5 μm. The differentiation of the F-DHJs into adipocytes is demonstrated using Oil Red O staining. In this induction condition, the differentiation of the F-DHJs into adipocytes does not occur. It was not possible to demonstrate a staining of the adipocyte cells.

[0203] Conclusion: no differentiation of DHJ fibroblasts into adipocytes is observed; thus, this model is not sufficiently sensitive to be able to be used as a tool for screening active agents that promote the differentiation of DHJ fibroblasts into adipocytes.

EXAMPLE 4: COMPARATIVE, OUTSIDE THE INVENTION: CELL MODELS OBTAINED FROM PAPILLARY OR RETICULAR FIBROBLASTS

[0204] 1) Process for Preparing Cell Models Outside the Invention

[0205] The process carried out is identical to that described above in example 1, point 3), in which the DHJ fibroblasts have been replaced either by papillary fibroblasts or by reticular fibroblasts.

[0206] At the end of this process this thus gives a cell model outside the invention obtained from papillary fibroblasts and a cell model outside the invention obtained from reticular fibroblasts.

[0207] 2) Study of the Use of Cell Models Outside the Invention as Tool for Screening Active Agents:

[0208] a. That Promote the Differentiation of Papillary or Reticular Fibroblasts into Adipocyte

[0209] The process carried out is identical to that described in example 1, point 4) a.

[0210] The detection of cells oriented towards an adipocyte differentiation profile is carried out under a microscope. Small refractive spheres signifying the presence of lipid vacuoles are observed inside a small number of cells in the cultures established from papillary and reticular fibroblasts (see FIG. 6). Counter-staining with Oil red O validates the fact that these vacuoles are lipid in nature.

[0211] Conclusion: a very small amount of differentiation of the papillary or reticular fibroblasts into adipocytes is observed; thus, the cell models obtained from papillary or reticular fibroblasts are not sufficiently sensitive to be able to be used as a tool for screening active agents that promote the differentiation of these fibroblasts into adipocytes.

[0212] b. That Promote the Differentiation of Papillary or Reticular Fibroblasts into Osteoblasts

[0213] The process carried out is identical to that described in example 1, point 4) b.

[0214] The detection of cells oriented towards an osteoblast differentiation profile is carried out by means of alizarin staining. This product stains calcified extracellular matrices red. No red staining is observed in the case of the cell model obtained from papillary fibroblasts, and therefore there is an absence of calcified extracellular matrix. In the case of the cell model obtained from reticular fibroblasts, very small traces of red staining indicating that the extracellular matrix is calcified are observed (see FIG. 7). Thus, the papillary and reticular fibroblasts have a very weak propensity to differentiate into osteoblasts.

[0215] Conclusion: a very small amount of differentiation of the papillary or reticular fibroblasts into osteoblasts is observed; thus, the cell models obtained from papillary or reticular fibroblasts are not sufficiently sensitive to be able to be used as a tool for screening active agents that promote the differentiation of these fibroblasts into osteoblasts.

[0216] c. That Promote the Differentiation of Papillary or Reticular Fibroblasts into chondroblasts

[0217] The process carried out is identical to that described in example 1, point 4) c.

[0218] The differentiation of the papillary or reticular fibroblasts into chondroblasts is demonstrated using staining with toluidine blue, supplemented with staining with safranin O. Pale blue and pale red staining is observed in the case of the cell models obtained from papillary and reticular fibroblasts, while dark blue staining is observed at the periphery, and also dark red staining in the case of the cell model obtained from fibroblasts of the DHJ. (see FIG. 8)

[0219] Conclusion: a very small amount of differentiation of the papillary or reticular fibroblasts into chondroblasts is observed; thus, the cell models obtained from papillary or reticular fibroblasts are not sufficiently sensitive to be able to be used as a tool for screening active agents that promote the differentiation of these fibroblasts into chondroblasts.

The table below summarizes the differentiation potential of the sub-populations of fibroblasts tested (Fp, Fr or F-DHJ) in adipocyte, osteoblast and chondroblast cells.

TABLE-US-00004 TABLE 2 Fp Fr F-DHJ Chondrocyte + +/− ++ Adipocyte + +/− +++ Osteoblast − +/− +++