DIRECT PATHWAY BIAS AND ALTERED STRIATAL NEUROGENESIS IN HUMAN IPSC MODELS OF 16P11.2 CNVS: EVIDENCE FROM SINGLE-CELL AND FUNCTIONAL ANALYSES

Medium spiny neurons (MSNs) regulate motor, cognitive, and social behaviours through direct (dMSN) and indirect (iMSN) basal ganglia pathways. While emerging genomic evidence links striatal circuit dysfunction to neurodevelopmental disorders (NDDs) and implicates MSNs in schizophrenia risk, mechanistic studies in human systems remain limited and have largely prioritised cortical excitatory and interneuron lineages.
We generated high-fidelity human iPSC-derived MSNs using an Activin A patterning strategy and benchmarked their developmental trajectories against human foetal and adult striatal single-cell atlases. We then modelled 16p11.2 copy number variants (CNVs) – duplications and deletions that confer risk for autism spectrum disorder, schizophrenia, intellectual disability, and related NDDs – using iPSCs from CNV carriers. Single-cell RNA sequencing across differentiation stages was integrated with cell-cycle and lineage marker assays, and MSN network function was assessed using calcium imaging and pharmacological modulation of direct-pathway signalling.
Both 16p11.2 duplications and deletions produced reciprocal shifts in neurogenesis kinetics and progenitor proliferation yet converged on a shared bias toward dMSN fate. This transcriptional reprogramming was evident across progenitors and MSN precursor states. CNV MSNs exhibited altered calcium dynamics and heightened responsiveness to direct-pathway stimulation, consistent with increased excitability within dMSN-enriched networks.
Our findings identify dMSN enrichment as a convergent cellular phenotype of opposing 16p11.2 CNVs, linking early developmental perturbations to functional changes in striatal circuitry. This work highlights MSNs as a key, underappreciated substrate for NDD pathogenesis and supports iPSC-derived striatal models as a platform for mechanism-led target discovery and therapeutic screening aimed at restoring pathway balance and excitability.