Replicative senescence in neural progenitors induces senescence-associated markers in midbrain organoids

Aging is the primary risk factor for Parkinson’s disease (PD), and astrocyte senescence, or astrosenescence, has emerged as a potential mechanism through which aging may progressively impair glial homeostatic neuronal support and render the brain more vulnerable to neurodegeneration. Yet human experimental models that specifically recapitulate astrosenescence within a PD-relevant genetic background are lacking. Here, we applied passage-induced replicative exhaustion to human iPSC-derived neuroepithelial stem cells (NESCs) from healthy donors and LRRK2 G2019S PD patients to establish an in vitro midbrain aging model. Extended passaging induced senescence-associated changes in NESCs, including reduced proliferative capacity, apoptosis resistance, reduction in telomerase activity, and sustained DNA damage response (DDR) activation, while preserving ventral midbrain identity and differentiation potential. Astrocytes derived from aged progenitors recapitulated multiple senescence hallmarks, including increase in SA-β-gal activity, nuclear lamina disruption, persistent DDR activation, and alteration in mitochondrial homeostasis. In human midbrain organoids (hMOs), passage-induced aging was associated with astrocyte-specific DDR activation, widespread lipidomic remodeling, and a selective reduction of dopaminergic neuron content in LRRK2 G2019S organoids compared to healthy donor-derived organoids. Multi-modal analysis of NESCs, astrocytes, and hMOs further revealed that the LRRK2 G2019S genetic background not only shifts baseline cellular states toward an aged-like profile but actively reshapes aging trajectories in a cell-type- and feature-dependent manner. Together, this work establishes a human iPSC-based midbrain model recapitulating key features of cellular senescence in both healthy and LRRK2 G2019S contexts and provides an in vitro platform to investigate the contribution of astrosenescence to PD-related neurodegeneration.