Vascularized human brain organoids as a model of the brain-peripheral axis in HIV-1 neuropathogenesis

Advances in stem cell biology and tissue engineering have enabled the generation of brain organoids from human induced pluripotent stem cells (hiPSCs), providing powerful platforms to model human brain development and neurodegenerative diseases. However, the lack of vascularization in conventional brain organoids limits nutrient and oxygen diffusion, thereby constraining their viability and physiological relevance. To overcome this limitation, we developed vascularized brain organoids using both normal hiPSCs and hiPSCs overexpressing the endothelial transcription factor ETS variant 2 (ETV2). Furthermore, this model also provides a unique opportunity to investigate how HIV-1 disrupts endothelial integrity within the CNS. Organoids were generated through co-culture differentiation using a modified protocol optimized to promote vascularization. Following characterization, organoids were exposed to HIV-1-infected PMPs in the presence or absence of ART. Viral replication was quantified using p24 ELISA and qRT-PCR assays. Our findings demonstrate the successful development of vascularized brain organoids (EndohBOs), which exhibit robust expression of CD31, Collagen IV, and GFAP footing, recapitulating key structural and cellular components of the human neurovascular unit. Notably, EndohBOs supported a higher number of transmigrated HIV-1-infected Primitive macrophage progenitor cells or Monocytes (PMPs) as evidenced by imaging. Interestingly, Antiretroviral therapy (ART) treatment failed to substantially reduce viral RNA or p24 levels, suggesting that despite ART exposure, viral replication persisted within EndohBOs. Overall, this vascularized brain organoid model provides a physiologically relevant human system to study the effects of HIV-1 and other neurotropic pathogens on the CNS. It also offers a promising platform for testing CNS-targeted therapeutic interventions aimed at restoring neurovascular integrity and mitigating HIV-induced barrier disruption.