Long‐Term Post‐Mitotic Culture of Human Fibroblasts Recapitulates and Accelerates Features of In Vivo Aging and Enables Dynamic Testing of Anti‐Aging Compounds

Biological aging drives cellular dysfunction and human disease, yet studying human aging dynamics remains challenging due to limited experimental platforms. Here we show that long‐term post‐mitotic culture of human fibroblasts recapitulates and accelerates features of in vivo aging. Longitudinal paired transcriptomic and epigenetic analyses of four female fibroblast lines revealed that in vitro aging reflects key signatures of in vivo primary fibroblast aging. Direct neuronal conversion of in vitro‐aged fibroblasts preserved biological age, enabling pseudo‐longitudinal modeling of neuronal aging through direct reprogramming. Single‐cell transcriptomics of in vitro‐aging fibroblasts revealed a time‐dependent increase in age‐heterogeneity, consistent with the previously reported age‐associated heterogeneity driven by the variable loss of transcriptional programs in vivo. Using this accelerated aging platform, we evaluated anti‐aging compounds: Metformin broadly attenuated transcriptomic and epigenetic aging, while Rapamycin showed limited efficacy. These findings align with pre‐clinical evidence, providing a platform to evaluate and predict therapeutic anti‐aging compound efficacy at molecular resolution. This system advances our understanding of aging mechanisms and facilitates the development of interventions against age‐related diseases.