A Human iPSC-Derived Tri-Culture Model of NeuroHIV: HIV Infection, Antiretroviral Drug Effects, Microglial Morphology, and Neuronal Calcium Dynamics

<jats:p>HIV-associated neurocognitive disorders (HAND) involve persistent central nervous system (CNS) viral reservoirs in microglia, driving chronic neuroinflammation, synaptic dysfunction, and neuronal injury. Thus, scalable, human-relevant multicellular CNS models are needed to elucidate neuroHIV pathogenesis and ART neurotoxicity. Here, we establish and characterize a fully human iPSC-derived (hiPSC) CNS tri-culture platform comprising isogenic neurons, astrocytes, and microglia that remains viable and functionally active for up to 32 days in vitro. Using a microglia-tropic fluorescent HIV reporter virus, GFP-HIV-AD8, we demonstrate selective infection of microglia with no detectable neuronal infection, consistent with human neuroHIV. HIV replication was quantified over 14 days post-infection and effectively suppressed by the first-generation integrase strand transfer inhibitor (INSTI)-containing regimen elvitegravir/tenofovir disoproxil fumarate/emtricitabine (EVG/TDF/FTC), and the second-generation INSTI-containing regimens dolutegravir/tenofovir disoproxil fumarate/emtricitabine (DTG/TDF/FTC) and bictegravir/tenofovir alafenamide/emtricitabine (BIC/TAF/FTC), at clinically achievable concentrations. HIV infection reduced microglial viability by approximately 70%, partially rescued by low-dose ART but worsened by high-dose DTG-containing regimens, suggesting concentration-dependent toxicity. In uninfected cultures, DTG induced microglial morphological changes consistent with a reactive or dysfunctional state. Calcium imaging revealed ART-specific neuronal effects: EVG/TDF/FTC reduced neuronal activity, whereas BIC/TAF/FTC enhanced it. In HIV-infected cultures, DTG shortened neuronal spike duration, suggesting HIV–ART interactions on synaptic physiology. Overall, second-generation INSTIs, particularly BIC/TAF/FTC, showed the most favorable CNS safety profile. Together, these findings establish a scalable iPSC-derived CNS tri-culture model for neuroHIV research, ART efficacy testing, and neurotoxicity assessment.</jats:p>