Human iPSC-Derived Organoid Model to Study the Neuropathology of HIV-1

Human immunodeficiency virus (HIV) affects an estimated 38 million people worldwide with nearly 1.6 million new infections every year. Despite the availability of combined antiretroviral therapy (cART), a significant percentage of people with HIV will develop neurological deficits. The lack of suitable in vitro models that can accurately recapitulate the human brain has hindered the understanding of HIV neuropathology.

Human induced pluripotent stem cells (hiPSCs) offer a promising solution, as they can be differentiated in vitro into primary human neural cell types such as astrocytes, neurons, oligodendrocytes, and microglia. hiPSCs are typically derived by introducing a set of pluripotency-associated genes, or “reprogramming factors,” into adult fibroblasts, converting them into pluripotent stem cells. These cells can propagate indefinitely and differentiate into all cell types of the body, including neurons, heart, pancreatic, and liver cells, making them highly valuable in regenerative medicine for replacing lost or diseased/damaged cells.

Advancements in 3D culture methods have enabled hiPSCs to generate complex in vitro models of the human brain, which are crucial for studying HIV neuropathology. Human cerebral organoids (hCOs) mimic the organization and cellular connections of the brain in vivo, allowing researchers to study HIV-central nervous system (CNS) interactions and identify disease mechanisms that cause HIV-induced cognitive impairment.

Derivation of hiPSCs and Generation of 3D Cerebral Organoids to Model HIV-1 Infection

In a recent publication by Donadoni et al, the researchers generated and characterized several hiPSC lines from healthy human donor dermal fibroblasts, which were then used to form 3D microglia-containing hCOs. Experiments were then conducted to investigate if the hCOs can be a model for both HIV-1 infection and suppression of HIV-1 replication after cART treatment.

Lifeline® human primary adult dermal fibroblasts (HDFa) isolated from different donors were cultured in FibroLife Basal Medium supplemented with FibroLife S2 LifeFactors. The HDFa were reprogrammed to hiPSCs using a non-integrating, self-replicating RNA-based reprogramming vector that expresses Oct-3/4, Klf-4, Sox2, Glis1, and c-Myc transcription factors. The hiPSCs were subsequently differentiated into hCOs, which formed four fully differentiated cell types: astrocytes, neuronal cells, microglia, and oligodendrocytes, as confirmed by immunohistochemistry and RT-PCR analysis.

Once characterized, the hCOs were infected with HIV-1, both with and without cART regimens. Viral infection was studied using cellular, molecular/biochemical, and virological assays to:

1. Determine the susceptibility of hCOs to HIV-1 infection.
2. Assess if the infected hCOs can be used to evaluate the efficacy of cART regimens to suppress HIV-1 replication.

Donadoni et al found that the hCOs were highly susceptible to HIV-1 infection, as demonstrated by viral p24-ELISA in culture media, RT-qPCR and RNAscope analysis of viral RNA, and ddPCR analysis of proviral HIV-1 in genomic DNA samples. HIV-1 infection also increased the expression of cleaved caspase-3, indicating higher apoptosis compared to uninfected controls. Furthermore, when the infected hCOs were treated with a cART regimen, replication and gene expression of HIV-1 were suppressed as early as 7 days post-infection. HIV-1 infection increased the expression levels of CCR5 and CXCR4. However, cART treatment suppressed the increase in CXCR4 but not CCR5.

The authors do acknowledge a few limitations of the hCOs used in the study, including the lack of brain microvascular endothelial cells (BMEC), which are central to forming the blood-brain barrier, and the absence of a functional immune system. While these factors limit the ability of hCOs to fully replicate the complex interactions between viral infections, immune responses, and the brain, the hiPSC-derived hCO model offers a better mechanism to study HIV-1 infection compared to current model systems. The 3D hCO model has enormous potential for investigating the consequences of HIV infection on the CNS, to both advance our understanding of the neuropathogenesis associated with HIV infection in the brain and test novel therapeutic and curative approaches in a more physiologically relevant system.

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