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

HIV-associated neurocognitive disorders (HAND) can persist despite effective ART and are associated with persistent central nervous system (CNS) viral reservoirs in microglia, chronic neuroinflammation, synaptic dysfunction, and neuronal injury. Thus, human-relevant multicellular CNS models are needed to elucidate neuroHIV pathogenesis and ART neurotoxicity. Here, we establish and characterize a fully human induced pluripotent stem cell (hiPSC)-derived CNS tri-culture model 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 infection of IBA1+ microglia with no detectable infection of IBA1− cells or TUBB3+ neurons. 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 concentrations based on reported plasma Cmax values. HIV infection reduced microglial viability by approximately 70%, partially rescued by lower in vitro ART concentrations but worsened by the supratherapeutic 3× Cmax condition of DTG-containing regimens. 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 under viral suppression, identifying an HIV–DTG-associated electrophysiological phenotype. Overall, BIC/TAF/FTC showed the most favorable profile across the cellular and electrophysiological endpoints assessed in this donor line. Together, these findings establish an hiPSC-derived CNS tri-culture model for controlled studies of neuroHIV virology, ART effects, and neuronal function.