Use of germ cells for preparing a micro hair follicle

Abstract

The invention relates to the use of germ cells for obtaining a micro hair follicle and to the use thereof for evaluating the effect of cosmetic, pharmaceutical or dermatological products and also for the prophylactic or therapeutic treatment of a state of reduced pilosity.

Claims

1. An in vitro process for preparing a micro hair follicle, comprising amplifying germ cells isolated from the bulge of a hair follicle in the telogen phase to obtain keratinocytes positive for CD200, CD29 and K15 markers in the presence of a ROCK inhibitor and then at least one step of culturing the keratinocytes positive for CD200, CD29 and K15 markers for a period of time sufficient to allow differentiation into a tubular structure of said keratinocytes positive for CD200, CD29 and K15 markers being brought into contact with the connective tissue sheath fibroblasts and/or dermal papilla fibroblasts.

2. The process according to claim 1, wherein the micro hair follicle is capable of renewing.

3. The process according to claim 1, in which the ROCK inhibitor is Y27632.

4. The process according to claim 1, in which an effective amount of the ROCK inhibitor is from 1 to 100 ?M.

5. The process according to claim 1, in which an effective amount of the ROCK inhibitor is from 5 to 25 ?M.

6. The process according to claim 1, in which the an effective amount of the ROCK inhibitor is 10 ?M.

7. The process according to claim 1, in which the germ cells are amplified in the presence of the ROCK inhibitor for at least 2 days.

8. The process according to claim 1, in which the germ cells are cultured in the presence of the ROCK inhibitor for at least 3 days.

9. The process according to claim 1, which comprises the following steps: a) isolating a hair follicle in the telogen phase from a scalp sample; b) separating the germ cells from the connective tissue sheath of the hair follicle; c) depositing the germ cells on a feeder support 3T3 fibroblasts that has been treated with mitomycin, in a culture medium; d) amplifying the germ cells into to obtain keratinocytes positive for CD200, CD29 and K15 markers at the surface of said support in the presence of a ROCK inhibitor; e) recovering the keratinocytes positives for the CD200, CD29 and K15 markers; and f) culturing the keratinocytes obtained in step e) in a 3D culture with connective tissue sheath fibroblasts and/or dermal papilla fibroblasts to allow their differentiation into the tubular structure.

10. The process according to claim 9, in which the said keratinocytes positives for the CD200, CD29 and K15 markers are recovered when they reach confluence while forming clusters.

11. The process according to claim 9, wherein in the step f), the number of keratinocytes placed in culture is between 1000 and 4000 cells per cm.sup.2.

12. The process according to claim 9, which comprises isolating the hair follicle in the telogen phase from the scalp sample by microdissection and further comprises between steps b) and c) separating the germ cells from the connective tissue and dermal part of the dermal papilla and culturing only the germ cells.

Description

DESCRIPTION OF THE FIGURES

(1) The figures make it possible to give a better illustration of the invention, without however limiting the scope thereof.

(2) FIG. 1: Germ cell localization

(3) FIG. 2: Separation of the connective tissue sheath cells and of the germ cells with a needle

(4) FIG. 3: Isolation of a telogen follicle containing the germ cells

(5) FIG. 4: Telogen follicle deposited on a feeder layer of 3T3 and migration of the first germ cells around the telogen follicle

(6) FIG. 5: Germ cells at confluence after 10 days of culture

(7) FIG. 6: Characterization of the cells at confluence, K15+, CD29+ and CD200+ keratinocytes

(8) FIG. 7: Proliferation of the germ cells with Rock inhibitor (FIG. 7a) and without Rock inhibitor (FIG. 7b)

(9) FIG. 8: Formation of the tubular structure responsible for the hair follicle after 3, 7 and 10 days of culture.

(10) The examples given below are presented as non-limiting illustrations of the invention.

EXAMPLE 1PREPARATION OF GERM CELLS

Experimental Protocol

(11) The germ cell region, called the bulge (FIG. 1), is extracted from the connective tissue sheath (FIG. 2) and then placed in a petri dish containing 3T3i cells (FIG. 3).

(12) i. Germ Cell Microdissection

(13) The hair follicles are extracted from a surgical residue of scalp. Said residue is first cut into 5 mm.sup.2 portions and then sectioned using a scalpel between the dermis and the hypodermis.

(14) The follicles are extracted using ophthalmic surgery forceps and are then sectioned just above the papilla with a scalpel. The bulb is then recovered. At this stage, the bulb comprises two compartments: the dermal compartment (dermal papilla and connective tissue sheath) and the matrix cells which form a cell mass.

(15) The epithelial part is separated from the dermal part using perfusion needles. Only the epithelial part is cultured.

(16) ii. Culture Conditions:

(17) The culture conditions have three main components:

(18) The Base Medium:

(19) Unless otherwise indicated, all of the media and buffers used in the examples are described in Bell et al. 1979, (P.N.A.S. USA, 76, 1274-1278), Asselineau and Prunieras, 1984, (British J. of Derm., 111, 219-222) or Asselineau et al., 1987, (Models in dermato., vol. III, Ed. Lowe & Maibach, 1-7).

(20) The MEM medium+10% FCS+7F (called Green 7F medium) has the following composition:

(21) TABLE-US-00001 MEM Final concentrations Foetal calf serum (FCS) 10% L-Glutamine 2 mM Sodium pyruvate 1 mM Penicillin - Streptomycin Penicillin 20 U/ml Streptomycin 20 ?g/ml Fungizone Penicillin 10 U/ml Streptomycin 10 ?g/ml Amphotericin-B 25 ng/ml Epidermal growth factor (EGF) 10 ng/ml Cholera toxin 10.sup.?10 M Hydrocortisone 0.4 ?g/ml Adenine hydrochloride 1.8 ? 10.sup.?4 M Triiodothyonine (T3) 2 ? 10.sup.?9 M Human transferrin 5 ?g/ml Bovine insulin 5 ?g/ml the culture supplements: growth factors and for instance 10 ?M of Y27632. the adhesion surface: the germ cells proliferate in the Green-based medium in the presence of a feeder layer of murine 3T3 fibroblasts arrested in the cell cycle by mitomycin treatment.
Culture-Amplification of Keratinocytes

(22) After microdissection, the hair follicles in the telogen phase are deposited in Petri dishes 60 mm in diameter, seeded beforehand with 1 million 3T3 cells, and covered with the complete Green 7F culture medium; a culture of germ cells at confluence is obtained.

(23) In order to generate the spheres, the cells are recovered at the subconfluent stage by enzymatic treatment. The spheres can be obtained in various ways with the conventional techniques already described (hanging drop, centrifugation, non-adhesive support). In the case of the hanging drop, 3000 cells are placed in the plates in spheno. They are recovered in 96-well plates after 48 h in order to monitor their progression.

(24) The telogen hair with the non-dissociated germ cell clump are isolated and deposited on a 3T3 feeder layer (FIG. 3). After 3 d of culture, the cells begin to proliferate in the form of a colony around the germ clump (FIG. 4). The cells reach subconfluence in 7 days (FIG. 5). They are characterized by a very small size and a strong proliferative capacity. In order to ensure amplification, the cells are then seeded into a T75 flask. Thus, after 3 weeks of culture of 3 bulbs, it is possible to generate approximately 40 million germ cells at passage 1.

(25) It was possible to observe that the germ cells are capable of very rapidly reaching confluence in the green 7F medium in the presence of the Rock Y27632 inhibitor at a concentration of 10 ?M (FIG. 7a)), whereas, in the same medium without addition of the rock inhibitor, the cells do not proliferate (FIG. 7b)).

(26) Thus, the germ cells cultured in 7F medium containing the rock inhibitor make it possible to obtain keratinocytes that are positive for the K15, CD29 and CD200 markers (FIG. 6) in sufficient amounts. Moreover, these cells are small in size, having a high nuclear/cytoplasmic ratio and a homogeneous phenotype.

(27) The cells which are obtained in a sufficient amount and which have the K15, CD29 and CD200 markers, homogeneously and reproducibly, are then placed in 3D culture in the presence of the fibroblasts from the connective tissue sheath and/or from the dermal papilla. These cells form spheres after 3 d of culture.

(28) After some 3 additional days of culture, these spheres change conformation, showing a polarized organization. A budding appears, which seems to indicate differentiation of the spheres into a tubular structure responsible for the formation of the hair follicle.