Biology subjects

Hart, R. P.

Publications and source records attributed to Hart, R. P..

At least 19 recordsLinked to original sources

bioRxiv2026

Identification of alcohol use disorder-associated and ethanol-responsive proteins and candidate biomarkers using human cortical organoids

Genome-wide association studies of alcohol use disorder (AUD) have identified >100 loci, yet the functional impact and the downstream molecular mechanisms are not well understood. As functional effectors of genes, proteins provide direct insight into disease mechanisms. However, most proteomic alterations in postmortem human brains of AUD likely reflect a combination of inherited susceptibility (AUD-associated), the consequences of alcohol exposure (ethanol-responsive), other environmental and lifestyle factors, as well as sample preparation. Here, we profiled the proteome of human microglia-containing cortical organoids derived from human induced pluripotent stem cells from individuals with AUD (n=11) and without (n=5), paired with or without intermittent ethanol exposure. To maximize statistical power, donors were selected based on high or low genetic liability for AUD as measured by polygenic scores. Among 8,952 proteins that passed quality control, 1,038 were AUD-associated and 718 were ethanol-responsive. AUD-associated proteins were enriched for neuronal signaling, immune, mitochondrial, and extracellular matrix pathways, whereas ethanol-responsive proteins were enriched for RNA processing, ribosome biogenesis, protein translation, vesicle trafficking, and membrane transport pathways. Twenty ethanol-responsive proteins were replicated in a postmortem human prefrontal cortex proteomic dataset. Using UK Biobank plasma proteomics data, we identified 26 candidate AUD-associated biomarkers and 9 candidate ethanol-responsive biomarkers. We also found that major proteomic variation was significantly correlated with AUD polygenic scores. Together, these findings reveal distinct proteomic signatures of inherited susceptibility to AUD and ethanol exposure, providing new insights into mechanisms underlying AUD and identifying candidate circulating biomarkers for further investigation.

cell biology↗

bioRxiv2026

Female iPSC X-chromosome inactivation (XCI) erosion and its transcriptomic effects during CRISPR gene editing and neural differentiation

Human induced pluripotent stem cells (hiPSC) and iPSC-differentiated neural cells, in combination with CRISPR editing, are commonly used for studying neurodevelopmental and other brain disorders. Female iPSCs undergo random X-chromosome inactivation (XCI) via epigenetic silencing by noncoding X inactive specific transcript (XIST). It is known that female iPSCs may lose XIST expression, leading to XCI erosion that affects both X-linked and autosomal gene expression. However, the effects of CRSIPR editing and neural differentiation on XCI erosion in iPSC-derived neurons and how this may confound a real-world transcriptomic analysis of differentially expressed genes (DEGs) are poorly understood. Here, leveraging bulk RNA-seq of hundreds of CRISPR-edited female iPSC lines from four donor lines for 66 genes and single-cell RNA-seq of iPSC-derived neurons of a subset of 42 edited genes, we investigated the effects of XCI erosion during CRISPR editing and in iPSC-derived neurons. We found that XCI erosion was variable in CRISPR-edited female iPSCs and largely preserved in iPSC-derived neurons. Like in iPSCs, XIST in neurons predominately influenced the expression of X-linked genes; however, its effect on autosomal genes was more pronounced in single neurons. Mechanistically, XIST epigenetically causes allelic imbalance of both X-linked and autosomal genes, with the former showing stronger allele-specific expression (ASE) bias. Notably, XIST-induced ASE bias exhibited a conserved positional pattern at loci affecting neurodevelopmental genes across different female lines and cell types. Finally, we demonstrated a confounding effect of XCI erosion on DEG analyses in iPSC-derived neurons. These results have significant implications in hiPSC modeling of neurodevelopmental and other brain disorders.

genomics↗

bioRxiv2025

Ethanol induces neuroimmune dysregulation and soluble TREM2 generation in a human iPSC neuron, astrocyte, microglia triculture model

Alcohol use disorders (AUDs) affect substantial populations worldwide and increase the risk of developing cognitive impairments and alcohol-associated dementia. While chronic inflammatory signaling likely plays an important role in alcohol-associated neurological sequalae, the precise mechanisms underlying alcohol-associated neuropathology remain enigmatic. We hypothesize that alcohol leads to neuroimmune dysregulation among neurons, astrocytes, and microglia; and is perpetuated by innate immune signaling pathways involving cell-cell signaling. To investigate how alcohol dysregulates neuroimmune interactions in a human context, we constructed a triculture model comprising neurons, astrocytes, and microglia derived from human induced pluripotent stem cells. After exposure to ethanol, we observed significant differential gene expression relating to innate immune pathways, inflammation, and microglial activation. Microglial activation was confirmed with morphological analysis and expression of CD68, a lysosomal-associated membrane protein and marker for phagocytic microglial activation. A striking finding in our study was the elevation of TREM2 expression and, specifically, TREM2 alternative splice variants that are predicted to give rise to soluble TREM2. TREM2 has been reported to be a risk factor for Alzheimers disease. These results suggest that ethanol exposure in the brain may lead to increased microglial activation and production of soluble isoform named TREM2219 through alternate splicing. Deciphering the molecular and cellular mechanisms underpinning ethanol-related neuroimmune dysregulation within a human context promises to shed light on the etiology of AUD-related disorders, potentially contributing to the development of effective therapeutic strategies. HighlightsO_LIWe prepared a "triculture" of human iPSC-derived neurons, astrocytes and microglia C_LIO_LIEthanol treatment produces substantial changes in gene expression with prominent effects on neuroimmune signaling C_LIO_LISeveral microglia-specific genes are induced by ethanol in tricultures but not in cultures of microglia alone C_LIO_LITREM2 expression is increased following ethanol treatment and results indicate a differential splicing of isoforms, encoding a soluble form of TREM2 C_LI

neuroscience↗

bioRxiv2025

High-throughput measurements of neuronal activity in single human iPSC-derived glutamate neurons

Induced pluripotent stem cell (iPSC)-derived neurons provide a promising platform for studying neuronal function and modeling central nervous system (CNS) diseases. However, functional analysis of large populations of iPSC-derived neurons has been challenging. Here, we developed a high throughput strategy targeting N-methyl-D-aspartate receptors (NMDA-R) to enhance neuronal activity and reveal functional phenotypes in human iPSC-induced glutamatergic neurons (iGlut). Using a genetically encoded calcium indicator (GCaMP8f), we first demonstrate that using artificial cerebrospinal fluid (ACSF) lacking Mg2+ (Mg2+-free) significantly increases neuronal firing, and that firing is enhanced by a potentiator (glycine) but inhibited by the NMDA-R antagonist AP-V. Similarly, multi-electrode array (MEA) recordings also show robust firing in Mg2+-free ACSF. Lastly, single-cell patch-clamp electrophysiology confirms the high firing rates in Mg2+-free ACSF across multiple iPSC donor lines and also reveals iPSC donor-specific tonic and bursting firing phenotypes. This new methodology provides a scalable, high-throughput method to study neuronal activity in iGlut neurons while preserving single-cell resolution. The strategy also reveals different functional phenotypes, enabling detailed characterization of iGlut neurons in diverse applications such as CNS disease modeling and drug screening. These findings establish a versatile framework for future studies of neuronal network dynamics and individual excitability in iPSC-derived neuronal cultures.

neuroscience↗

bioRxiv2024

Defective DcpS Decapping Manifests in Creatine Deficiency Syndrome and Neurological impairment

Biallelic mutations in the DCPS gene disrupting the decapping activity of the scavenger decapping protein DcpS, leads to neurodevelopmental deficiencies and intellectual disability. However, the molecular basis for the neurogenesis defects in these individuals remains unknown. Here we show that cells derived from individuals with a DCPS mutation harbor a creatine deficiency and a corresponding elevation of the creatine precursor, guanidinoacetate (GAA). The altered metabolite levels are a consequence of a reduction in both the mRNA and protein levels for the enzyme that converts GAA into creatine, guanidinoacetate methyltransferase. Importantly, the compromised neurogenesis and neurite outgrowth phenotypes observed during the differentiation of DcpS mutant patient derived induced pluripotent stem cell differentiation into neurons was reversed upon supplementation of creatine monohydrate. These findings suggest creatine deficiency as the major underlying factor for the neurogenetic defect detected in DcpS mutant cells and a potential driver of the neurological deficiencies in affected individuals.

cell biology↗

bioRxiv2024

Integrated Single-Cell Multiomic Profiling of Caudate Nucleus Suggests Key Mechanisms in Alcohol Use Disorder

Alcohol use disorder (AUD) induces complex transcriptional and regulatory changes across multiple brain regions including the caudate nucleus, which remains understudied. Using paired single-nucleus RNA-seq and ATAC-seq on caudate samples from 143 human postmortem brains, including 74 with AUD, we identified 17 distinct cell types. We found that a significant portion of the alcohol-induced changes in gene expression occurred through altered chromatin accessibility. Notably, we identified novel transcriptional and chromatin accessibility differences in medium spiny neurons, impacting pathways such as RNA metabolism and immune response. A small cluster of D1/D2 hybrid neurons showed distinct differences, suggesting a unique role in AUD. Microglia exhibited distinct activation states deviating from classical M1/M2 designations, and astrocytes entered a reactive state partially regulated by JUND, affecting glutamatergic synapse pathways. Oligodendrocyte dysregulation, driven in part by OLIG2, was linked to demyelination and increased TGF-{beta}1 signaling from microglia and astrocytes. We also observed increased microglia-astrocyte communication via the IL-1{beta} pathway. Leveraging our multiomic data, we performed cell type-specific expression quantitative trait loci analysis, integrating that with public genome-wide association studies to identify AUD risk genes such as ADAL and PPP2R3C, providing a direct link between genetic variants, chromatin accessibility, and gene expression in AUD. These findings not only provide new insights into the genetic and cellular mechanisms in the caudate related to AUD but also demonstrate the broader utility of large-scale multiomic studies in uncovering complex gene regulation across diverse cell types, which has implications beyond the substance use field.

neuroscience↗

bioRxiv2024

Scaled and Efficient Derivation of Loss of Function Alleles in Risk Genes for Neurodevelopmental and Psychiatric Disorders in Human iPSC

Translating genetic findings for neurodevelopmental and psychiatric disorders (NPD) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, here we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop-codons (iSTOP) that lead to mRNA nonsense-mediated-decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 NPD genes. Using RNAseq, we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Interestingly, for three schizophrenia risk genes (SETD1A, TRIO, CUL1), despite the high efficiency of base editing, we only obtained heterozygous LoF alleles, suggesting their essential roles for cell growth. We replicated the reported neural phenotypes of SHANK3-haploinsufficiency and found CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.

genetics↗

bioRxiv2024

Polygenic Risk for Alcohol Use Disorder Affects Cellular Responses to Ethanol Exposure in a Human Microglial Cell Model

Polygenic risk scores (PRS) assess genetic susceptibility to Alcohol Use Disorder (AUD), yet their molecular implications remain underexplored. Neuroimmune interactions, particularly in microglia, are recognized as significant contributors to AUD pathophysiology. We investigated the interplay between AUD PRS and ethanol in human microglia derived from iPSCs from individuals with high- or low-PRS (HPRS or LPRS) of AUD. Ethanol exposure induced elevated CD68 expression and morphological changes in microglia, with differential responses between HPRS and LPRS microglial cells. Transcriptomic analysis revealed expression differences in MHCII complex and phagocytosis-related genes following ethanol exposure; HPRS microglial cells displayed enhanced phagocytosis and increased CLEC7A expression, unlike LPRS microglial cells. Synapse numbers in co-cultures of induced neurons with microglia after alcohol exposure were lower in HRPS co-cultures, suggesting possible excess synapse pruning. This study provides insights into the intricate relationship between AUD PRS, ethanol, and microglial function, potentially influencing neuronal functions in developing AUD.

neuroscience↗

bioRxiv2023

Dysregulated neuroimmune interactions and sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

Human immunodeficiency virus type-1 (HIV-1) associated neurocognitive disorder (HAND) affects up to half of HIV-1 positive patients with long term neurological consequences, including dementia. There are no effective therapeutics for HAND because the pathophysiology of HIV-1 induced glial and neuronal functional deficits in humans remains enigmatic. To bridge this knowledge gap, we established a model simulating HIV-1 infection in the central nervous system using human induced pluripotent stem cell (iPSC) derived microglia combined with sliced neocortical organoids. Upon incubation with two replication-competent macrophage-tropic HIV-1 strains (JRFL and YU2), we observed that microglia not only became productively infected but also exhibited inflammatory activation. RNA sequencing revealed a significant and sustained activation of type I interferon signaling pathways. Incorporating microglia into sliced neocortical organoids extended the effects of aberrant type I interferon signaling in a human neural context. Collectively, our results illuminate the role of persistent type I interferon signaling in HIV-1 infected microglial in a human neural model, suggesting its potential significance in the pathogenesis of HAND. Highlights of the workO_LIHIV-1 productively infects iPSC-derived microglia and triggers inflammatory activation. C_LIO_LIHIV-1 infection of microglia results in sustained type I interferon signaling. C_LIO_LIMicroglia infected by HIV-1 incorporate into sliced neocortical organoids with persistent type I interferon signaling and disease risk gene expression. C_LI

neuroscience↗

bioRxiv2023

Upregulated GIRK2 counteracts ethanol-induced changes in excitability and respiration in human neurons

Genome-wide association analysis (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified non-coding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G protein-coupled inwardly-rectifying potassium channel that regulates neuronal excitability. How changes in GIRK2 affect human neuronal excitability and the response to repeated ethanol exposure is poorly understood. Here, we studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multi-electrode-arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 days of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-dependent changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons. SIGNIFICANCE STATEMENTAlcohol use disorder (AUD) is a major health problem that has worsened since COVID, affecting over 100 million people worldwide. While it is known that heritability contributes to AUD, specific genes and their role in neuronal function remain poorly understood, especially in humans. In the current manuscript, we focused on the inwardly-rectifying potassium channel GIRK2, which has been identified in an AUD-endophenotype genome-wide association study. We used human excitatory neurons derived from healthy donors to study the impact of GIRK2 expression. Our results reveal that elevated GIRK2 counteracts ethanol-induced increases in glutamate response and intracellular calcium, as well as deficits in activity-dependent mitochondrial respiration. The role of GIRK2 in mitigating ethanol-induced hyper-glutamatergic and mitochondrial offers therapeutic promise for treating AUD.

neuroscience↗

bioRxiv2022

Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium (GIRK2) channel subunit 2, have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants ( Affected: AF) and four control subjects without variants ( Unaffected: UN). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.

neuroscience↗

bioRxiv2022

The amyloid precursor protein modulates the position and length of the axon initial segment offering a new perspective on Alzheimer's disease genetics

The small A{beta} peptide has been hypothesized to be the main driver of Alzheimers disease (AD). A{beta} is a proteolytic cleavage product of a larger protein, the amyloid precursor protein (APP), whose normal functions remain largely unexplored. We report here activities of the full-length APP protein that relate directly to the etiology of AD. Increasing neuronal activity leads to a rapid increase in App gene expression. In both cultures of mouse cortical neurons and human iPSC-derived neurons, elevated APP protein changes the structure of the axon initial segment (AIS), the site of action potential initiation. In neurons with elevated APP, the AIS shortens in length and shifts in position away from the cell body. Both changes would be expected to reduce neuronal excitability. The AIS effects are due to the cell-autonomous actions of APP; exogenous A{beta} - either fibrillar or oligomeric - has no effect. The findings relate directly to AD in several ways. In culture, APP carrying the Swedish familial AD mutation (APPSwe) induces stronger AIS changes than wild type APP. Ankyrin G and {beta}IV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, and APPSwe binds more avidly than wild type APP. Finally, neurons in the frontal cortex of humans with sporadic AD reveal histologically elevated levels of APP protein that invade the domain of the AIS, whose length is significantly shorter than that found in healthy control neurons. The findings offer an alternative explanation for the effects of at least some familial AD mutations. SignificanceIn familial Alzheimers disease (AD) the linkage between the genetics of APP, the neuropathology of the amyloid plaques and the symptoms of dementia are one of the strongest pieces of evidence supporting the amyloid cascade hypothesis - a conceptualization that marks the A{beta} peptide as the root cause of AD. Yet, formally, the genetics only point to APP, not its A{beta} breakdown product. We report here that the full-length APP protein affects the properties of the axon initial segment and through these changes serves as a dynamic regulator of neuronal activity. We propose that this newly discovered APP function offers a different, A{beta}-independent, view of the genetic evidence.

neuroscience↗

bioRxiv2021

Type I Interferon Signaling Drives Microglial Dysfunction and Senescence in Human iPSC Models of Down Syndrome and Alzheimers Disease

Microglia are critical for brain development and play a central role in Alzheimers disease (AD) etiology. Down syndrome (DS), also known as trisomy 21, is the most common genetic origin of intellectual disability and the most common risk factor for AD. Surprisingly, little information is available on the impact of trisomy of human chromosome 21 (Hsa21) on microglia in DS brain development and AD in DS (DSAD). Using our new induced pluripotent stem cell (iPSC)-based human microglia-containing cerebral organoid and chimeric mouse brain models, here we report that DS microglia exhibit enhanced synaptic pruning function during brain development. Consequently, electrophysiological recordings demonstrate that DS microglial mouse chimeras show impaired synaptic functions, as compared to control microglial chimeras. Upon being exposed to human brain tissue-derived soluble pathological tau, DS microglia display dystrophic phenotypes in chimeric mouse brains, recapitulating microglial responses seen in human AD and DSAD brain tissues. Further flow cytometry, single-cell RNA- sequencing, and immunohistological analyses of chimeric mouse brains demonstrate that DS microglia undergo cellular senescence and exhibit elevated type I interferon signaling after being challenged by pathological tau. Mechanistically, we find that shRNA-mediated knockdown of Hsa21encoded type I interferon receptor genes, IFNARs, rescues the defective DS microglial phenotypes both during brain development and in response to pathological tau. Our findings provide first in vivo evidence supporting a paradigm shifting theory that human microglia respond to pathological tau by exhibiting accelerated senescence and dystrophic phenotypes. Our results further suggest that targeting IFNARs may improve microglial functions during DS brain development and prevent human microglial senescence in DS individuals with AD.

neuroscience↗

bioRxiv2021

Analyses of the Autism-associated Neuroligin-3 R451C Mutation in Human Neurons Reveals a Gain-of-Function Synaptic Mechanism

Mutations in many synaptic genes are associated with autism spectrum disorders (ASDs), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C-substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C-mutation on human neurons has not been investigated. Here, we generated human knock-in neurons with the NLGN3 R451C-mutation. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C-mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses. No significant cell death and endoplasmic reticulum stress were detected. Importantly, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons that corresponded to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C-mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASDs.

neuroscience↗

bioRxiv2021

Optimized Splitting of RNA Sequencing Data by Species

Gene expression studies using chimeric xenograft transplants or co-culture systems have proven to be valuable to uncover cellular dynamics and interactions during development or in disease models. However, the mRNA sequence similarities among species presents a challenge for accurate transcript quantification. To identify optimal strategies for analyzing mixed-species RNA sequencing data, we evaluate both alignment-dependent and alignment-independent methods. Alignment of reads to a pooled reference index is effective, particularly if optimal alignments are used to classify sequencing reads by species, which are re-aligned with individual genomes, generating >97% accuracy across a range of species ratios. Alignment-independent methods, such as Convolutional Neural Networks, which extract the conserved patterns of sequences from two species, classify RNA sequencing reads with over 85% accuracy. Importantly, both methods perform well with different ratios of human and mouse reads. Our evaluation identifies valuable and effective strategies to dissect species composition of RNA sequencing data from mixed populations.

bioinformatics↗

bioRxiv2021

mRNA-decapping associated DcpS enzyme controls critical steps of neuronal development

Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development, and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.

neuroscience↗

bioRxiv2019

Strategies for Integrating Single-Cell RNA Sequencing Results With Multiple Species

Single-cell RNA sequencing (scRNAseq) is a robust technology for parsing gene expression in individual cells from a tissue or other complex source. One application involves experiments where cells from multiple species are recovered from a single sample, such as when human cells are transplanted into an animal model. We transplanted microglial precursor cells into newborn mouse brain and then recovered unenriched cortical tissue six months later. Dissociated cells were assessed by scRNAseq. The default method for analyzing these results begins by aligning sequencing reads with a mixture of both mouse and human reference genomes. While this clearly identifies the human cells as a distinct cluster, the clustering is artificially driven by expression from non-comparable gene identifiers from different species. We devised a method for translating expression counts from human to mouse and evaluated four algorithms for parsing mixed-species scRNAseq data. Our optimal approach split raw sequencing reads according to the best alignment score in each genome, and then re-aligned reads only with the appropriate genome. After gene symbol translation, pooled results indicate that cell types are more appropriately clustered and that differential expression analysis identifies species-specific patterns. This method should be applicable to any mixed-species scRNAseq experiment.\n\nSummary of optimal strategyO_LIMixed-species scRNAseq data are aligned with mixture of mouse and human reference genomes\nC_LIO_LIThe BAM file is scanned to find the best alignment score for each sequencing read identifier; these are used to split the paired FASTQ files into two sets of files\nC_LIO_LIEach set of species-specific, paired FASTQ files is re-aligned with only the appropriate reference genome\nC_LIO_LIRaw counts imported into Seurat\nC_LIO_LIThe human counts table is translated to mouse gene symbols using a custom HomoloGene translation table\nC_LIO_LIResults are merged and analyzed\nC_LI

bioinformatics↗

bioRxiv2019

Xenotransplantation of Human PSC-derived Microglia Creates a Chimeric Mouse Brain Model that Recapitulates Features of Adult Human Microglia

Microglia, the brain-resident macrophages, exhibit highly dynamic functions in neurodevelopment and neurodegeneration. Human microglia possess unique features as compared to mouse microglia, but our understanding of human microglial functions is largely limited by an inability to obtain human microglia under homeostatic states. We developed a human pluripotent stem cell (hPSC)-based microglial chimeric mouse brain model by transplanting hPSC-derived primitive macrophage precursors into neonatal mouse brains. The engrafted human microglia widely disperse in the brain and replace mouse microglia in corpus callosum at 6 months post-transplantation. Single-cell RNA-sequencing of the microglial chimeric mouse brains reveals that xenografted hPSC-derived microglia largely retain human microglial identity, as they exhibit signature gene expression patterns consistent with physiological human microglia and recapitulate heterogeneity of adult human microglia. Importantly, the engrafted hPSC-derived microglia exhibit dynamic response to cuprizone-induced demyelination and species-specific transcriptomic differences in the expression of neurological disease-risk genes in microglia. This model will serve as a novel tool to study the role of human microglia in brain development and degeneration.

neuroscience↗