Bakkar K, Le Balch M, Collin C, Sellathurai T, Thibaut S, Michelet J.F.

L’Oréal Research and Innovation, Advanced Research,

Aulnay-sous-Bois, France

Références1.T. Kageyama, C. Yoshimura, D. Myasnikova, K. Kataoka, T. Nittami, S. Maruo, J. Fukuda. Biomaterials, 2018, 154:291-300.2. M. Ohyama, O. Veraitch. J. Dermatol. Sciences, 2013, 70:78-873. G. Lindner, R. Horland , I. Wagner, B. Ataç, R. Lauster. J. Biotechnol., 2011, 152(3):108-1124. J. Lee, R. Böscke, P-C. Tang, B-H. Hartman, S. Heller, K-R. Koehler. Cell Report, 2018, 22:242-254.

Matrix cells (Mx) isolation and amplification

Figure 1. We developed a new method to isolate and to cultivate human hair matrix cells.

A. Extraction of matrix with a very delicate microdissection technique. Matrix cells are placed onto a feeder layer (irradiated human dermal fibroblasts) in a classical Green medium contai-ning 10 µM Rock inhibitor (Y-27632) for amplification.

B. Small hair matrix cells at passage 1 (P1).

C. Matrix cells form spontaneously clusters at P1.Figure 3.

A. The primary dermal papilla fibroblasts (DP) were trypsinized and seeded in a 6 well low attachment plate and cultured in William’s E medium at 37°C under 5% CO

2.

B. After 3-4 days, the characteristic cell clumping occurred.

C. At day 4 to 6, neo dermal papilla-like spheroids spontaneously self-formed and detached from the plate surface. These spheroids have a strong alkaline phosphatase activity (*) and a diameter close to a micro-dissected hair follicle dermal papilla (around 200 µm /**).

A COMBINATION OF HAIR MATRIX CELLS AND DERMAL PAPILLA SPHEROIDS SELF ORGANIZES TO FORM

HAIR FOLLICLE ORGANOID IN VITRO

INTRODUCTIONMammalian hair follicle morphogenesis results of highly complex and coordinated interactions between epithelial and mesenchymal cells, which when injected into mouse skin conduct to hair follicle neo-genesis.Recently, several hair follicle organoids were reported: - in vitro 3D hair follicle germ structures from mouse embryonic cells were obtained and resulted in de novo hair follicles growth when injected (1). - Preliminary results showed that in vitro combinations of human outer root sheath keratinocytes and dermal papilla fibroblasts led to long rod organoid structures (2,3). - More surprisingly, hair shaft growths were produced in vitro from pluripotent stem cells derived from mouse embryonic cells (2).The challenge to produce such organoids from human adult hair follicle progenitor cells remains to be take up in the goal to pave the way of regenerative medicine. We developed new methods to amplify human hair follicle matrix cells and to produce neo dermal papilla spheroids. An original combination of both drives to the growth of hair organoids with K85 positive cells.

Mx vs ORS proliferation status (Ki67 staining)Figure 2. ORS keratinocytes as well as matrix cells were grown in Green medium containing 10 µM Rock inhibitor (Y-27632) onto irradiated human dermal fibroblasts until passage 2 (P2). Cell nucleus were stained with Hoescht.

A. As described, the nuclear marker of proliferation Ki67 (green) is strongly expressed into the nucleus of matrix cells of the hair follicle (*) as well as into the P2 matrix cells in vitro as compared to the P2 ORS keratinocytes where the staining is weaker.

B. Neither the matrix cells nor the ORS keratinocytes expressed the K85 (kera-tin 85/green being located into the hair follicle in Ki67 negative matrix cells, precortex and cuticule *) according to the indefinite proliferative state effect induced conditionally by the ROCK inhi-bitor Y-27632. ORS and matrix cells are K85 negative.

C. The hair follicle ORS (*) strongly expressed the basal differentiation mar-ker Keratin 5 (K5) as compared to the weak expression in the matrix cell com-partment (**) . A strong K5 staining is observed in the P2 ORS keratinocytes in vitro as compared to the very weak expression into the undifferentiated P2 matrix cells.

Hair follicle neo-dermal papilla-like spheroid generation

Mx vs ORS pre-cortex/cuticule differentiation marker (K85 staining)

Mx vs ORS basal keratinocyte marker (K5 staining)

In vitro hair follicle organoid formation

Figure 4.

A. Sequential increases in length of hair follicle organoid were observed from day 2 to day 10. Formation of organoid after adding matrix cells to a neo-dermal papilla in a fully defined serum free medium (William’s E).

B. Mosaic K85 expression in the hair follicle organoid (green). Ki67 is detected in the matrix cells surrounding the dermal papilla.

C. Positive versican staining confirms the mesenchymal pole of the organoid sustaining the dynamic growth of matrix cells.

D. The laminin 511 expression (green) at the dermal papilla/matrix cells junction demonstrates the strong interaction between both compartments as described in the bulbar region.

CONCLUSION For the first time, we were able to isolate and to amplify in vitro human adult hair matrix cells. In addition, we set up a method to obtain self aggregated neo-dermal papilla spheroids in a fully defined culture medium. The combination of these human adult matrix cells with the neo-dermal papilla spheroids conducted to the morphogenesis of a hair follicle organoid. This structure is polarized as demonstrated with the Ki67 staining in the matrix cells located in the lower part of the follicle organoid and the proteoglycan versican in the mesenchymal part. In addition, the laminin 511 showed a strong interaction between both compartments. With K85 staining, all these results encourage us to propose this new organoid as a mimic model of the human bulbar region. Although further characterization is still required, it gives us new perspectives to assess the effect of actives to study hair growth aspects. This preliminary study opens new options to envision future applications in the regenerative medicine field.

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