Primary human astrocytes were differentiated from human neuroprogenitor cells (NPCs, ABC-TC372-1), purchased from Accegen Biotech (Fairfield, NJ, USA) using the protocol developed by Hammond et al., 2002 [20]. The NPCs were confirmed to express the neural stem-cell marker Nestin and passed routine mycoplasma contamination tests and the test results were negative. Briefly, NPCs were plated on poly-D-lysine (PDL)-treated 6-well plates with glass coverslips and cultured in 2 mL of 90% KnockOut DMEM, 10% fetal calf serum (FCS), 0.1% L-Glutamine, and 0.1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Half of the media was replaced every 48 hours. NPCs were allowed to differentiate for four weeks until they exhibited a full astrocyte phenotype. Aliquots of differentiating NPCs were characterized every 7 days. Confirmation of the astrocyte phenotype was performed based on morphology and quantitative real time-polymerase chain reaction (qRT-PCR) analysis of Glial Fibrillary Acidic Protein (GFAP) transcripts four weeks post-differentiation and results are presented in Fig. 1A,B.

Fig. 1.

Generation and characterization of primary astrocytes. (A) Confocal images of NPCs and differentiated astrocytes expressing the astrocytic marker GFAP (red) at day 1 (left) and day 28 (right). (B) Quantification of GFAP expression by NPCs and Astrocytes through qRT-PCR. (C) Flow cytometry of Astrocytes stained for CD4-FITC and CXCR4-APC (right), versus isotype control (left). (D) Flow cytometry of primary astrocytes stained for CCR5 and CXCR4 coreceptor (right), versus isotype control (left). Numbers reflect percentage of cells in that quartile. N = 4, scale bar = 100 µm. Abbreviations: DAPI, 4^′,6-diamidino-2-phenylindole; GFAP, Glial Fibrillary Acidic Protein; NPCs, neuroprogenitor cells; FITC, Fluorescein isothiocyanate; CCR5, C-C chemokine receptor type 5; CXCR4, C-X-C chemokine receptor type 4; APC, allophycocyanin; qRT-PCR, quantitative real-time polymerase chain reaction.

Healthy donor-derived human iPSC line (BYS0113, ATCC, Manassas, VA, USA) was cultured on dishes coated with hESC-qualified Matrigel in mTeSR medium (STEMCELL Technologies, Vancouver, Canada). Neural progenitors were generated from iPSC cultures following the protocol described by Jin et al. [21]. These neuroprogenitors were then differentiated into astrocytes over three weeks using the STEMDiff Astrocytes differentiation kit (STEMCELL Technologies, Vancouver, Canada), as per the manufacturer’s instructions. The differentiated astrocytes were subsequently matured for an additional three weeks using the STEMDiff Astrocytes maturation kit (STEMCELL Technologies, Vancouver, Canada), also following the manufacturer’s instructions. The matured iPSC-derived astrocytes (iAstrocytes) were used for experiments within 2–3 weeks post-maturation. For the generation of macrophages and microglia (myeloid) precursors from the human iPSC line (ATCC, BYS0113), we utilized the STEMDiff Hematopoietic differentiation kit (STEMCELL Technologies, Vancouver, Canada), following the manufacturer’s instructions. Myeloid precursors were differentiated into macrophages over a period of two weeks in a medium composed of DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 2 mM GlutaMAX (Gibco, Grand Island, NY, USA) and 100 ng/mL macrophage colony-stimulating factor (M-CSF) (Peprotech, Cranbury, NJ, USA), according to Haenseler et al. [22]. Similarly, myeloid precursors were differentiated into microglia over three weeks in a medium composed of DMEM/F12 supplemented with 2 mM GlutaMAX (Gibco), 100 ng/mL Interleukin-34 (IL-34) (Peprotech, Cranbury, NJ, USA), and 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (Millipore, Burlington, MA, USA), as previously established [22]. Once in maturation medium, iPSC-derived cells lose their ability to proliferate and become terminally differentiated. STR authentication and karyotyping of the iPSC line were performed by ATCC (Manassas, VA, USA). Cells were regularly tested for mycoplasma contamination the test results were negative, indicating that the cells were free of contamination. Cells were used within 30 passages for this study.

Flow cytometry was employed to characterize the expression of surface markers on human primary astrocytes crucial for binding and fusion, including CD4, C-C chemokine receptor type 5 (CCR5), and CXCR4. Astrocytes were dissociated using TripLE (Gibco, Grand Island, NY, USA) non-enzymatic dissociation reagent, transferred to clear flow tubes, and centrifuged at 1000 RPM for 5 minutes. After aspirating TripLE, cells were incubated with monoclonal wash solution (90% PBS, 10% FCS, 0.1% sodium azide) containing CD4-FITC (Fluorescein isothiocyanate), CCR5-phycoerythrin (PE), and C-X-C chemokine receptor type 4-allophycocyanin (CXCR4-APC) antibodies. Following a 45-minute incubation at 4 °C, cells were washed and fixed with 4% paraformaldehyde (PFA) (ThermoFisher Scientific, Waltham, MA, USA). Stained cells were analyzed using a FACSymphony A5 SE (BD Biosciences, Franklin Lakes, NJ, USA) with isotype-stained cells serving as controls for analysis.

To confirm the differentiation of primary astrocytes from NPCs, cells were stained for GFAP, an astrocyte-specific marker. Astrocytes plated on coverslips were fixed in 4% PFA for at least 15 minutes and permeabilized in a buffer containing 90% PBS, 10% FCS, and 0.1% Triton X-100. Next, cells were incubated with 1:1000 dilution of chicken anti-GFAP primary antibody (Abcam, Cambridge, UK) at 4 °C for 24 hours, followed by washing and staining with 1:500 dilution of the secondary antibody, Goat anti-Chicken Cy5 (Jackson ImmunoResearch Labs, West Grove, PA, USA). Slides were washed twice in PBS, stained with 4^′,6-diamidino-2-phenylindole (DAPI), and mounted on coverslips using glycerol-based mounting media. Images were acquired using a Nikon A1-R confocal microscope (Nikon, Tokyo, Japan), and maximum intensity Z-projections were generated using ImageJ-FIJI software (version 2.14.0, National Institutes of Health, Bethesda, MD, USA). The images shown are representative of cultures generated from three independent experiments.

HEK-293T cells (CRL3216, ATCC, Manassas, VA, USA) were transfected with 5 µg of HIV-1 viral plasmids (pNL43 for X4-tropic, pNL43-YU2 Env or pNL43-BaL Env for R5-tropic) using PolyJet transfection reagent (SignaGen, Frederick, MD, USA) as described [23]. To identify productively infected cells, HIV-1 proviral plasmids were constructed to express fluorescent reporters: enhanced green fluorescent protein (EGFP) in plasmids pNL43 and pNL43-YU2 Env-EGFP, or near-infrared fluorescent protein (iRFP) in plasmid pNL43-BaL Env-iRFP. Forty-eight hours post-transfection, the supernatant was harvested, centrifuged to remove cellular debris, filtered through a 0.2-micron syringe filter (ThermoFisher Scientific, Waltham, MA, USA), and stored at –80 °C. To enhance astrocyte tropism, pseudotyped viruses were generated by cotransfecting 1.50 µg of Vesicular Stomatitis Virus Glycoprotein (pVSV-G-Env) expression plasmid with the above-mentioned proviral DNA [24]. Pseudotyped HIV particles interact with ubiquitous phosphatidylserine and low-density lipoprotein receptors (LDLRs) on the target cell surface, inducing endocytosis of the virion into the astrocyte [25]. All viruses were titrated in U87MG CD4+ CCR5+ cells (ARP-4035, NIH-AIDS Reagents Program, Germantown, MD, USA) to determine the multiplicity of infection (MOI). The cell species identification was carried out by ATCC (Manassas, VA, USA) through Cytochrome oxidase 1 (CO1) gene barcoding. All cell lineages utilized in this study were routinely tested for mycoplasma contamination with all outcomes being negative.

Four weeks post-differentiation, astrocytes were infected with 0.1 or 1 MOI of virus. Eighteen hours post-infection, cells were washed with PBS to remove unbound virus and maintained in astrocyte medium. Supernatant was collected every 48 hours until 10–15 days post-infection, and coverslips were collected for imaging. The remaining cells were harvested for RNA extraction using the MirVana kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s recommendations. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). cDNA was synthesized from 300 ng of total RNA using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific, Waltham, MA, USA) in a total reaction volume of 20 µL. qRT-PCR was performed using TaqMan Universal PCR master mix (ThermoFisher Scientific, Waltham, MA, USA) and the appropriate TaqMan assays or primers (refer to Table 1) with 2 µL of the cDNA reaction mixture. PCR assays were conducted on an ABI ViiA 7 real-time PCR system (Applied Biosystems, Waltham, MA, USA) with the following cycling conditions: initial activation of Taq DNA polymerase at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Results were normalized to the expression of the endogenous control Ribosomal Protein Lateral Stalk Subunit P0 (RPLP0).

To assess the role of cell-associated infection of human astrocytes, we co-cultured mature iAstro either with HIV- infected iMac or iMG in a 1:1 ratio. Briefly, iMac and iMG cultured in 6 well-plates were infected with HIV-BaL-Env-iRFP MOI 1 for 18 hrs, washed and maintained in medium for 10 days. Cells were then harvested, counted and added to mature iAstro previously labeled with Calcein and Hoechst (Thermo-Fisher Scientific, Waltham, MA, USA) per manufacturer’s recommendation. Co-cultures (1:1 ratio) were maintained for an additional 7 days and coverslips were harvested and fixed for at least 15 min in 4% PFA, and permeabilized. Cells were incubated with 1: 1000 dilution of rabbit Iba-1 primary antibody (Fujifilm Wako Chemicals, Richmond, VA, USA) at 4 °C for 24 hrs, followed by washing and staining with secondary antibody, Goat Ab to rabbit-Cy3 (Jackson ImmunoResearch Labs, West Grove, PA, USA), in a 1:500 dilution of buffer. The slides were washed twice in PBS and mounted on coverslips using glycerol-based mounting media. The remaining cells in the wells were harvested with TripLE, washed, fixed with 4% PFA and subjected to flow cytometry to quantitate infectivity.

Culturing and differentiation of NPCs into astrocytes were characterized by morphological changes and the expression of GFAP, an astrocyte-specific marker, over a period of four weeks (Fig. 1A,B). Subsequently, we assessed the expression of the HIV receptor (CD4) and coreceptors (CCR5 and CXCR4) using flow cytometry and specific markers. The results indicate a lack of CD4 and CCR5 expression, while more than 75% of cells express CXCR4, compared to the isotype control (Fig. 1C,D). These findings suggest that fully differentiated primary astrocytes do not express the canonical HIV receptor CD4 or the CCR5 co-receptor on their cell surface. Instead, over 75% of the cells express CXCR4, indicating that neurotropic viruses (R5-tropic) likely enter these cells through alternative pathways.

To evaluate the susceptibility of primary astrocytes to HIV infection and their ability to produce infectious virions, we exposed these cells to both R5- and X4-tropic HIV strains. Astrocytes were infected with HIV-1 YU2 EGFP (CCR5-utilizing strain) and HIV-1 NL43 EGFP (CXCR4-utilizing strain) reporter viruses with and without VSV-G-Env complementation, as described in the methods. Infectivity was monitored by assessing EGFP expression in cells using microscopy in conjunction with GFAP staining (Fig. 2A,B). Results indicate very low levels of infection (0.1%) by both NL43 and YU2 strains with their native Env protein. In contrast, VSV-G-Env complemented virus infected astrocytes very efficiently ($>$80%) at an MOI of 0.1, suggesting a potential entry-level defect. Dual staining of GFAP confirmed that all cells in the culture were mature astrocytes, with no contamination from NPCs or other cell types (Fig. 2A,B). Additionally, the synthesis of early and late viral proteins, indicative of productive infection, was assessed by measuring multiply spliced (MS) variants of viral RNA via qRT-PCR (Fig. 2C). Our findings confirm that primary human astrocytes can be infected by different HIV-1 strains via mechanisms independent of CD4 or other canonical chemokine receptors. Furthermore, cells infected with both wild-type and VSV-G-Env-complemented viruses (YU2 and NL43) exhibited the synthesis of both Gag and multiply spliced RNA transcripts, demonstrating productive infection.

Fig. 2.

Cell-free HIV-1 infection of primary astrocytes and viral replication. (A) Confocal images of primary astrocytes stained with GFAP (red, Cy5) and Nucleus (DAPI, blue) upon exposure to HIV-1 NL43-EGFP, HIV-1 NL43-EGFP complemented with VSV-G-Env, or no virus (mock). (B) Confocal images of primary astrocytes stained with GFAP (red, Cy5) and DAPI (blue) upon exposure to HIV-1 NL43(YU2)-EGFP, HIV-1 NL43(YU2)-EGFP (green) complemented with VSV-G-Env, or no virus (mock). (C) Quantification of Gag transcript and multiply spliced transcripts in astrocytes exposed to HIV-1 at day 15, post-infection by qRT-PCR. (D) Assessment of cumulative infectious particles released in the supernatant over the period of 15 days post-infection. N = 4, scale bar = 100 µm. White arrows show HIV-infected astrocytes. Abbreviations: EGFP, enhanced green fluorescent protein; HIV-1, Human Immunodeficiency Virus; YU2-VSV-G, YU2 strain pseutotyped with the envelope glycoprotein of vesicular stomatitis virus; MS, multiply spliced; VSV-G-Env, envelope glycoprotein of vesicular stomatitis virus.

Next, we investigated whether the virus particles produced by infected astrocytes were infectious. Supernatants collected at various time points post-infection were used to infect HIV-permissive U87MG cells (Fig. 2D). U87MG cells infected with different dilutions of virus generated by astrocytes showed EGFP expression, indicating that astrocytes are capable of producing infectious virus particles up to 15 days post-infection. These results collectively suggest that once infected, astrocytes may serve as long-lived viral reservoirs.

iPSCs are emerging as promising tools for developing more reliable in vitro models to study neuropathogenesis [24, 25]. These cells can be cultured in chemically defined media to generate various cell lineages resembling those found in human brain tissue, including astrocytes and microglia [26]. In this study, iPSC-derived astrocytes, referred to as iAstro, were infected with R5-tropic HIV virus with and without the VSV-G-Env complementation. Specifically, iAstro were exposed to HIV-1 NL43 BaL-near-iRFP reporter virus with and without VSV-G-Env complementation. Infectivity was assessed by monitoring iRFP expression in cells using microscopy in conjunction with GFAP staining (Fig. 3A). These results confirm very low levels of infection (0.1%) by HIV-NL43 BaL-iRFP with its native Env protein, whereas the VSV-G-Env complemented virus infected the cells very efficiently ($>$90%) at an MOI of 1 (Fig. 3A). HIV-1 entry into the brain primarily involves infected monocytes, macrophages and/or microglia. To investigate the role of infected myeloid cells in establishing infection in astrocytes within the CNS, we utilized iPSC technology to generate iPSC-derived astrocytes (iAstro) and autologous iPSC-derived macrophages (iMac) or microglia (iMG). First, iMac and iMG were infected with HIV-1 NL43 Bal-iRFP virus, and infection was monitored over 10 days. Post-infection, both cell types were fixed and assessed for reporter gene expression as a measure of infection. As shown in Fig. 3B, approximately 20% of iMac and 10% of iMG were infected by HIV-1 NL43 Bal-iRFP. Next, we co-cultured calcein-labeled iAstro with infected iMac and iMG for up to 7 days to assess the ability of infected macrophages/microglia to trans infect astrocytes. Both macrophages and microglia closely interacted with astrocytes regardless of their infectious status (Fig. 3C). In both cases, infection was restricted to a few cells. To quantify this interaction, we lifted the cells from co-culture on day 10 and performed flow cytometry, allowing for cell sorting and quantification. After gating for cells, we identified the calcein-positive population (iAstro) and quantified the percentage of events positive for iRFP (Fig. 3D,E). Our analysis revealed that less than 2% of astrocytes interacting with HIV-infected iMac were HIV-positive (Fig. 3D), whereas less than 1% of astrocytes interacting with infected iMG were HIV-positive (Fig. 3E). Together, these results confirm that astrocytes, whether primary or iPSC-derived, can be infected by cell-free or cell-associated HIV, albeit at very low levels.

Fig. 3.

Cell-associated infection of astrocytes. (A) Confocal images of iPSC-derived astrocytes (iAstro) stained with GFAP (red, Cy3) and DAPI (blue) upon exposure to cell-free HIV-1 NL43(BaL)-iRFP, HIV-1 NL43(BaL)-iRFP (white) complemented with VSV-G-Env at 1 MOI, or no virus (mock). (B) Representative images of iPSC-derived macrophages (iMac) and microglia (iMG) upon infection with HIV-1 NL43(BaL)-iRFP (pink) at 1 MOI. (C) HIV-infected iMac and iMG were harvested 7 days post infection and co-cultured with calcein-labeled iAstro (green) for additional 10 days. Representative confocal images depict HIV-infected (white) iMac and iMG (Iba-1+, red) in close contact with iAstro (calcein+, green). Representative flow cytometry plot to identify calcein-positive iAstrocytes in cell suspension from co-culture with (D) macrophages or (E) microglia. iAstrocytes population was gated (upper panel) and further separated by iRFP expression to assess the percentage of iAstrocytes that were positive for HIV-infection (lower panel). Scale bar = 50 µm in (A,C), and scale bar = 150 µm in (B). Abbreviations: iPSC, induced pluripotent stem cells; MOI, multiplicity of infection; iRFP, near-infrared fluorescent protein; SSC-H, side scatter pulse-height.