HIV-1 infection of genetically engineered iPSC-derived central nervous system-engrafted microglia in a humanized mouse model

Abstract

The central nervous system (CNS) is a major human immunodeficiency virus type 1 reservoir. Microglia are the primary target cell of HIV-1 infection in the CNS. Current models have not allowed the precise molecular pathways of acute and chronic CNS microglial infection to be tested with in vivo genetic methods. Here, we describe a novel humanized mouse model utilizing human-induced pluripotent stem cell-derived microglia to xenograft into murine hosts. These mice are additionally engrafted with human peripheral blood mononuclear cells that served as a medium to establish a peripheral infection that then spread to the CNS microglia xenograft, modeling a trans-blood-brain barrier route of acute CNS HIV-1 infection with human target cells. The approach is compatible with iPSC genetic engineering, including inserting targeted transgenic reporter cassettes to track the xenografted human cells, enabling the testing of novel treatment and viral tracking strategies in a comparatively simple and cost-effective way vivo model for neuroHIV.

Keywords: HIV encephalitis; HIV-1; HIV-associated neurocognitive disorder; induced pluripotent stem cell; latent reservoir; microglia.

Figure 1:. Overview of in vitro experiments:

(A) (top) Timeline of iPSC-HPC-iMG differentiation with (bottom) corresponding, stage-specific morphological appearances of cell cultures at low power magnification (4x − 20x, as indicated) of (top) conditional reporter transgene insertion at chromosome 19 AAVS1 (Adeno-Associated Virus Integration Site 1) targeting locus. (bottom). (B,C) Schematic of (B) HIV-1 JFRL-Cre genome including Cre coding cassette followed by IRES internal ribosomal entry sequence to drive nef expression for conditional recombination of the dsRed-GFP reporter and (C) dsRed to eGFP switch in HIV-Cre infected cells. (D) Conditional transgene expression: with (top) HIV-1 JFRL-Cre infected and bottom (uninfected) MSE2104 iPSC-derived microglia (iMG). Notice robust ubiquitous dsRed and, in a subset of cells, GFP expression. Cells were harvested 48h post-infection with 20ng p24 HIV-1 JRFL-Cre virus. (E) Approximately 3% of GFP+ MSE2104 iMG with 20ng p24 HIV-1 JRFL Cre. (F) Recombination-sensitive 401bp PCR product extending into 5’ of GFP cassette, as indicated. Notice the dosage-sensitivity of recombination to virus levels (p24 range 0–20ng) in cell culture, as indicated.

Figure 2:. Humanized mouse model with central and peripheral xenograft.

(A) Timeline and overview, a central hiPSC-HPC xenograft into the neonatal brain is followed by peripheral hu-PMBC administration (i.p.) in young host animals, followed four weeks later by central or peripheral infection with HIV. (B) 4x magnification of whole hemisphere view of DAPI-counterstained (blue) coronal brain section from adult animal neonatally engrafted with dsRed+ hIPSC-HPC microglial progenitors. Notice large numbers of dsRed-expressing cells at particularly high densities in the medial temporal lobe, including the hippocampus. (C) 40x magnification from coronal sections at upper layers I and/or II of the adult cerebral cortex of neonatally engrafted animals confirms numerous dsRed-expressing human cells and myeloid markers Iba1 and P2RY12 purinergic receptors. DAPI, counterstain. Scale bars (A) 500, (B) 25 μm.

Figure 3:. Neonatally engrafted hIPSC-HPC microglia become HIV infected after peripheral infection.

(A) (y-axis) Quantitative PCR-based blood HIV RNA copy numbers per ml cheek blood, (x-axis) in individual mice no. 1–9 neonatally engrafted with hIPSC-HPC, including with (no. 1–5) and without (no. 6–9) additional huPMBC transplant as an adult. All Animals had received a single dose of HIV (250ng p24 antigen) i.p., four weeks before the first blood draw. N= 2–3 blood draws/animal, data shown as mean ± S.E.M. Notice robust titers are limited to animals that received hu-PMBC. (B,C) Representative sections from the adult rostromedial cerebral cortex of neonatally hiPSC-HPC and adult hu-PBMC engrafted mice, (top) HIV infected (bottom) uninfected animals. Sections were counterstained nucleophilic dye DAPI (blue) and show human iMG expressing dsRed reporter transgene, with additional co-staining by (B) immunohistochemistry with HuNu human-specific nuclear antigen to mark human cells and HIVp24 antigen (green) and (C) HIV gag-pol RNA FISH (green) with the latter showing above background signal in the infected mouse. Scale bars, 50 μm.