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Opinion statement The adult mammalian heart has limited capacity for regeneration, and any major injury such as a myocardial infarction results in the permanent loss of up to 1 billion cardiomyocytes. The field of cardiac cell therapy aims to replace these lost contractile units with de novo cardiomyocytes to restore lost systolic function and prevent progression to heart failure. Arguably, the ideal cell for this application is the human cardiomyocyte itself, which can electromechanically couple with host myocardium and contribute active systolic force. Pluripotent stem cells from human embryonic or induced pluripotent lineages are attractive sources for cardiomyocytes, and preclinical investigation of these cells is in progress. Recent work has focused on the efficient generation and purification of cardiomyocytes, tissue engineering efforts, and examining the consequences of cell transplantation from mechanical, vascular, and electrical standpoints. Here we discuss historical and contemporary aspects of pluripotent stem cell-based cardiac cell therapy, with an emphasis on recent preclinical studies with translational goals.

Spatial transcriptomics enables the precise mapping of gene expression patterns within tissue architecture, offering unprecedented insights into cellular interactions, tissue heterogeneity, and disease pathology that are unattainable with traditional transcriptomic approaches. We present a tool for processing spatial transcriptomics data, SCALPEL (Spatial Cell Analysis, Labeling, Processing, and Expression Linking). SCALPEL is specifically designed to support the analysis of large, atlas-level datasets. Our new workflow features advanced 3D segmentation optimized for dense and heterogeneous tissues, refined filtering criteria, and transcriptome-based doublet detection to remove low-quality or artifactual cells. Cell type label transfer from existing taxonomies is further improved through updated filtering thresholds. Spatial domain detection is incorporated to capture local transcriptomic organization, and tissue sections are registered to the Allen Mouse Brain Common Coordinate Framework version 3 (CCFv3) for precise anatomical alignment. Genome-wide expression imputation from single-cell RNA-sequencing (scRNAseq) further enriches the dataset. Crucially, we benchmark the performance of this updated pipeline against a previously published version of our whole-mouse-brain (WMB) dataset (Yao et al., 2023b), demonstrating substantial improvements in cell number, expression profile clarity, and spatial registration. These advances provide a robust foundation for downstream spatial analyses and set a new standard for large-scale spatial transcriptomics studies.

Substance use disorders (SUD) and drug addiction are major threats to public health, impacting not only the millions of individuals struggling with SUD, but also surrounding families and communities. One of the seminal challenges in treating and studying addiction in human populations is the high prevalence of co-morbid conditions, including an increased risk of contracting a human immunodeficiency virus (HIV) infection. Of the ~15 million people who inject drugs globally, 17% are persons with HIV. Conversely, HIV is a risk factor for SUD because chronic pain syndromes, often encountered in persons with HIV, can lead to an increased use of opioid pain medications that in turn can increase the risk for opioid addiction. We hypothesize that SUD and HIV exert shared effects on brain cell types, including adaptations related to neuroplasticity, neurodegeneration, and neuroinflammation. Basic research is needed to refine our understanding of these affected cell types and adaptations. Studying the effects of SUD in the context of HIV at the single-cell level represents a compelling strategy to understand the reciprocal interactions among both conditions, made feasible by the availability of large, extensively-phenotyped human brain tissue collections that have been amassed by the Neuro-HIV research community. In addition, sophisticated animal models that have been developed for both conditions provide a means to precisely evaluate specific exposures and stages of disease. We propose that single-cell genomics is a uniquely powerful technology to characterize the effects of SUD and HIV in the brain, integrating data from human cohorts and animal models. We have formed the Single-Cell Opioid Responses in the Context of HIV (SCORCH) consortium to carry out this strategy.

Background Alzheimer’s disease (AD) is clinically characterized by a progressive cognitive decline associated with stereotyped accumulation of amyloid‐beta (Aβ) plaques and hyperphosphorylated Tau (pTau) tangles across brain regions. While histopathology has revealed patterns of regional involvement, the molecular and cellular events that accompany and potentially drive this progression remain incompletely understood. Method We applied single nucleus RNA sequencing (RNAseq), ATAC‐seq, and Multiome profiling to over 7 million high‐quality nuclei from 10 brain regions—spanning medial and lateral entorhinal cortices, hippocampus, multiple temporal and frontal cortical areas, and primary visual cortex—sampled from the same cohort of 84 aged human donors across the AD spectrum (3 regions from all donors, 7 from those without severe co‐morbidities). We predicted the cell‐type for each nucleus by mapping to an expanded BRAIN initiative cell‐type taxonomy, which included AD‐associated non‐neuronal states and ∼70 brain region‐specific neuronal types. These datasets were paired with regional quantitative measurements of Aβ (6e10), pTau (AT8), and other protein pathologies, as well as cellular stains for neurons, microglia, and astrocytes. Result We inferred two distinct protein pathology accumulation patterns across brain regions: neocortical areas accumulated Aβ prior to pTau, whereas hippocampus and entorhinal cortex had early and, in some cases, substantial pTau burden independent of Aβ. Among neocortical regions, accumulation of AT8 beyond the temporal medial lobe strongly associated with dementia. In analyzing cell‐type abundance differences associated with higher levels of pTau pathology, we identified shared motifs of selective neuronal loss consistent with our previous observations from the middle temporal gyrus. These included loss of L2/3 and L5 intratelencephalic excitatory neurons and several types of inhibitory interneurons (e.g., Vip, Sst, Pvalb). These same vulnerable inhibitory types were also reduced in hippocampal and entorhinal regions, when present, and we observed parallel increases in astrocyte, microglial, and oligodendrocyte precursor cells. Additionally, we observed a decrease in specific, regionally specialized neuron subtypes in the hippocampus, entorhinal cortex, and visual cortex. Conclusion Our multimodal, multi‐region single‐cell atlas reveals common and region‐specific patterns of cellular vulnerability in AD. These cell‐types, particularly those commonly affected in distinct neural circuits, could serve as candidate therapeutic targets and biomarkers.

Alzheimer's disease (AD) is clinically characterized by a progressive cognitive decline associated with stereotyped accumulation of amyloid-beta (Aβ) plaques and hyperphosphorylated Tau (pTau) tangles across brain regions. While histopathology has revealed patterns of regional involvement, the molecular and cellular events that accompany and potentially drive this progression remain incompletely understood. We applied single nucleus RNA sequencing (RNAseq), ATAC-seq, and Multiome profiling to over 7 million high-quality nuclei from 10 brain regions-spanning medial and lateral entorhinal cortices, hippocampus, multiple temporal and frontal cortical areas, and primary visual cortex-sampled from the same cohort of 84 aged human donors across the AD spectrum (3 regions from all donors, 7 from those without severe co-morbidities). We predicted the cell-type for each nucleus by mapping to an expanded BRAIN initiative cell-type taxonomy, which included AD-associated non-neuronal states and ∼70 brain region-specific neuronal types. These datasets were paired with regional quantitative measurements of Aβ (6e10), pTau (AT8), and other protein pathologies, as well as cellular stains for neurons, microglia, and astrocytes. We inferred two distinct protein pathology accumulation patterns across brain regions: neocortical areas accumulated Aβ prior to pTau, whereas hippocampus and entorhinal cortex had early and, in some cases, substantial pTau burden independent of Aβ. Among neocortical regions, accumulation of AT8 beyond the temporal medial lobe strongly associated with dementia. In analyzing cell-type abundance differences associated with higher levels of pTau pathology, we identified shared motifs of selective neuronal loss consistent with our previous observations from the middle temporal gyrus. These included loss of L2/3 and L5 intratelencephalic excitatory neurons and several types of inhibitory interneurons (e.g., Vip, Sst, Pvalb). These same vulnerable inhibitory types were also reduced in hippocampal and entorhinal regions, when present, and we observed parallel increases in astrocyte, microglial, and oligodendrocyte precursor cells. Additionally, we observed a decrease in specific, regionally specialized neuron subtypes in the hippocampus, entorhinal cortex, and visual cortex. Our multimodal, multi-region single-cell atlas reveals common and region-specific patterns of cellular vulnerability in AD. These cell-types, particularly those commonly affected in distinct neural circuits, could serve as candidate therapeutic targets and biomarkers.

The telencephalon of the mammalian brain comprises multiple regions and circuit pathways that play adaptive and integrative roles in a variety of brain functions. There is a wide array of GABAergic neurons in the telencephalon; they play a multitude of circuit functions, and dysfunction of these neurons has been implicated in diverse brain disorders. In this study, we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished by utilizing 611,423 single-cell transcriptomes from the comprehensive and high-resolution transcriptomic and spatial cell type atlas for the adult whole mouse brain we have generated, supplemented with an additional single-cell RNA-sequencing dataset containing 99,438 high-quality single-cell transcriptomes collected from the pre- and postnatal developing mouse brain. We present a hierarchically organized adult telencephalic GABAergic neuronal cell type taxonomy of 7 classes, 52 subclasses, 284 supertypes, and 1,051 clusters, as well as a corresponding developmental taxonomy of 450 clusters across different ages. Detailed charting efforts reveal extraordinary complexity where relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types can often be found in distant and diverse brain regions indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Additionally, we find various spatial dimensions of both discrete and continuous variations among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal and most pallidal GABAergic neuronal types emerge simultaneously during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell type taxonomy can serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

Sleep apnea is a respiratory disorder characterized by periods of sleep during which no effective breaths are taken. Recurrent episodes of apnea caused by upper airway obstruction and/or cessation of central respiratory signaling results in repeated bouts of hypoxemia. Sleep apnea is quite common, affecting approximately 10% of adults in western nations, but a large proportion of patients remain undiagnosed and untreated. Continuous positive air pressure devices, or CPAP therapy, are effective therapeutics to maintain upper airway patency and prevent apneas, but are not universally available nor tolerated by sleep apnea patients. Obesity, the single biggest risk factor for the development of sleep apnea, is even more common and its prevalence is expected to rise. Therefore, sleep apnea represents a considerable health burden for the world population. Patients with sleep apnea have a greater risk of developing cardiovascular complications, such as hypertension and stroke, and as a result, much research has focused on the mechanisms of damage to the cardiovascular system. However, OSA is also known to impact the central nervous system. Some proportion of sleep apneas are generated by a failure of the central respiratory network to signal properly. Furthermore, patients with untreated sleep apnea experience mild cognitive impairments and are reported to have alterations in the activity of a number of brain regions. Even so, the mechanisms by which sleep apnea affects the central nervous system remain largely unknown. As both a critical regulator of learning and memory and one of only two structures in the mammalian brain capable of adult neurogenesis, the hippocampus may be particularly at risk in untreated sleep apnea. Indeed, individuals with sleep apnea experience mild cognitive impairment associated with changes to the hippocampus. Although oxygen homeostasis is a well-recognized factor that can influence multiple hippocampal processes, the impact of intermittent hypoxia (IH), a principal consequence of sleep apnea, on hippocampal neurophysiology remains unclear. I hypothesized that intermittent hypoxia would cause dysfunction in multiple stages of adult neural development to impair circuit function of the dentate gyrus (DG), thus contributing to injury of the hippocampus. The studies related in this dissertation utilize behavioral, electrophysiological, and immunohistological techniques to describe the effects of chronic intermittent hypoxia (IH) exposure on the neurophysiology of the murine hippocampus. IH impaired spatial memory in the Barnes maze apparatus and correlated with attenuated long-term potentiation (LTP) in the DG. Immunohistological analyses revealed that IH differentially perturbs adult neurogenesis by decreasing the number of new-born neurons, while simultaneously increasing neuroprogenitor cell proliferation. Although administration of the superoxide anion scavenger antioxidant, MnTMPyP, mitigated LTP suppression and prevented adult born neuron loss, IH-dependent proliferation of neuroprogenitor cells was unaffected. These data demonstrate that IH disrupts multiple processes in the DG that are both dependent on, and independent of, reactive oxygen species (ROS). These novel findings identify IH-induced changes in cellular and functional correlates of hippocampal learning and memory that likely contribute to cognitive deficits in sleep apnea. Further work is required to determine the non-ROS-mediated mechanisms that affect the neural progenitor pool of the hippocampus and whether either mechanism could serve as a future target for sleep apnea therapeutics.

Image-based spatial transcriptomics platforms are powerful tools often used to identify cell populations and describe gene expression in intact tissue. Spatial experiments return large, high-dimensional datasets and several open-source software packages are available to facilitate analysis and visualization. The outputs of spatial transcriptomics platforms are typically imperfect. For example, local variations in transcript detection probability are common. Software tools to characterize imperfections and their impact on downstream analyses are lacking so the data quality is assessed manually, a laborious and often a subjective process. Here we describe imperfections in a dataset of 641 fresh-frozen adult mouse brain sections collected using the Vizgen MERSCOPE. Common imperfections included the local loss of tissue from the section, tissue outside the imaging volume due to detachment from the coverslip, transcripts missing due to dropped images, varying detection probability through space, and differences in transcript detection probability between experiments. We describe the incidence of each imperfection and the likely impact on the accuracy of cell type labels. We develop MerQuaCo, open-source code that detects and quantifies imperfections without user input, facilitating the selection of sections for further analysis with existing packages. Together, our results and MerQuaCo facilitate rigorous, objective assessment of the quality of spatial transcriptomics results.

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single-cell RNA-sequencing (scRNA-seq) dataset of ~7 million cells profiled, and a spatially resolved transcriptomic dataset of ~4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

Alzheimer's disease (AD) is the most common cause of dementia in older adults. Neuropathological and imaging studies have demonstrated a progressive and stereotyped accumulation of protein aggregates, but the underlying molecular and cellular mechanisms driving AD progression and vulnerable cell populations affected by disease remain coarsely understood. The current study harnesses single cell and spatial genomics tools and knowledge from the BRAIN Initiative Cell Census Network to understand the impact of disease progression on middle temporal gyrus cell types. We used image-based quantitative neuropathology to place 84 donors spanning the spectrum of AD pathology along a continuous disease pseudoprogression score and multiomic technologies to profile single nuclei from each donor, mapping their transcriptomes, epigenomes, and spatial coordinates to a common cell type reference with unprecedented resolution. Pseudo-progression analysis showed two major epochs corresponding with a slow early increase in pathology and a later exponential increase that correlated with cognitive decline. The early phase included inflammatory microglial and reactive astrocyte component, as well as a selective loss of Sst+ inhibitory neuron types in superficial cortical layers, loss of myelinating oligodendrocytes, and up-regulation of a re-myelination program by OPCs. The later phase involved loss of excitatory neurons and Pvalb and Vip neuron subtypes also predominantly in superficial layers. These cell vulnerabilities were also seen in prefrontal cortex and replicated by other independent studies when integrated with the BRAIN Initiative reference. Study data and exploratory tools are freely available to accelerate progress in AD research at SEA-AD.org.Competing Interest StatementThe authors have declared no competing interest.Footnotes* The revised manuscript now features: 1) Description of a single nucleus RNA sequencing dataset generated from a new cortical region, the dorsolateral prefrontal cortex (DLPFC) 2) An integrated atlas across 10 publicly available single cell studies that also profiled the DLPFC in AD donors 3) New data and analysis of MERFISH spatial analyses 4) Additional analyses on non-neuronal cells* http://sea-ad.org* https://adknowledgeportal.synapse.org/Explore/Studies/DetailsPage?Study=syn26223298* https://registry.opendata.aws/allen-sea-ad-atlas/