Considerations for the development of iPSC-derived cell therapies: a review of key challenges by the JSRM-ISCT iPSC Committee.

Since their first production in 2007, human induced pluripotent stem cells (iPSCs) have provided a novel platform for the development of various cell therapies targeting a spectrum of diseases, ranging from rare genetic eye disorders to cancer treatment. However, several challenges must be tackled for iPSC-based cell therapy to enter the market and achieve broader global adoption. This white paper, authored by the Japanese Society for Regenerative Medicine (JSRM) - International Society for Cell Therapy (ISCT) iPSC Committee delves into the hurdles encountered in the pursuit of safe and economically viable iPSC-based therapies, particularly from the standpoint of the cell therapy industry. It discusses differences in global guidelines and regulatory frameworks, outlines a series of quality control tests required to ensure the safety of the cell therapy, and provides details and important considerations around cost of goods (COGs), including the impact of automated advanced manufacturing.

Cross-platform validation of neurotransmitter release impairments in schizophrenia patient-derived NRXN1-mutant neurons.

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Addiction associated N40D mu-opioid receptor variant modulates synaptic function in human neurons.

The OPRM1 A118G single nucleotide polymorphism (SNP rs1799971) gene variant encoding the N40D µ-opioid receptor (MOR) has been associated with dependence on opiates and other drugs of abuse but its mechanism is unknown. The frequency of G-allele carriers is ~40% in Asians, ~16% in Europeans, and ~3% in African-Americans. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy. Here we generated homozygous N40D subject-specific induced inhibitory neuronal cells (iNs) from seven human-induced pluripotent stem (iPS) cell lines from subjects of European descent (both male and female) and probed the impact of N40D MOR regulation on synaptic transmission. We found that D40 iNs exhibit consistently stronger suppression (versus N40) of spontaneous inhibitory postsynaptic currents (sIPSCs) across multiple subjects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were recapitulated in two sets of independently engineered isogenic N40D iNs. In addition, we employed biochemical analysis and observed differential N-linked glycosylation of human MOR N40D. This study identifies neurophysiological and molecular differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.

Characterization hiPSC-derived neural progenitor cells and neurons to investigate the role of NOS1AP isoforms in human neuron dendritogenesis.

Abnormal dendritic arbor development has been implicated in a number of neurodevelopmental disorders, such as autism and Rett syndrome, and the neuropsychiatric disorder schizophrenia. Postmortem brain samples from subjects with schizophrenia show elevated levels of NOS1AP in the dorsolateral prefrontal cortex, a region of the brain associated with cognitive function. We previously reported that the long isoform of NOS1AP (NOS1AP-L), but not the short isoform (NOS1AP-S), negatively regulates dendrite branching in rat hippocampal neurons. To investigate the role that NOS1AP isoforms play in human dendritic arbor development, we adapted methods to generate human neural progenitor cells and neurons using induced pluripotent stem cell (iPSC) technology. We found that increased protein levels of either NOS1AP-L or NOS1AP-S decrease dendrite branching in human neurons at the developmental time point when primary and secondary branching actively occurs. Next, we tested whether pharmacological agents can decrease the expression of NOS1AP isoforms. Treatment of human iPSC-derived neurons with d-serine, but not clozapine, haloperidol, fluphenazine, or GLYX-13, results in a reduction in endogenous NOS1AP-L, but not NOS1AP-S, protein expression; however, d-serine treatment does not reverse decreases in dendrite number mediated by overexpression of NOS1AP isoforms. In summary, we demonstrate how an in vitro model of human neuronal development can help in understanding the etiology of schizophrenia and can also be used as a platform to screen drugs for patients.

Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self-renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3-D microgeometries can support hESC colony formation, self-renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions.

Tbx6 is a determinant of cardiac and neural cell fate decisions in multipotent P19CL6 cells.

Multipotent P19CL6 cells differentiate into cardiac myocytes or neural lineages when stimulated with dimethyl sulfoxide (DMSO) or retinoic acid (RA), respectively. Expression of the transcription factor Tbx6 was found to increase during cardiac myocyte differentiation and to decrease during neural differentiation. Overexpression of Tbx6 was not sufficient to drive P19CL6 cells to a cardiac myocyte fate or to accelerate DMSO-induced differentiation. In contrast, knockdown of Tbx6 dramatically inhibited DMSO-induced differentiation of P19CL6 cells to cardiac myocytes, as evidenced by the loss of striated muscle-specific markers and spontaneous beating. Tbx6 knockdown was also accompanied by almost complete loss of Nkx2.5, a transcription factor involved in the specification of the cardiac myocyte lineage, indicating that Nkx2.5 is downstream of Tbx6. In distinction to its positive role in cardiac myocyte differentiation, Tbx6 knockdown augmented RA-induced differentiation of P19CL6 cells to both neurons and glia, and accelerated the rate of neurite formation. Conversely, Tbx6 overexpression attenuated differentiation to neural lineages. Thus, in the P19CL6 model, Tbx6 is required for cardiac myocyte differentiation and represses neural differentiation. We propose a model in which Tbx6 is a part of a molecular switch that modulates divergent differentiation programs within a single progenitor cell.

A model to evaluate the cytotoxicity of the fungal volatile organic compound 1-octen-3-ol in human embryonic stem cells.

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol ("mushroom alcohol"), a major fungal volatile organic compound (VOC) associated with mushroom and mold odors. Using an airborne exposure technique, human embryonic stem cells were exposed for 1 h to different concentrations (0-1,000 ppm) of racemic 1-octen-3-ol and its enantiomers, (R)-(-)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. Cytotoxicity was assayed using both the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and the fluorescently tagged Calcein AM-mediated "live and dead" assay. Racemic 1-octen-3-ol and (S)-(+)-1-octen-3-ol exhibited greater cytotoxicity to the undifferentiated human cell line H1 than did (R)-(-)-1-octen-3-ol. The inhibition concentration 50 (IC(50)) values assessed by the MTS assay for racemic 1-octen-3-ol, (S)-(+)-1-octen-3-ol and (R)-(-)-1-octen-3-ol were, respectively, 109, 98, and 258 ppm. These IC(50) values were 40-80-fold lower than that of vapor phase toluene, an industrial chemical used as a positive control in this study. Our report pioneers the modeling of human embryonic stem cells as an in vitro approach to screen the potential toxicity of fungal VOCs. Human embryonic stem cells exposed to 1-octen-3-ol, and its enantiomers in the vapor phase showed more cytotoxicity than those exposed to toluene.

Establishment and characterization of two iPSC lines derived from healthy controls.

We have generated two iPSC lines from skin biopsies of two healthy individuals. Skin fibroblasts were derived and reprogrammed using a Sendai virus-based approach. The resulting iPSC lines have normal karyotype, express stemness markers and can generate endoderm, mesoderm and ectoderm in vitro. These iPSC lines can be used as healthy controls in differentiation paradigms as well as backbone for gene editing experiments.

Defining research priorities in dystonia.

OBJECTIVE: Dystonia is a complex movement disorder. Research progress has been difficult, particularly in developing widely effective therapies. This is a review of the current state of knowledge, research gaps, and proposed research priorities. METHODS: The NIH convened leaders in the field for a 2-day workshop. The participants addressed the natural history of the disease, the underlying etiology, the pathophysiology, relevant research technologies, research resources, and therapeutic approaches and attempted to prioritize dystonia research recommendations. RESULTS: The heterogeneity of dystonia poses challenges to research and therapy development. Much can be learned from specific genetic subtypes, and the disorder can be conceptualized along clinical, etiology, and pathophysiology axes. Advances in research technology and pooled resources can accelerate progress. Although etiologically based therapies would be optimal, a focus on circuit abnormalities can provide a convergent common target for symptomatic therapies across dystonia subtypes. The discussions have been integrated into a comprehensive review of all aspects of dystonia. CONCLUSION: Overall research priorities include the generation and integration of high-quality phenotypic and genotypic data, reproducing key features in cellular and animal models, both of basic cellular mechanisms and phenotypes, leveraging new research technologies, and targeting circuit-level dysfunction with therapeutic interventions. Collaboration is necessary both for collection of large data sets and integration of different research methods.

Distinct cardiogenic preferences of two human embryonic stem cell (hESC) lines are imprinted in their proteomes in the pluripotent state.

Although both the H1 and HES2 human embryonic stem cell lines (NIH codes: WA01 and ES02, respectively) are capable of forming all three germ layers and their derivatives, various lines of evidence including the need to use different protocols to induce cardiac differentiation hint that they have distinct preferences to become chamber-specific heart cells. However, a direct systematic comparison has not been reported. Here we electrophysiologically demonstrated that the distributions of ventricular-, atrial- and pacemaker-like derivatives were indeed different (ratios=39:61:0 and 64:33:3 for H1 and HES2, respectively). Based on these results, we hypothesized the differences in their cardiogenic potentials are imprinted in the proteomes of undifferentiated H1 and HES2. Using multiplexing, high-resolution 2-D Differential In Gel Electrophoresis (DIGE) to minimize gel-to-gel variations that are common in conventional 2-D gels, a total of 2000 individual protein spots were separated. Of which, 55 were >2-fold differentially expressed in H1 and HES2 (p<0.05) and identified by mass spectrometery. Bioinformatic analysis of these protein differences further revealed candidate pathways that contribute to the H1 and HES2 phenotypes. We conclude that H1 and HES2 have predetermined preferences to become ventricular, atrial, and pacemaker cells due to discrete differences in their proteomes. These results improve our basic understanding of hESCs and may lead to mechanism-based methods for their directed cardiac differentiation into chamber-specific cardiomyocytes.

Functional consequences of overexpressing the gap junction Cx43 in the cardiogenic potential of pluripotent human embryonic stem cells.

Gap junctions, encoded by the connexin (Cx) multi-gene family, couple adjacent cells and underlie cell-cell communications. Previous mouse studies suggest that Cxs play an important role in development but their role in human cardiogenesis is undefined. Human embryonic stem cells (hESC) provide a unique model for studying human differentiation. Lentivirus-mediated stable overexpression of Cx43 in hESC (Cx43-hESC) did not affect colony morphology, karyotype and expression of pluripotency genes such as Oct4 but completely suppressed the formation of spontaneously beating, cardiomyocyte-containing clusters in embryoid bodies (EBs). Unlike control hEBs, the transcripts of several mesodermal markers (kallikrein, delta-globin, and CMP), ventricular myosin light chain and cardiac troponin I were absent or delayed. Transcriptomic and pathway analyses showed that 194 genes crucial for movement, growth, differentiation and maintenance were differentially expressed in Cx43-hESC. We conclude that Cx43 mediates the expression of an array of genes involved in human cardiogenesis, in addition to intercellular communication.

hsa-let-7c miRNA Regulates Synaptic and Neuronal Function in Human Neurons.

Non-coding RNA, including microRNA (miRNA) serves critical regulatory functions in the developing brain. The let-7 family of miRNAs has been shown to regulate neuronal differentiation, neural subtype specification, and synapse formation in animal models. However, the regulatory role of human let-7c (hsa-let-7c) in human neuronal development has yet to be examined. Let-7c is encoded on chromosome 21 in humans and therefore may be overexpressed in human brains in Trisomy 21 (T21), a complex neurodevelopmental disorder. Here, we employ recent developments in stem cell biology to show that hsa-let-7c mediates important regulatory epigenetic functions that control the development and functional activity of human induced neuronal cells (iNs). We show that overexpression of hsa-let-7c in human iNs derived from induced pluripotent stem (iPS), as well as embryonic stem (ES), cells leads to morphological as well as functional deficits including impaired neuronal morphologic development, synapse formation and synaptic strength, as well as a marked reduction of neuronal excitability. Importantly, we have assessed these findings over three independent genetic backgrounds, showing that some of these effects are subject to influence by background genetic variability with the most robust and reproducible effect being a striking reduction in spontaneous neural firing. Collectively, these results suggest an important function for let-7 family miRNAs in regulation of human neuronal development and raise implications for understanding the complex molecular etiology of neurodevelopmental disorders, such as T21, where let-7c gene dosage is increased.

Cyclin D and cdk4 are required for normal development beyond the blastula stage in sea urchin embryos.

cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.

Ethanol-mediated activation of the NLRP3 inflammasome in iPS cells and iPS cells-derived neural progenitor cells.

BACKGROUND: Alcohol abuse produces an enormous impact on health, society, and the economy. Currently, there are very limited therapies available, largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. RESULTS: Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs, but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition, a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover, a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells. CONCLUSIONS: This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models.

Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds.

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo. Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼ 3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼ 38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance.

Increased nicotine response in iPSC-derived human neurons carrying the CHRNA5 N398 allele.

Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk of addiction-associated phenotypes in humans yet little is known the underlying neural basis. Induced pluripotent stem cells (iPSCs) were derived from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in CHRNA5. To understand the impact of these nicotinic receptor variants in humans, we differentiated these iPSCs to dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Results show that N398 variant human DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. While both variants exhibited physiological properties consistent with mature neuronal function, the N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of nicotine in both DA and glutamatergic neurons. Glutamatergic N398 neurons responded to lower nicotine doses (0.1 µM) with greater frequency and amplitude but they also exhibited rapid desensitization, consistent with previous analyses of N398-associated nicotinic receptor function. This study offers a proof-of-principle for utilizing human neurons to study gene variants contribution to addiction.

Human embryonic stem cells: genetic manipulation on the way to cardiac cell therapies.

Almost 7 years after their first derivation from human embryos, a pressing urgency to deliver the promises of therapies based on human embryonic stem cells (hESC) has arisen. Protocols have been developed to support long-term growth of undifferentiated cells and partially direct differentiation to specific cell lineages. The stage has almost been set for the next step: transplantation in animal models of human disease. Here, we review the state-of-the-art with respect to the transplantation of embryonic stem cell-derived heart cells in animals. One problem affecting progress in this area and functional analysis in vivo in general, is the availability of genetically marked hESC. There are only a few cell lines that express reporter genes ubiquitously, and none is associated with particular lineages; a major hurdle has been the resistance of hESC to established infection and chemical transfection methodologies to introduce ectopic genes. The methods that have been successful are reviewed. We also describe the processes for generating a new, genetically-modified hESC line that constitutively expresses GFP as well as some of its characteristics, including its ability to form cardiomyocytes with electrophysiological properties of ventricular-like cells.

A P19Cl6 GFP reporter line to quantify cardiomyocyte differentiation of stem cells.

The clinical application of stem cell therapies is still limited by the ability to produce defined, differentiated cell populations in large numbers in culture. High throughput screens to identify factors which enhance differentiation to particular lineages and promote expansion of precursors in culture are dependent on the development of sensitive and reproducible assays for screening. Here we describe a bioassay to identify factors with cardiomyogenic activity which enhance the yield of cardiomyocytes from undifferentiated stem cells. The assay is based on a Green Fluorescent Protein (GFP) reporter under the transcriptional control of the 250 bp MLC-2v promoter expressed in pluripotent P19 embryonal carcinoma cells. We show that reporter expression is limited to developing cardiomyocytes and can be used to determine quantitatively the number of ventricular cardiomyocytes formed in cultures under inducing or non-inducing conditions. This assay differs from all others described previously in that it has an easily quantifiable readout, there is negligible background differentiation in the absence of exogenous cardiogenic factors and it is carried out feeder cell-free. Thus, it is entirely independent of competing differentiation inhibitory factors, such as leukemia inhibitory factor. Patch clamp electrophysiology of the GFP-positive cells confirmed their functional ventricular phenotype and indicated that selection on the basis of GFP would provide cells suitable for transplantation.

Spontaneous ATM Gene Reversion in A-T iPSC to Produce an Isogenic Cell Line.

A spontaneously reverted iPSC line was identified from an A-T subject with heterozygous ATM truncation mutations. The reverted iPSC line expressed ATM protein and was capable of radiation-induced phosphorylation of CHK2 and H2A.X. Genome-wide SNP analysis confirmed a match to source T cells and also to a distinct, non-reverted iPSC line from the same subject. Rearranged T cell receptor sequences predict that the iPSC culture originated as several independently reprogrammed cells that resolved into a single major clone, suggesting that gene correction likely occurred early in the reprogramming process. Gene expression analysis comparing ATM(-/-) iPSC lines to unrelated ATM(+/-) cells identifies a large number of differences, but comparing only the isogenic pair of A-T iPSC lines reveals that the primary pathway affected by loss of ATM is a diminished expression of p53-related mRNAs. Gene reversion in culture, although likely a rare event, provided a novel, reverted cell line for studying ATM function.

Persistent infection by HSV-1 is associated with changes in functional architecture of iPSC-derived neurons and brain activation patterns underlying working memory performance.

BACKGROUND: Herpes simplex virus, type 1 (HSV-1) commonly produces lytic mucosal lesions. It invariably initiates latent infection in sensory ganglia enabling persistent, lifelong infection. Acute HSV-1 encephalitis is rare and definitive evidence of latent infection in the brain is lacking. However, exposure untraceable to encephalitis has been repeatedly associated with impaired working memory and executive functions, particularly among schizophrenia patients. METHODS: Patterns of HSV-1 infection and gene expression changes were examined in human induced pluripotent stem cell (iPSC)-derived neurons. Separately, differences in blood oxygenation level-dependent (BOLD) responses to working memory challenges using letter n-back tests were investigated using functional magnetic resonance imaging (fMRI) among schizophrenia cases/controls. RESULTS: HSV-1 induced lytic changes in iPSC-derived glutamatergic neurons and neuroprogenitor cells. In neurons, HSV-1 also entered a quiescent state following coincubation with antiviral drugs, with distinctive changes in gene expression related to functions such as glutamatergic signaling. In the fMRI studies, main effects of schizophrenia (P = .001) and HSV-1 exposure (1-back, P = 1.76 × 10(-4); 2-back, P = 1.39 × 10(-5)) on BOLD responses were observed. We also noted increased BOLD responses in the frontoparietal, thalamus, and midbrain regions among HSV-1 exposed schizophrenia cases and controls, compared with unexposed persons. CONCLUSIONS: The lytic/quiescent cycles in iPSC-derived neurons indicate that persistent neuronal infection can occur, altering cellular function. The fMRI studies affirm the associations between nonencephalitic HSV-1 infection and functional brain changes linked with working memory impairment. The fMRI and iPSC studies together provide putative mechanisms for the cognitive impairments linked to HSV-1 exposure.