Brain Organoid‐on‐a‐Chip: Model System for Predicting cART induced Neurotoxicity and Neuropsychiatric Effects

Abstract

The current combination of antiretroviral therapy (cART) therapy comes with its benefits along with its side effect, e.g. neurotoxicity issues that lead to the progressive loss of neurons in HIV‐infected individuals and leads to neuropsychiatric adverse effects (NP‐AE)(e.g. depression, anxiety, and mood disorders, etc.) on a long‐term basis. Furthermore, due to the limitation of the resemblance of animal models to human biology (due to species differences) and unavailability of the robust humanized HIV‐1 infected mouse model. We have proposed a new strategy to generate human induced pluripotent stem cells (hiPSCs)‐derived brain organoids in a controlled manner by combining stem cell biology with lab‐on‐a‐chip technology, thereby creating a brain‐organoid‐on‐a‐chip system (Shown in Fig. 1). The innovative part of this project is the organoid vascularization by growing them directly on the microfluidic chip in Matrigel under the controlled milieu of growth factors/ nutrient molecules, thus, facilitating the angiogenesis like process. The organoids were generated as per published Lancaster protocol with minor lab modification and characterized for pluripotent markers (NANOG, OCT‐4, SOX2, KLF4, etc.) using RT‐PCR assay and immunofluorescence for the characterization of the key markers of human brain via the expression of neuronal progenitor’s (SOX2), a cortical proliferating neuronal (TUJ1), forebrain (FOXG1) and hippocampus (PROX1) neuronal lineage markers. Using the developed brain organoids, we have determined the effect of combination cART therapy on the increased risk of neurotoxicity using the CSF concentration of marketed cART regimen, i.e. EVF‐13.85 ng/ml, TDF‐5 ng/ml; FTC‐109 ng/ml; DOR‐35.4 ng/ml; 3TC‐46 ng/ml; EVG‐25 ng/ml; TAF‐20 ng/ml) up to day 7. Results showed that organoids treated with single component or cART combination, resulted in significant loss of dendrites, increased beading, shrinkage of neuropil and lower cell viability for day 7 > day 1 in cART treated samples compared to control samples. Similarly, we also have analyzed other marketed cART combination, they also showed loss of MAP‐2 staining (neuron marker for neurogenesis) for day‐7 treated samples. As the literature suggests, Efavirenz (non‐nucleoside reverse transcriptase inhibitors; NNRTIs) and Dolutegravir (Integrase Inhibitors) have shown more neuropsychic adverse effect in HIV patient’s early or later stages of the treatment. We also, checked the effect of EVF toxicity using two different concentration (i.e. 10 ng/ml and 30 ng/ml) dose up to 7 days and measured the cell number and neurite growth as a toxicity parameter. Our results showed that 30 ng/ml treated samples showed loss of neurite length in a very significant manner even on day 3 and continued until day 7. However, for lower concentration (10 ng/ml), day 7 showed significant loss in neurite length compared to day 1. For future studies, we will be studying the long term c ART toxicity and try to establish a correlation of the molecular markers and gene expression profiles (as mentioned in Fig. 2), as an indicator for cART‐associated neuropsychiatric adverse events (NP‐AEs).Support or Funding InformationSOP‐Office of Science Research Award and start‐up funds from SOP, TTUHSC, TX.Schematic diagram of organoid‐on‐a‐chip model to study cART toxicity leading to NP‐AE’sFigure 1Key factors (neurotransmitters or molecular biomarkers) associated with cART toxicity leading to NP‐AE’s to be studied using organoid‐on‐a‐chip modelFigure 2