Human iPSC-derived exosomes for amelioration of Huntington's disease through mitochondrial, synaptic, and anti-apoptotic mechanisms.

Huntington's disease (HD) is driven by expanded CAG repeats in the huntingtin gene, resulting in mutant huntingtin (mHtt) aggregation, mitochondrial dysfunction, neuronal loss, and neuroinflammation. Although stem cell-based therapies provide potent regenerative, anti-inflammatory, and neuroprotective effects, their clinical translation remains constrained by tumorigenicity and poor engraftment efficiency. Therefore, we investigated whether exosomes derived from human induced pluripotent stem cells (iPSC-exo) could serve as a safe, cell-free therapeutic approach by leveraging their paracrine mechanisms. iPSC-exo were isolated from iPSC-conditioned medium and characterized by nanoparticle tracking analysis, Western blotting, and ExoView assays, confirming exosomal size (∼100 nm) and expression of canonical markers (CD63, CD81, ALIX, HSP70/90). Treatment of HD neural stem cells with iPSC-exo significantly reduced mHtt aggregation, as shown by EM48 immunostaining and Western blot analysis. iPSC-exo also restored mitochondrial membrane potential, improved mitochondrial morphology, and upregulated CREB while suppressing c-Jun expression, indicating enhanced mitochondrial and stress resilience. Furthermore, iPSC-exo promoted neurite outgrowth and synaptic maturation, reflected by increased MAP2 and PSD95 levels and reduced Tuj1 expression, signifying neuronal progression toward a mature phenotype. Western blot analysis revealed downregulation of BAX, cleaved caspase-3, NF-κB, and JNK, alongside upregulation of BCL-2 and TrkB, demonstrating suppression of apoptosis and inflammation with concurrent activation of survival pathways. These multifaceted effects collectively alleviated mHtt aggregation, as confirmed by EM48 immunostaining and Western blot analysis. Together, our findings demonstrate that iPSC-exo mitigate HD pathology by improving mitochondrial function, neuronal differentiation, and anti-apoptotic signaling, thereby reducing mHtt accumulation.