Generation of 3D Neurospheres for Modeling HIV-1 Infection and Stem Cell EV Analysis

Abstract

Human Immunodeficiency Virus Type 1 (HIV-1) is a single stranded RNA retrovirus that affects 36.9 million people globally. Patients infected with HIV-1 may develop neurological symptoms as a result of central nervous system (CNS) cell damage. To better understand viral impact on the brain, we adapted an in vitro model of the CNS, 3D neurospheres, composed of multiple cell types. Here, we wanted to demonstrate HIV-1 infection of neurospheres, as well as explore the potential of stem cell (iPSC and MSC) EVs to protect neurospheres from cellular damage. Therefore, we infected neurospheres with HIV-1, treated with cART, and probed for HIV-1 marker proteins pr55, p24, and nef. We also verified the presence of astrocytes, microglia, and neurons in the neurosphere after infection through western blotting for cellular markers. To further investigate the potential neuroprotective effects of EVs, we induced cellular damage through irradiation in the neurospheres and observed increased differentiation of astrocytes and microglia and adherence of cells after EV treatment. Lastly, we tested for MMP proteins important for the degradation of the extracellular matrix and found that MMP9 was present in iPSC EVs, suggesting a mechanism by which the EVs may penetrate the neurosphere and enter cells. Overall, we show that neurospheres may be a potential future platform for more comprehensive in vitro CNS HIV-1 research and that stem cell EVs may exert neuroprotective effects on damaged cells.