Women living with HIV give birth to ~1.5M infants each year, 80% of them exposed to ARVs in utero. Most ARVs can cross the placenta, but their safety has not been fully elucidated in the context of pregnancy. The Tsepamo study reported a signal for risk of neural tube defects in infants exposed to the InSTI dolutegravir (DTG) from conception. Many ARVs affect mitochondria, which could impact embryonic development. Our objective was to characterize and compare the effects of 14 ARV regimens on cultured human embryonic stem cells (hESCs), with respect to pluripotency, and cellular and mitochondrial health.
CA1S hESCs were cultured (n=5 independent experiments) in the presence of 0.1% DMSO or 1X Cmax of the following regimens: DTG, raltegravir (RAL), bictegravir (BIC), cobicistat-boosted elvitegravir (EVG/COBI), or efavirenz (EFV) on a TDF/FTC backbone; DTG, RAL, BIC, EVG/COBI, or rilpivirine (RPV) on a TAF/FTC backbone; DTG, RAL, or ritonavir-boosted darunavir (DRVr) on an ABC/3TC backbone; cabotegravir (CAB)/RPV. After 3 days, cells were assessed via flow cytometry using markers for mitochondrial mass, intermembrane potential, reactive oxygen species (ROS), cell viability, and apoptosis. Two markers of pluripotency, specifically SSEA-3 (lost early in differentiation) and TRA-1-60 (a later marker), were also assessed. Regimens were grouped according to base ARV and compared to DMSO control using Kruskal-Wallis with Dunn’s correction.
HESCs exposed to DTG or BIC had a 3-fold reduction in cell counts (p≤0.005) compared to controls. BIC exposure resulted in a 5-fold decrease in viability (p=0.026) and a 6-fold increase in apoptosis (p=0.01). In regards to pluripotency, exposure to regimens containing DTG or CAB resulted in a >80% loss of SSEA-3 expression compared to controls (p≤0.02). There were no significant differences between regimens with respect to mitochondrial mass, intermembrane potential, ROS, or loss of TRA-1-60 expression. No effects were detected for the backbones, RAL, EVG/COBI, EFV, RPV, or DRVr.
These data indicate that exposure to some ARV regimens at pharmacological concentrations, especially DTG or BIC, appear toxic to cultured hESCs. Our results further suggest that exposure to the InSTIs DTG and CAB can induce hESC differentiation. Given the increasing use of DTG and other InSTIs, it is imperative to investigate their long-term safety in the context of pregnancy and embryonic development.