Replicative senescence of neural progenitors induces astrocyte senescence in 2D cultures and human midbrain organoids

Abstract

Astrocyte senescence (astrosenescence) has emerged as a potential mechanism through which ageing may progressively impair glial homeostatic neuronal support and render the brain more vulnerable to neurodegenerative diseases such as Parkinsons disease (PD). 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 a healthy donor and LRRK2-G2019S PD patients to establish an in vitro midbrain ageing model. Extended passaging induced senescence-associated changes in NESCs, including reduced proliferative capacity, decreased cleaved caspase 3 (CC3) and telomerase mRNA expression, and DNA damage response (DDR) activation, while preserving differentiation potential. Astrocytes derived from aged progenitors showed an increase in SA-{beta}-gal activity, loss of Lamin B1, DDR activation, and an altered mitochondrial morphology. In human midbrain organoids (hMOs), passage-induced ageing was associated with astrocyte-specific DDR activation and lipidomic remodelling. A multi-modal integrative analysis further revealed that, despite the variable penetrance of individual senescence-associated markers across lines, passage condition constitutes the primary discriminant of the collective cellular state, supporting the view that replicative senescence as a biological program is detectable only when individual readouts are aggregated across modalities. Together, this work establishes a human iPSC-based platform 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.