Self-assembled complete hair follicle organoids by coculture of neonatal mouse epidermal cells and dermal cells in Matrigel.

Abstract

Background3D organoid cultures of hair follicles (HFs) are powerful models that mimic native HF for both in-depth study of HF disease and precision therapy. However, few studies have investigated the complete structure and properties of HF organoids. To investigate and characterize the complete HF organoids self-assembled by coculture of neonatal mouse epidermal cells (MECs) and dermal cells in Matrigel.MethodsFresh epidermal and dermal cells from newborn mice (n=4) were isolated, and cocultured (1:1 ratio) in Matrigel using DMEM/F12 medium for 1 week. During the culture, an inverted microscope was used to observe the morphology of the 3D constructs. After 1 week, hematoxylin-eosin (HE) and immunofluorescence (IF) staining of HF-related markers (K5, K73, AE13, and K10), HF stem cell markers (K15, CD34, CD49f), skin-derived precursor-related marker (Nestin), and dermal papillae (DP)-specific markers (SOX2 and ALP) was performed in the harvested constructs to identify the HF organoids.ResultsEpidermal and dermal cells self-assembled into HF organoids comprising an infundibular cyst-like structure, a lower segment-like structure, and a bulb-like structure from tail to root. The HF organoid had multiple, well-defined compartments similar to native anagen HF. Of the three segments, K73 was expressed in the inner root sheath-like layer, AE13 was localized in the hair shaft-like structure, K5, K15, CD34, and CD49f were present in the outer root sheath-like layer, Nestin labeled the connective tissue sheath-like layer, and SOX2 and ALP were expressed in the DP-like structure. Furthermore, K10 and K73 were expressed in the infundibular cyst-like structure. The expression of these molecular proteins was consistent with native anagen HF.ConclusionsThe complete HF organoid regenerated in Matrigel has specific compartments and is an excellent model to study HF disease and precision therapy.