HEP14-activated PKC-ERK1/2 pathway boosts HEP14-empowered hADSCs for ovarian regeneration and functional restoration

Ovarian insufficiency (OI), including both premature ovarian insufficiency (POI) and age-related natural-aging ovarian insufficiency (ARNA-OI), significantly affects women’s health and remains persistent challenge in clinical treatment. In this study, bulk and scRNA-sequencing analysis indicated that HEP14 alters gene expression patterns in hADSCs and promotes the survival, antifibrotic, and proangiogenic capabilities, as well as the transdifferentiation potential of hADSCs. The key factors involved in these processes were upregulated by the HEP14-activated PKC-ERK1/2 signaling pathway. In both doxorubicin-induced POI and age-related natural-aged ARNA-OI mouse models, the combined administration of h-hADSCs and HEP14 encapsulated in PLGA microspheres (HEP14/PLGA) substantially enhanced ovarian regeneration and restoration of gonadal hormone levels through autocrine, paracrine, or endocrine mechanisms. Importantly, this combined therapy markedly improved the fertility of POI mice. These results demonstrate that the therapeutic potential of this combined therapy for POI and ARNA-OI, opening new avenues for clinical research and treatment strategies. The isolation of hADSCs from human subcutaneous adipose tissue was carried out using the previously described method with modifications. hADSCs were cultured in DMEM/F-12 medium (Gibco) supplemented 10% fetal bovine serum (Gibco), 1%Glumax (Gibco), 1%NEAA(Gibco) and 1% penicillin/ streptomycin (Gibco) at 37 °C in a humidified 5% CO2 incubator. Primary hADSCs treated with HEP14 (h-ADSCs) or vehicle (c-hADSCs) for 48h at passage 4 were used as samples for performing scRNA-Sequencing.