From Spheroid to Sprouting: Microenvironmental Modulation Boosts Hair Follicle Organoid Morphogenesis in 3D Culture

Alopecia represents a global therapeutic challenge, with the challenge of achieving de novo folliculogenesis in alopecic regions rather than reactivating telogen-phase hair follicles to enhance hair density. The successful in vitro cultivation of hair follicle organoids (HFOs) and the effective enhancement of HFOs sprouting rates are critical advancements in addressing this problem. While R-spondin1 (RSPO1) demonstrates in vivo efficacy in activating quiescent follicular stem cells and enhancing hair density, its capacity to augment HFOs sprouting efficiency remains to be elucidated. In this study, we regulate the growth microenvironment of HFOs using RSPO1, enhancing the HFOs sprouting rate and exploring its mechanism. HFO-induced dermal papilla cells (DPCs) induction was quantified via alkaline phosphatase (ALP) assay, with RSPO1-mediated hair-regenerative gene/protein modulation assessed through triple-method analysis (qPCR/WB/IF). Sequencing delineated RSPO1-affected molecular pathways. Our study demonstrates that RSPO1 significantly promotes HFOs sprouting rate and upregulates the gene/protein expression of DPCs. Furthermore, RSPO1 not only potentiates Wnt/β-catenin-mediated organoid patterning but also coordinates a signaling triad involving PI3K-Akt, Rap1 and NF-κB to drive synchronized hair follicle sprouting and stratified pilogenic differentiation. Our 3D organoid-based expansion strategy enables definitive treatment of alopecia through in vitro hair follicle expansion, achieving functional hair neogenesis in patients with baldness.