iPSC-derived Neural Progenitor Research Articles

Shared and unique 3D genomic features of substance use disorders across multiple cell types.

Recent genome-wide association studies (GWAS) have revealed shared genetic components among alcohol, opioid, tobacco and cannabis use disorders. However, the extent of the underlying shared causal variants and effector genes, along with their cellular context, remain unclear. We leveraged our existing 3D genomic datasets comprising high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq and RNA-seq across >50 diverse human cell types to focus on genomic regions that coincide with GWAS loci. Using stratified LD regression, we determined the proportion of genomewide SNP heritability attributable to the features assayed across our cell types by integrating recent GWAS summary statistics for the relevant traits: alcohol use disorder (AUD), tobacco use disorder (TUD), opioid use disorder (OUD) and cannabis use disorder (CanUD). Statistically significant enrichments (P<0.05) were observed in 14 specific cell types, with heritability reaching 9.2-fold for iPSC-derived cortical neurons and neural progenitors, confirming that they are crucial cell types for further functional exploration. Additionally, several pancreatic cell types, notably pancreatic beta cells, showed enrichment for TUD, with heritability enrichments up to 4.8-fold, suggesting genomic overlap with metabolic processes. Further investigation revealed significant positive genetic correlations between T2D with both TUD and CanUD (FDR<0.05) and a significant negative genetic correlation with AUD. Interestingly, after partitioning the heritability for each cell type's cis-regulatory elements, the correlation between T2D and TUD for pancreatic beta cells was greater (r=0.2) than the global genetic correlation value. Our study provides new genomic insights into substance use disorders and implicates cell types where functional follow-up studies could reveal causal variant-gene mechanisms underpinning these disorders.

Haloperidol, Olanzapine, and Risperidone Induce Morphological Changes in an In Vitro Model of Human Hippocampal Neurogenesis.

Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs). Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone). Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment. Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action.

Comparison of Extracellular Vesicles from Induced Pluripotent Stem Cell-Derived Brain Cells.

The pathophysiology of many neuropsychiatric disorders is still poorly understood. Identification of biomarkers for these diseases could benefit patients due to better classification and stratification. Exosomes excreted into the circulatory system can cross the blood-brain barrier and carry a cell type-specific set of molecules. Thus, exosomes are a source of potential biomarkers for many diseases, including neuropsychiatric disorders. Here, we investigated exosomal proteins produced from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neural stem cells, neural progenitors, neurons, astrocytes, microglia-like cells, and brain capillary endothelial cells. Of the 31 exosome surface markers analyzed, a subset of biomarkers were significantly enriched in astrocytes (CD29, CD44, and CD49e), microglia-like cells (CD44), and neural stem cells (SSEA4). To identify molecular fingerprints associated with disease, circulating exosomes derived from healthy control (HC) individuals were compared against schizophrenia (SCZ) patients and late-onset Alzheimer's disease (LOAD) patients. A significant epitope pattern was identified for LOAD (CD1c and CD2) but not for SCZ compared to HC. Thus, analysis of cell type- and disease-specific exosome signatures of iPSC-derived cell cultures may provide a valuable model system to explore proteomic biomarkers for the identification of novel disease profiles.

Cognitive and inflammatory heterogeneity in severe mental illness: Translating findings from blood to brain

Recent findings link cognitive impairment and inflammatory-immune dysregulation in schizophrenia (SZ) and bipolar (BD) spectrum disorders. However, heterogeneity and translation between the periphery and central (blood-to-brain) mechanisms remains a challenge. Starting with a large SZ, BD and healthy control cohort (n = 1235), we aimed to i) identify candidate peripheral markers (n = 25) associated with cognitive domains (n = 9) and elucidate heterogenous immune-cognitive patterns, ii) evaluate the regulation of candidate markers using human induced pluripotent stem cell (iPSC)-derived astrocytes and neural progenitor cells (n = 10), and iii) evaluate candidate marker messenger RNA expression in leukocytes using microarray in available data from a subsample of the main cohort (n = 776), and in available RNA-sequencing deconvolution analysis of postmortem brain samples (n = 474) from the CommonMind Consortium (CMC). We identified transdiagnostic subgroups based on covariance between cognitive domains (measures of speed and verbal learning) and peripheral markers reflecting inflammatory response (CRP, sTNFR1, YKL-40), innate immune activation (MIF) and extracellular matrix remodelling (YKL-40, CatS). Of the candidate markers there was considerable variance in secretion of YKL-40 in iPSC-derived astrocytes and neural progenitor cells in SZ compared to HC. Further, we provide evidence of dysregulated RNA expression of genes encoding YKL-40 and related signalling pathways in a high neuroinflammatory subgroup in the postmortem brain samples. Our findings suggest a relationship between peripheral inflammatory-immune activity and cognitive impairment, and highlight YKL-40 as a potential marker of cognitive functioning in a subgroup of individuals with severe mental illness.

Proteomic-miRNA Biomics Profile Reveals 2D Cultures of Human iPSC-Derived Neural Progenitor Cells More Sensitive than 3D Spheroid System Against the Experimental Exposure to Arsenic.

The iPSC-derived 3D models are considered to be a connective link between 2D culture and in vivo studies. However, the sensitivity of such 3D models is yet to be established. We assessed the sensitivity of the hiPSC-derived 3D spheroids against 2D cultures of neural progenitor cells. The sub-toxic dose of Sodium Arsenite (SA) was used to investigate the alterations in miRNA-proteins in both systems. Though SA exposure induced significant alterations in the proteins in both 2D and 3D systems, these proteins were uncommon except for 20 proteins. The number and magnitude of altered proteins were higher in the 2D system compared to 3D. The association of dysregulated miRNAs with the target proteins showed their involvement primarily in mitochondrial bioenergetics, oxidative and ER stress, transcription and translation mechanism, cytostructure, etc., in both culture systems. Further, the impact of dysregulated miRNAs and associated proteins on these functions and ultrastructural changes was compared in both culture systems. The ultrastructural studies revealed a similar pattern of mitochondrial damage, while the cellular bioenergetics studies confirm a significantly higher energy failure in the 2D system than to 3D. Such a higher magnitude of changes could be correlated with a higher amount of internalization of SA in 2D cultures than in 3D spheroids. Our findings demonstrate that a 2D culture system seems better responsive than a 3D spheroid system against SA exposure.

Stable and efficient generation of functional iPSC-derived neural progenitor cell rosettes through regulation of collective cell-cell behavior.

Although the potential of stem cells to differentiate into several cell types has shown promise in regenerative medicine, low differentiation efficiency and poor reproducibility significantly limit their practical application. We developed an effective and robust differentiation strategy for the efficient and robust generation of neural progenitor cell rosettes from induced pluripotent stem cells (iPSCs) incorporating botulinum hemagglutinin (HA). Treatment with HA suppressed the spontaneous differentiation of iPSCs cultured under undirected differentiation conditions, resulting in the preservation of their pluripotency. Moreover, treatment with HA during neural progenitor differentiation combined with dual SMAD inhibition generated a highly homogeneous population of PAX6-and SOX1-expressing neural progenitor cells with 8.4-fold higher yields of neural progenitor cells than untreated control cultures. These neural progenitor cells formed radially organized rosettes surrounding the central lumen. This differentiation method enhanced the generation of functional iPSC-derived neural progenitor cell rosettes throughout the culture vessel, suggesting that the regulation of collective cell-cell behavior using HA plays a morphogenetically important role in rosette formation and maturation. These findings show the significance of HA in the suppression of spontaneous differentiation through spatial homogeneity. The study proposes a novel methodology for the efficient derivation of functional iPSC-derived neural progenitor cell rosettes.

Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome

Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.

Human iPSC-derived neural progenitor cells secreting GDNF provide protection in rodent models of ALS and retinal degeneration.

Human induced pluripotent stem cells (iPSCs) are a renewable cell source that can be differentiated into neural progenitor cells (iNPCs) and transduced with glial cell line-derived neurotrophic factor (iNPC-GDNFs). The goal of the current study is to characterize iNPC-GDNFs and test their therapeutic potential and safety. Single-nuclei RNA-seq show iNPC-GDNFs express NPC markers. iNPC-GDNFs delivered into the subretinal space of the Royal College of Surgeons rodent model of retinal degeneration preserve photoreceptors and visual function. Additionally, iNPC-GDNF transplants in the spinal cord of SOD1G93A amyotrophic lateral sclerosis (ALS) rats preserve motor neurons. Finally, iNPC-GDNF transplants in the spinal cord of athymic nude rats survive and produce GDNF for 9months, with no signs of tumor formation or continual cell proliferation. iNPC-GDNFs survive long-term, are safe, and provide neuroprotection in models of both retinal degeneration and ALS, indicating their potential as a combined cell and gene therapy for various neurodegenerative diseases.

A Human Cerebral Organoid Model of Neural Cell Transplantation.

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.

Mesenchymal properties of iPSC-derived neural progenitors that generate undesired grafts after transplantation

Although neural stem/progenitor cells derived from human induced pluripotent stem cells (hiPSC-NS/PCs) are expected to be a cell source for cell-based therapy, tumorigenesis of hiPSC-NS/PCs is a potential problem for clinical applications. Therefore, to understand the mechanisms of tumorigenicity in NS/PCs, we clarified the cell populations of NS/PCs. We established single cell-derived NS/PC clones (scNS/PCs) from hiPSC-NS/PCs that generated undesired grafts. Additionally, we performed bioassays on scNS/PCs, which classified cell types within parental hiPSC-NS/PCs. Interestingly, we found unique subsets of scNS/PCs, which exhibited the transcriptome signature of mesenchymal lineages. Furthermore, these scNS/PCs expressed both neural (PSA-NCAM) and mesenchymal (CD73 and CD105) markers, and had an osteogenic differentiation capacity. Notably, eliminating CD73+ CD105+ cells from among parental hiPSC-NS/PCs ensured the quality of hiPSC-NS/PCs. Taken together, the existence of unexpected cell populations among NS/PCs may explain their tumorigenicity leading to potential safety issues of hiPSC-NS/PCs for future regenerative medicine.

Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.

Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity.

Tracking human neurologic disease status in mouse brain/plasma using reporter-tagged, EV-associated biomarkers

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease caused by a retrotransposon insertion in intron 32 of the TAF1 gene. This insertion causes mis-splicing of intron 32 (TAF1-32i) and reduced TAF1 levels. TAF1-32i transcript is unique to XDP patient cells and can be detected in their extracellular vesicles (EVs). We engrafted patient and control iPSC-derived neural progenitor cells (hNPCs) into the striatum of mice. To track TAF1-32i transcript spread by EVs, we transduced the brain-implanted hNPCs with a lentiviral construct called ENoMi, which consists of a re-engineered tetraspanin scaffold tagged with bioluminescent and fluorescent reporter proteins under an EF-1α promoter. Alongside this improved detection in ENoMi-hNPCs-derived EVs, their surface allows specific immunocapture purification, thereby facilitating TAF1-32i analysis. Using this ENoMi-labeling method, TAF1-32i was demonstrated in EVs released from XDP hNPCs implanted in mouse brains. Post-implantation of ENoMi-XDP hNPCs, TAF1-32i transcript was retrieved in EVs isolated from mouse brain and blood, and levels increased over time in plasma. We compared and combined our EV isolation technique to analyze XDP-derived TAF1-32i with other techniques, including size exclusion chromatography and Exodisc. Overall, our study demonstrates the successful engraftment of XDP patient-derived hNPCs in mice as a tool for monitoring disease markers with EVs.

Variant-to-gene mapping followed by cross-species genetic screening identifies GPI-anchor biosynthesis as a regulator of sleep.

Genome-wide association studies (GWAS) in humans have identified loci robustly associated with several heritable diseases or traits, yet little is known about the functional roles of the underlying causal variants in regulating sleep duration or quality. We applied an ATAC-seq/promoter focused Capture C strategy in human iPSC-derived neural progenitors to carry out a "variant-to-gene" mapping campaign that identified 88 candidate sleep effector genes connected to relevant GWAS signals. To functionally validate the role of the implicated effector genes in sleep regulation, we performed a neuron-specific RNA interference screen in the fruit fly, Drosophila melanogaster, followed by validation in zebrafish. This approach identified a number of genes that regulate sleep including a critical role for glycosylphosphatidylinositol (GPI)-anchor biosynthesis. These results provide the first physical variant-to-gene mapping of human sleep genes followed by a model organism-based prioritization, revealing a conserved role for GPI-anchor biosynthesis in sleep regulation.

Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome.

Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system.

Morphological Characterization of Astrocytes in a Xenograft of Human iPSCDerived Neural Precursor Cells.

Transplantation of a mixed astrocyte and neuron culture is of interest in the development of cell therapies for neurodegenerative diseases. In this case, an assessment of engraftment requires a detailed morphological characterization, in particular an analysis of the neuronal and glial populations. In the experiment performed, human iPSC-derived neural progenitors transplanted into a rat striatum produced a mixed neuron and astrocyte population in vivo by the sixth month after transplantation. The morphological characteristics and neurochemical profile of the xenografted astrocytes were similar to those of mature human astroglia. Unlike neurons, astrocytes migrated to the surrounding structures and the density and pattern of their distribution in the striatum and cerebral cortex differed, which indicates that the microenvironment affects human glia integration. The graft was characterized by the zonal features of glial cell morphology, which was a reflection of cell maturation in the central area, glial shaft formation around the transplanted neurons, and migration to the surrounding structures.

P12-29 Establishment of 3D neurosphere cultures from human iPSC-derived neural progenitor cells

P12-29 Establishment of 3D neurosphere cultures from human iPSC-derived neural progenitor cells

Rare CACNA1H and RELN variants interact through mTORC1 pathway in oligogenic autism spectrum disorder

Oligogenic inheritance of autism spectrum disorder (ASD) has been supported by several studies. However, little is known about how the risk variants interact and converge on causative neurobiological pathways. We identified in an ASD proband deleterious compound heterozygous missense variants in the Reelin (RELN) gene, and a de novo splicing variant in the Cav3.2 calcium channel (CACNA1H) gene. Here, by using iPSC-derived neural progenitor cells (NPCs) and a heterologous expression system, we show that the variant in Cav3.2 leads to increased calcium influx into cells, which overactivates mTORC1 pathway and, consequently, further exacerbates the impairment of Reelin signaling. Also, we show that Cav3.2/mTORC1 overactivation induces proliferation of NPCs and that both mutant Cav3.2 and Reelin cause abnormal migration of these cells. Finally, analysis of the sequencing data from two ASD cohorts—a Brazilian cohort of 861 samples, 291 with ASD; the MSSNG cohort of 11,181 samples, 5,102 with ASD—revealed that the co-occurrence of risk variants in both alleles of Reelin pathway genes and in one allele of calcium channel genes confer significant liability for ASD. Our results support the notion that genes with co-occurring deleterious variants tend to have interconnected pathways underlying oligogenic forms of ASD.

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

SummaryFragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

Interleukin-17a Induces Neuronal Differentiation of Induced-Pluripotent Stem Cell-Derived Neural Progenitors From Autistic and Control Subjects

Prenatal exposure to maternal immune activation (MIA) has been suggested to increase the probability of autism spectrum disorder (ASD). Recent evidence from animal studies indicates a key role for interleukin-17a (IL-17a) in promoting MIA-induced behavioral and brain abnormalities reminiscent of ASD. However, it is still unclear how IL-17a acts on the human developing brain and the cell types directly affected by IL-17a signaling. In this study, we used iPSC-derived neural progenitor cells (NPCs) from individuals with ASD of known and unknown genetic cause as well as from neurotypical controls to examine the effects of exogenous IL-17a on NPC proliferation, migration and neuronal differentiation, and whether IL-17a and genetic risk factors for ASD interact exacerbating alterations in NPC function. We observed that ASD and control NPCs endogenously express IL-17a receptor (IL17RA), and that IL-17a/IL17RA activation modulates downstream ERK1/2 and mTORC1 signaling pathways. Exogenous IL-17a did not induce abnormal proliferation and migration of ASD and control NPCs but, on the other hand, it significantly increased the expression of synaptic (Synaptophysin-1, Synapsin-1) and neuronal polarity (MAP2) proteins in these cells. Also, as we observed that ASD and control NPCs exhibited similar responses to exogenous IL-17a, it is possible that a more inflammatory environment containing other immune molecules besides IL-17a may be needed to trigger gene-environment interactions during neurodevelopment. In conclusion, our results suggest that exogenous IL-17a positively regulates the neuronal differentiation of human NPCs, which may disturb normal neuronal and synaptic development and contribute to MIA-related changes in brain function and behavior.

Retroviral infection of human neurospheres and use of stem Cell EVs to repair cellular damage

HIV-1 remains an incurable infection that is associated with substantial economic and epidemiologic impacts. HIV-associated neurocognitive disorders (HAND) are commonly linked with HIV-1 infection; despite the development of combination antiretroviral therapy (cART), HAND is still reported to affect at least 50% of HIV-1 infected individuals. It is believed that the over-amplification of inflammatory pathways, along with release of toxic viral proteins from infected cells, are primarily responsible for the neurological damage that is observed in HAND; however, the underlying mechanisms are not well-defined. Therefore, there is an unmet need to develop more physiologically relevant and reliable platforms for studying these pathologies. In recent years, neurospheres derived from induced pluripotent stem cells (iPSCs) have been utilized to model the effects of different neurotropic viruses. Here, we report the generation of neurospheres from iPSC-derived neural progenitor cells (NPCs) and we show that these cultures are permissive to retroviral (e.g. HIV-1, HTLV-1) replication. In addition, we also examine the potential effects of stem cell derived extracellular vesicles (EVs) on HIV-1 damaged cells as there is abundant literature supporting the reparative and regenerative properties of stem cell EVs in the context of various CNS pathologies. Consistent with the literature, our data suggests that stem cell EVs may modulate neuroprotective and anti-inflammatory properties in damaged cells. Collectively, this study demonstrates the feasibility of NPC-derived neurospheres for modeling HIV-1 infection and, subsequently, highlights the potential of stem cell EVs for rescuing cellular damage induced by HIV-1 infection.