Nitric oxide reduces HCMV spread in cortical organoids.
Modeling defects in the CNS during congenital HCMV infection have been largely limited to 2-dimensional cell culture systems due to species specificity. However, the emergence of 3-dimensional cortical organoids has allowed investigation of congenital infection in a tissue model with features of fetal forebrain (
21–23). Brains from congenitally infected fetuses have necrotic regions that contain infiltrating macrophages and microglia (
7). These immune cells produce nitric oxide via NOS2 in response to pathogenic infection (
24–26), suggesting that nitric oxide is present in the fetal brain during congenital infection. However, the impact of nitric oxide on HCMV infection and tissue within the fetal brain is unknown. To determine the role of nitric oxide during HCMV infection in developing neural tissue, we began our studies by defining the effect of HCMV and nitric oxide on cortical organoids. We and others demonstrated previously that nitric oxide inhibits HCMV replication in cultured human fibroblasts and epithelial cells using the spontaneous-release nitric oxide donor diethylenetriamine NONOate (DETA/NO) to mimic nitric oxide production by NOS2 (
48,
51). Since macrophages and microglia are not typically present in cortical organoids, we used DETA/NO to determine the impact of nitric oxide on this tissue. Cortical organoids were differentiated from an iPSC line derived from a heathy individual (
52,
53). Organoids were cultured to day 35 of development, at which time they have developed defined brain region identities (
54). Organoids were mock infected or infected using HCMV strain TB40/E encoding enhanced green fluorescent protein (TB40/E-eGFP) at a multiplicity of infection (MOI) of 500 infectious units (IU) per microgram of tissue. Viral stocks were produced using MRC-5 human fibroblasts (TB40/E
Fb-eGFP). Organoids were treated at 2 h postinfection (hpi) and every 24 h with 400 μM DETA/NO or vehicle. Organoids were treated with DETA (spent donor) as an additional control. DETA (spent donor) was prepared by incubating medium containing 400 μM DETA/NO at 37°C for 72 h (
48). This incubation releases nitric oxide from DETA/NO and leaves only the parental backbone and nitric oxide oxidation products, which serve to distinguish effects of nitric oxide from these variables. GFP expression was not observed in mock-infected organoids at 11 days postinfection (dpi) (
Fig. 1A). We observed an increase in GFP fluorescence and spread from 4 dpi to 11 dpi in vehicle and DETA-treated, HCMV-infected organoids (
Fig. 1A), indicating efficient HCMV replication. In DETA/NO-treated, HCMV-infected organoids, GFP was dramatically decreased compared to vehicle and DETA (
Fig. 1A). We quantified the mean fluorescent intensity (MFI) normalized to the cross-sectional surface area and observed an average 79% decrease during DETA/NO exposure compared to vehicle (
Fig. 1B). DETA/NO-treated organoids maintained a size similar to that seen under vehicle and DETA conditions, suggesting that nitric oxide did not impact organoid growth (
Fig. 1B). These data demonstrate that nitric oxide, but not DETA backbone or nitric oxide oxidation products, reduces HCMV spread in cortical organoids.
Macrophages and microglia produce nanomolar to micromolar levels of nitric oxide in response to infection (
26,
27). We estimated steady-state nitric oxide levels to ensure that concentrations were within physiological conditions. We modeled steady-state nitric oxide release over 24 h from 200 and 400 μM DETA/NO using COPASI software (
Fig. 1C) (
55). The maximum steady-state nitric oxide concentrations from 200 and 400 μM were predicted to be 1.3 μM and 1.8 μM, respectively (
Fig. 1C). These results demonstrate that cortical organoid exposure to concentrations of nitric oxide observed during infection is sufficient to reduce HCMV spread in cortical organoids.
Neural rosette structures and tissue organization are disrupted in nitric oxide-exposed cortical organoids.
Similar to the human brain, cortical organoids develop 3-dimensional structure with distinct multicellular layer identities (
54). HCMV infection of cortical organoids increases cell death and disrupts tissue morphology and organization, specifically neural rosette structures (
21–23). To determine if nitric oxide inhibition of HCMV spread could alleviate this disruption, we evaluated cell viability and neural rosette structure. Consistent with our previous studies (
21), we observed that GFP fluorescence was largely confined to the periphery of the organoid, with limited spread to the inner layers (
Fig. 2A). We first labeled for fragmented DNA using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in cryosectioned organoids because nitric oxide is known to induce DNA damage (
Fig. 2A). We quantified TUNEL relative to Hoechst total DNA signal (
Fig. 2B). In uninfected organoids, the TUNEL/Hoechst ratio was significantly increased by 2.3-fold in DETA/NO-treated organoids compared to vehicle, which did not occur in control DETA (spent donor) conditions (
Fig. 2B). We observed an increased ratio of TUNEL/Hoechst across all conditions in HCMV-infected organoids similar to that seen in mock DETA/NO-treated samples (
Fig. 2B). We cannot rule out the possibility that the upper limit of detection was reached for this assay. We next measured cell viability. Organoids were infected and treated as described above. At 11 dpi, organoids were dissociated to obtain single cells and viability was quantified by trypan blue exclusion. In contrast to TUNEL, mock-infected organoids had no difference in cell viability between conditions (
Fig. 1C). All HCMV-infected organoids had decreased cell viability compared to mock-infected ones (
Fig. 2C). The addition of DETA/NO further reduced viability, yet these differences were not statistically significant. Collectively, our data suggest that nitric oxide does increase DNA damage in uninfected samples with no detectible impact on viability. In contrast, infection increased TUNEL regardless of nitric oxide exposure as well as reduced overall viability.
Tissue sections also revealed substantial changes to organoid organization, including regions lacking TUNEL and Hoechst (
Fig. 2A). The areas were present in all organoid conditions but were more evident in mock and HCMV-infected organoids exposed to nitric oxide (
Fig. 2A, arrows). As the regions appeared similar to radial neural rosette structures formed by SOX2-expressing neural progenitor cells (NPCs), we labeled for SOX2, a transcription factor necessary for progenitor maintenance and a marker for NPCs (
56–59). We observed that mock-infected, vehicle-treated organoids displayed SOX2-expressing cells organized in layered radial neural rosette morphology (
Fig. 3A and
B, arrowheads). As previously described (
21), rosette structures were disrupted in HCMV-infected, vehicle-treated organoids (
Fig. 3A and
B, arrows). SOX2-expressing cells also formed rosette-like structures in nitric oxide-exposed cortical organoids. However, the structures lacked the morphology and layering of a typical rosette, and the overall organization of the structures was compromised (
Fig. 3B, arrowheads versus arrows). DETA (spent donor) alone in mock- and HCMV-infected organoids exhibited an intermediate phenotype, with some sections displaying more typical rosette morphology and others showing disrupted rosette-like structures (
Fig. 3B, arrowheads versus arrows). Our data indicate that nitric oxide, and possibly its oxidation products, disrupts cortical organoid organization. Overall, these results suggest that nitric oxide inhibits viral spread in cortical organoids yet, regardless of infection, disrupts structure and organization.
Nitric oxide attenuates HCMV replication and alters markers of NPC proliferation.
SOX2-expressing NPC rosettes were disrupted in nitric oxide exposed organoids (
Fig. 3A and
B). Therefore, we explored the impact of nitric oxide on NPC development and function during infection. NPCs are susceptible to HCMV infection and are significantly disrupted in HCMV-infected organoids (
21,
22). We used 2-dimensional cultures of NPCs differentiated from the same healthy iPSC line from which the cortical organoids were derived. NPCs were cultured as neurospheres before dissociation and plating (
60).
Nitric oxide can have cytotoxic and cytostatic effects based on concentration. Therefore, we assessed these effects in uninfected NPCs. Cells were plated subconfluently at 5 × 10
5 cells/well and treated with a range of 100 to 400 μM DETA/NO or vehicle or not treated. Cell viability was quantified again using trypan blue exclusion at 96 h posttreatment. Cell viability at 100 and 200 μM concentrations were similar to vehicle and no treatment conditions (
Fig. 4A). However, viability of cells in monolayer cultures decreased at higher concentrations. We observed an initial increase in live-cell number in untreated and vehicle-treated NPCs; however, this increase did not occur in 100 and 200 μM DETA/NO, suggesting that nitric oxide induces cytostasis at specific concentrations. We observed a decrease in live-cell count at 300 and 400 μM concentrations (
Fig. 4A).
We next examined the impact of nitric oxide on HCMV replication. To increase infection efficiency, we used TB40/E-eGFP produced in ARPE-19 epithelial cells (TB40/E
Epi-eGFP) (
61). NPCs were plated, incubated for 3 days, and infected with TB40/E
Epi-eGFP at an MOI of 3 IU/cell. At 2 hpi, cultures were treated with 200 μM DETA/NO, DETA (spent donor), or vehicle control, which were replaced every 24 h. As previously described, the maximum steady-state nitric oxide concentration from 200 μM DETA/NO was estimated to be 1.3 μM (
Fig. 1C). Viral DNA levels were quantified at 2, 48, and 96 hpi (
Fig. 4B). Titers of cell-free virus from 96 hpi were determined on ARPE-19 epithelial cells (
Fig. 4B). Viral DNA levels increased by 0.8 log from 2 to 96 hpi, indicating productive viral replication. Nitric oxide reduced viral DNA levels by 0.6 log and viral titers by 0.9 log at 96 hpi (
Fig. 4B). TB40/E
Epi-eGFP is known to be more highly cell associated. Therefore, we quantified titers of both cell-associated and cell-free virus from 96 hpi (
Fig. 4C). Cell-free titers were decreased by ~0.9 log compared to cell-associated titers in both vehicle and DETA (spent donor) control. Nitric oxide reduced cell-associated and cell-free titers by 1 log and 0.8 log, respectively; however, this difference was not statistically significant (
Fig. 4C). To further elucidate when nitric oxide impacts viral replication, we determined levels of viral proteins using Western blot analysis. Nitric oxide did not impact IE1 (immediate early) levels (
Fig. 4D and
E). However, IE2 (immediate early), UL44 (early), and pp28 (late) levels were decreased after 48 hpi during nitric oxide exposure (
Fig. 4D and
E). Together these data suggest that nitric oxide attenuates HCMV replication in NPCs after the onset of viral DNA synthesis, and this result is consistent with our previous studies in human fibroblasts and epithelial cells (
48).
Nitric oxide is cytostatic or cytotoxic to NPCs in a concentration-dependent manner (
Fig. 4A), which can have consequences for cellular differentiation (
62,
63). To investigate nitric oxide-induced cytostasis, we measured steady-state levels of the cell cycle regulators cyclin-dependent kinase inhibitor p21
CIP1/WAF1, which induces cell cycle arrest, and cyclin B, which promotes cell cycle progression (
Fig. 5A). Plated NPCs were infected as described above at an MOI of 3 IU/cell or mock-infected and treated at 2 hpi with 200 μM DETA/NO or vehicle control. In uninfected NPCs, p21
CIP1/WAF1 levels were increased during nitric oxide exposure by 3.4-, 4.1-, 3.9-, and 3.6-fold at 24, 48, 72, and 96 hpi, respectively, relative to vehicle (
Fig. 5A and
B). In contrast, levels remained unchanged in HCMV-infected cells (
Fig. 5A and
B). No changes were observed for cyclin B levels (
Fig. 5A and
B). These data suggest that nitric oxide exposure induces p21
CIP1/WAF1 expression, which likely contributes to cytostasis of uninfected NPCs. Expression of p21
CIP1/WAF1 and cell cycle exit are tightly linked with the onset of cellular differentiation (
62,
63). We hypothesized that nitric oxide-induced cytostasis may interfere with NPC maintenance and differentiation in mock and HCMV-infected cultures. SOX2 is a master regulator required for maintaining multipotency and progenitor identity of NPCs (
56–59,
64). Downregulation of SOX2 is associated with cell cycle exit, decreased proliferation, and terminal differentiation (
59,
65). Studies have demonstrated that SOX2 levels are reduced in an IE1-dependent manner during HCMV infection of embryonic-derived NPCs (
13,
19). We initiated our studies by investigating SOX2 levels during nitric oxide exposure. Plated NPCs were mock or HCMV infected as described above at an MOI of 3 IU/cell and treated at 2 hpi with 200 μM DETA/NO or vehicle control, and SOX2 levels were determined at 96 hpi by Western blot analysis (
Fig. 5C). As we were interested in the separate impact of nitric oxide on uninfected and infected cultures, we set DETA/NO treatment relative to vehicle for each condition. SOX2 levels were decreased by 0.3-fold relative to vehicle in uninfected NPCs. This reduction was more dramatic in HCMV-infected NPCs, with a 0.6-fold decrease during nitric oxide exposure. Our data demonstrate that nitric oxide exposure reduces SOX2 levels and cellular proliferation and increases p21
CIP1/WAF1 levels. Taken together, these results suggest that nitric oxide disrupts regulators of multipotency and differentiation.
Mitochondrial function is compromised during nitric oxide exposure.
During differentiation, NPCs undergo a metabolic shift from mainly glycolytic to oxidative phosphorylation involving mitochondrial respiration (
66,
67). Nitric oxide can disrupt mitochondrial respiration directly by inhibiting complex I and IV of the electron transport chain or indirectly through aconitase inhibition (
28–30). To determine if nitric oxide decreases respiration, we examined mitochondrial function using an extracellular flux assay (
Fig. 6). NPCs were plated, incubated for 3 days, infected at an MOI of 3 IU/cell or mock infected, and treated at 2 hpi with 200 μM DETA/NO, DETA (spent donor), or vehicle control. At 24 hpi, a mitochondrial stress assay was performed. Briefly, basal oxygen consumption rate (OCR) of cells (
Fig. 6A) is measured before sequential injections of oligomycin (ATP synthase inhibitor), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; uncoupler of mitochondria), and rotenone/antimycin A (complex I and III inhibitors, respectively). This assay measures basal (green), ATP-linked (blue), and maximal (yellow) OCR (
Fig. 6A). The difference between basal and maximal OCR is the spare capacity of the cells. A representative plot of the mitochondrion stress assay is shown in
Fig. 6B, with quantification of OCR shown in
Fig. 6C to
F.
Basal OCR of HCMV-infected cells was significantly increased by 79% compared to uninfected NPCs (
Fig. 6C), indicating that infection increases mitochondrial respiration by 24 hpi in NPCs. However, nitric oxide exposure reduced basal OCR by 33% in infected NPCs but not in uninfected cells. ATP-linked OCR also increased by 84% in vehicle-treated, HCMV-infected cells compared to vehicle-treated, uninfected NPCs (
Fig. 6D). This rate was reduced by 31% upon nitric oxide exposure of HCMV-infected cells. No differences were observed in mock-infected conditions. Maximal respiration of NPCs was increased by 39% during infection (
Fig. 6E), indicating an increase in the maximum range of the cells’ response to energy demands. However, in contrast to basal and ATP-linked OCR, maximal respiration was reduced in both uninfected and HCMV-infected NPCs by a respective 55% and 54% during exposure to nitric oxide. Likewise, the spare respiratory capacity of the cells was reduced by a respective 89% and 76% in mock and HCMV-infected conditions during nitric oxide exposure (
Fig. 6F). OCRs of the DETA (spent donor) did not deviate from those of vehicle for the uninfected or HCMV-infected groups and were not included in the statistical analysis (
Fig. 6C to
F). These data demonstrate that nitric oxide significantly suppresses HCMV-mediated increase of basal and ATP-linked respiration. Furthermore, and regardless of infection, nitric oxide reduces the capacity of NPCs to respond to energy demands through mitochondrial respiration regardless of infection.
Nitric oxide limits neuron and glial lineages during NPC differentiation.
Reduced mitochondrial respiration, decreased SOX2 expression, and cytostasis led us to hypothesize that nitric oxide restricts neuron and glial cell differentiation from NPCs. To test this hypothesis, we investigated the effect of nitric oxide with and without infection on neural differentiation at 14 days post differentiation. Cells were cultured in the absence of growth factors to allow spontaneous differentiation (
16,
60). We treated them with 200 μM DETA/NO every 48 h to reduce the chance of cytotoxicity due to prolonged exposure and reduced the MOI to avoid loss of cultures due to lytic replication during the 14 days. NPCs were plated for 3 days and infected at an MOI of 0.05 IU/cell or mock infected. Subsequently, cells were treated at 2 hpi and every 48 h with DETA/NO, DETA (spent donor), or vehicle control up to 11 dpi. We assessed markers of neural populations using immunofluorescence (
Fig. 7A). We used the marker Nestin to identify the progenitor population because SOX2 is reduced during HCMV infection of embryonic stem cell-derived NPCs (
13). Further, we identified neuron populations by labeling for Tuj1, a class III beta-tubulin specific to neurons, and glial populations by labeling for glial fibrillary acidic protein (GFAP), a type III intermediate filament protein expressed by glial cells (
Fig. 7A). In mock-infected vehicle-treated cultures, we observed expression of each developmental marker, indicating a mix of neural populations. We observed Nestin-positive cells contained small processes with large cell bodies (
Fig. 7A), while Tuj1-positive cells had long, netted neurites with small cell bodies (
Fig. 7A). GFAP-positive cells had long projections with a large cell body (
Fig. 7A). HCMV-infected cultures contained single nuclei and multinucleated GFP-positive cell bodies (
Fig. 7B), indicating syncytium formation regardless of nitric oxide exposure. We attempted to quantify progenitor, neuron, and glial populations using immunofluorescence, since our goal was to evaluate changes in marker expression. However, we were unable to use this approach due to culture density and overlapping projections (
Fig. 7A).
To overcome this limitation, we switched to flow cytometry to quantify differentiated neural populations, which also allowed us to identify double- and triple-marker-positive cells (
68). As described above, NPCs were plated from dissociated iPSC-derived NPC neurospheres and completed using different NPC passages. The passage numbers are presented as iPSC/NPC neurosphere passage numbers. Cells from passage 45/22 (P45/22), P61/12, and P66/9 were used for these studies. NPCs were HCMV or mock infected, and flow cytometry was performed at 11 dpi. Cells were labeled with Nestin-, Tuj1-, and GFAP-conjugated fluorescent antibodies. Gates were placed to isolate live, single-cell events, and GFP
+ events were identified by first gating on the unstained, uninfected population (
Fig. 8A). Forward and side scatter gates were extended to include the larger GFP
+ cells. It is possible that syncytia were lost despite the increased gate size; however, we reasoned that these cell bodies would be equally lost in all conditions. Uninfected cells had no GFP
+ events. GFP
+ events varied in infected cultures depending on the passage of NPCs, despite infection at the same MOI (
Fig. 9A). For cells from P45/22, 54.7% of the live, single-cell events were GFP
+. In P61/12 and P66/9 cells, GFP
+ events were 1.6 and 5.7%, respectively. As GFP levels varied between experiments, we grouped GFP
+ and GFP
− events to analyze the whole culture population for each experiment. We used fluorescence minus one (FMO) controls (
Fig. 8B) to define Tuj1/GFAP, Tuj1/Nestin, and GFAP/Nestin populations, and our gating strategy is shown in
Fig. 8C.
We first determined frequencies of Tuj1
+ Nestin
− GFAP
− events, which represent neuron populations, and Tuj1
+ Nestin
+ GFAP
− events, which likely represent an immature neuron population, as they express both Tuj1 and Nestin (
Fig. 9B and
C). We quantified these populations by gating on live, single-cell, Tuj1
+ events from Tuj1/GFAP populations (
Fig. 8C, Q1). Tuj1
+ Nestin
− GFAP
− (neurons) frequencies in the mock-infected vehicle-treated control groups varied between passage number. For P45/22 cultures, the frequency of Tuj1
+ Nestin
− GFAP
− (neurons) events was 25% compared with 45% and 41% quantified in P61/12 and P66/9 cultures, respectively (
Fig. 9B and
C). This variation indicates intrinsic differences in the spontaneous differentiation capacity of the NPCs, and we suspect that these differences also contribute to the variability in susceptibility to infection (
Fig. 9A). Upon exposure to nitric oxide, we observed a substantial decrease in the percent of Tuj1
+ Nestin
− GFAP
− (neurons) events in P45/22 cultures in mock- and HCMV-infected populations (
Fig. 9C). Less dramatic reductions were observed in P61/12 and P66/9 (
Fig. 9C), suggesting that sensitivity to nitric oxide is influenced by intrinsic differences between passages and developmental states. The percent of Tuj1
+ Nestin
+ GFAP
− (immature neurons) events for mock-infected, vehicle-treated cells were more similar between P45/22 and P66/9 at a respective 31% and 26% (
Fig. 9B and
C). This population was increased in P45/22 and P66/9 cultures during nitric oxide exposure regardless of infection (
Fig. 9C). For P61/12, the percent of Tuj1
+ Nestin
+ GFAP
− (immature neurons) events for mock-infected, vehicle-treated cultures was 42% and no differences were observed during nitric oxide exposure (
Fig. 9C). Again, these data suggest that differentiation capacity between cell passages influences their susceptibility to nitric oxide. Cells exposed to DETA (spent donor) had similar frequencies of Tuj1
+ Nestin
− GFAP
− cells (neurons). For P45/22 and P66/9, there was an increased percent of Tuj1
+ Nestin
+ GFAP
− (immature neurons) compared to vehicle, though not to the level of nitric oxide-exposed cells (
Fig. 9C). Overall, the results suggest that nitric oxide limits differentiation of immature neurons (Tuj1
+ Nestin
+ GFAP
−) to neurons (Tuj1
+ Nestin
− GFAP
−).
We next examined the percent Tuj1
+ Nestin
+ GFAP
+ and Tuj1
+ Nestin
− GFAP
+ cells (
Fig. 9D) relative to live, single cells by gating on Tuj1
+ GFAP
+ events (
Fig. 8C, Q2). Tuj1
+ Nestin
+ GFAP
+ populations likely represent progenitor-like cells (
69,
70). The identity of Tuj1
+ Nestin
− GFAP
+ populations is unknown, but they may represent cells in transition to a neuron or glial state after downregulation of Nestin (
69). Frequencies of Tuj1
+ Nestin
+ GFAP
+ (progenitor-like) events varied between passages for mock-infected, vehicle-treated cultures with levels being most similar between P45/22 and P66/9 (
Fig. 9B and
D). We observed an increase in the P66/9 progenitor-like population in uninfected nitric oxide exposed cultures that was not observed in other passages (
Fig. 9D). P61/12 had substantially less Tuj1
+ Nestin
+ GFAP
+ (progenitor-like) events in mock-infected vehicle-treated cultures (
Fig. 9B and
D). DETA (spent donor) treatment decreased frequencies of Tuj1
+ Nestin
+ GFAP
+ events in P45/22 and P66/9, suggesting some impact of nitric oxide oxidation products on this progenitor-like population (
Fig. 9D). Tuj1
+ Nestin
− GFAP
+ (transition state) populations were also variable between passages in mock-infected vehicle-treated cultures at 15.3%, 2.2%, and 5.2% for P45/22, P61/12, and P66/9, respectively (
Fig. 9B and
D). This population was reduced during infection of P45/22, and nitric oxide exposure also reduced these frequencies regardless of infection (
Fig. 9D). Overall, our data indicate a limited impact of nitric oxide on Tuj1
+ Nestin
+ GFAP
+ (progenitor-like) and Tuj1
+ Nestin
− GFAP
+ (transition state) populations.
Percent of Tuj1
− Nestin
− GFAP
+ cells, representing glial populations, were obtained by gating on Tuj1
− GFAP
+ (
Fig. 8C, Q3) and then gating on Tuj1
− Nestin
− GFAP
+ (
Fig. 9E). We again observed differences in the frequency of glial populations between the passages of mock-infected, vehicle-treated groups with a respective 4.8%, 0.2%, and 2.3% for P45/22, P61/12, and P66/9 (
Fig. 9B and
E). For P45/22 and P66/9, Tuj1
− Nestin
− GFAP
+ (glial) populations in infected, vehicle-treated cultures were modestly decreased compared with mock-infected, vehicle-treated cultures. We observed a limited impact from the spent donor on this population (
Fig. 9E), suggesting that oxidation products do not alter glial populations. Nitric oxide decreased Tuj1
− Nestin
− GFAP
+ (glial) frequencies regardless of infection (
Fig. 9E), suggesting that nitric oxide limits glial differentiation. Frequencies of Tuj1
− Nestin
+ GFAP
+ cells, which may represent immature glial cells, were below 1% and not included in further analysis.
Finally, levels of Tuj1
− Nestin
+ GFAP
− cells, representing progenitor populations (
Fig. 9B and
F) were obtained by gating on Tuj1
− GFAP
− events and Nestin
+ events (
Fig. 8C, Q4). Levels of Tuj1
− Nestin
+ GFAP
− (progenitor) cells were notably higher in P66/9 than P45/22 and P61/12 (
Fig. 9B and
F). Nitric oxide decreased progenitor populations in P66/9 mock-infected vehicle-treated cultures from 7.1% to 3%, with a similar reduction observed in infected cultures (
Fig. 9F). The frequencies of progenitor populations for P45/22 and P61/12 were too low to observe differences (
Fig. 9F). Taken together, our data suggest that nitric oxide, and to some degree oxidation products, disrupts NPC differentiation of both neuron (Tuj1
+ Nestin
− GFAP
−) and glial (Tuj1
− Nestin
− GFAP
+) populations with a trend toward increased immature neurons (Tuj1
+ Nestin
+ GFAP
−) regardless of infection.