Identity crisis: exploring the boundaries of cell type identification in the age of single-cell transcriptomics

Food in history and culture Once upon a time, not all that long ago, human identity was generally viewed rather simply. It was assumed that identity achieved its final form in the course of childhood and adolescence, culminating in the famous Eriksonian “identity crisis,” the successful resolution of which ushered in a competent adulthood. While experts disputed just when and how the larger aspects of individual identity congealed – gender identity for instance – and argued as well about the relationship between individual and group identity, identity was not seen as something adults actively worked on or typically experienced conflict over. In recent years, prodded by feminist and queer theorists, students of identity have radically changed their views. Increasingly they see human identity as a continual work in progress, constructed and altered by the totality of life experience. While much of the work in support of this belief concentrates on the larger aspects of identity – especially gender, ethnicity, and sexual preference – in fact human identity involves many other categories. Identity is constructed in complex ways, more or less consciously and overtly. Some aspects of identity, in particular those listed above, are applicable both to individual identity and a person's identification as a member of a cohesive and coherent group. Other aspects of individual identity are more subtle, perhaps less prone to being problematized, and not linked to group membership in any obvious way.

INTRODUCTION: The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. METHODS: We combine high-dimensional flow cytometry with single cell transcriptomics to comprehensively purify and functionally characterize NSPCs from the developing human brain during mid-gestation. We utilize index sorting based on the combinatorial expression of cell-surface markers (immunophenotype) to directly link surface-marker expression to the expressed transcriptomes of single cells, allowing for rigorous validation of sort purity. We further demonstrate the functional properties of each identified cell type in vitro and in vivo. RESULTS: CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain.THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+ THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a novel transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell, which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. CONCLUSIONS: The results presented here are strong proof-of-concept that distinct cell types from the developing brain can be prospectively isolated based on the expression of multiple surface markers. The combined results from scRNA-seq, in vitro differentiation, and in vivo transplantation offers the most rigorous interrogation of NSPCs’ transcriptomic identities and functional behavior. The modular nature of our antibody panel allows for the easy expansion or simplification of the panel, based on the cell-type of interest, thus providing a valuable tool for future investigation of both molecular and functional NSPC heterogeneity.

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.

Replicating the native mechanical environment of cells in vitro is essential for accurately capturing their physiological behaviour as it occurs in vivo. While astrocytes have been extensively studied within the brain, leptomeningeal cells (LMCs), which reside in the arachnoid and pia mater layers of the meninges, have received comparatively less attention despite their essential structural and immunological functions at the brain-meningeal interface. These two tissues are in direct contact yet exhibit stark differences in mechanical properties; the brain possesses a Young's modulus in the low kilopascal (kPa) range, whereas the leptomeninges exhibit stiffness in the low megapascal (MPa) range. This pronounced mechanical contrast raises critical questions regarding the mechanosensitivity of LMCs and their adaptive responses to different substrate stiffnesses. In this study, the mechanobiological responses of primary human LMCs and astrocytes were investigated when cultured on substrates that either replicated or deviated from their native mechanical environments. Nanoindentation, morphological analysis, and protein expression profiling were employed to assess mechanosensitive behaviours. The results demonstrate that both LMCs and astrocytes are responsive to mechanical stimuli, though their capacity to adapt varies. LMCs are particularly sensitive to changes in substrate stiffness, especially when the mechanical properties diverge from their native environment. In contrast, astrocytes maintain more stable behaviour across a wider range of stiffnesses. However, both cell types exhibit diminished physiological relevance when grown on rigid gigapascal (GPa) plastic surfaces, underscoring the importance of using physiologically relevant stiffness in vitro. These findings establish a foundational understanding of how LMCs respond to mechanical alterations in two-dimensional cultures that mimic both the native and non-native stiffness environments, providing new insight into the mechanosensitivity of this overlooked but functionally significant cell type. STATEMENT OF SIGNIFICANCE: The leptomeninges, made up of the pia and arachnoid layers, protect the brain while also regulating cerebrospinal fluid, immune responses, and blood flow, helping maintain overall brain function. Despite being in direct contact, the brain and leptomeninges exhibit starkly different mechanical properties. The brain has a Young's modulus in the low kPa range, whereas the leptomeninges are significantly stiffer, with a Young's modulus in the MPa range. This sharp mechanical contrast raises important questions about how LMCs respond to their mechanical environment. Given that LMCs remain an understudied cell population, understanding their response to mechanical cues could provide new insights into their role in brain function and disease.

Atomic force microscopy studies of living bacterial cells in native soil and permafrost

The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties1–3. 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 by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-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. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification 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 integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.

How closely human organoids recapitulate cell-type diversity and cell-type maturation of their target organs is not well understood. We developed human retinal organoids with multiple nuclear and synaptic layers. We sequenced the RNA of 158,844 single cells from these organoids at six developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable ‘developed’ state at a rate similar to human retina development in vivo and the transcriptomes of organoid cell types converged towards the transcriptomes of adult peripheral retinal cell types. The expression of disease-associated genes was significantly cell-type specific in adult retina and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in adult human retinas.

Differences in Potassium Channel Composition Underlie Distinct Action Potential Kinetics in Transcriptomically Identified Neocortical Mouse Cell Types

The terrorist attacks of 9/11 have not only demonized and defamed the Muslim Americans but have also labeled the entire community as terrorists and extremists. The attacks have left a negative and detrimental image of Muslim Americans as terrorists, who till today are struggling and combating this stereotype and negative image. This paper explores the concepts of identity and identity crisis as they are communicated through Kamila Shamsie’s ‘Burnt Shadows’. These two fundamental concepts serve to be constructive lenses in the wake of the terrorist attacks of 9/11, as post 9/11 discourses have for the most part focused on Muslim American identity crisis, negative representation of the Islamic faith as fundamentalist Islam and representing Muslims as terrorists and extremists. The terrorist attacks of 9/11 will be represented as a site of paraxial engagement with both social and individual self-understandings and that representation of various aspects of identity in the aftermath of 9/11 allows for the process of identity negotiation which evolves phases of realization of the need to reinterpret identity and finally moving on to the phase of reclaiming identity. This paper will reveal how the identity crisis faced by the protagonist of the novel leads to an identity confusion surrounding the tragic incident of 9/11 and its aftermath where the convergence of fiction and reality presents the intricacy of defining the incident and of relating the self to the ruthless and inhuman hostility and eventually revealing the loss of a stable identity which leads to the protagonist’s struggle for reinterpreting and reclaiming his true identity.

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.

The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs1. Retinal cell types may have evolved to accommodate these varied needs, but this has not been systematically studied. Here we generated and integrated single-cell transcriptomic atlases of the retina from 17 species: humans, two non-human primates, four rodents, three ungulates, opossum, ferret, tree shrew, a bird, a reptile, a teleost fish and a lamprey. We found high molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (RGCs) and Müller glia), with transcriptomic variation across species related to evolutionary distance. Major subclasses were also conserved, whereas variation among cell types within classes or subclasses was more pronounced. However, an integrative analysis revealed that numerous cell types are shared across species, based on conserved gene expression programmes that are likely to trace back to an early ancestral vertebrate. The degree of variation among cell types increased from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified rodent orthologues of midget RGCs, which comprise more than 80% of RGCs in the human retina, subserve high-acuity vision, and were previously believed to be restricted to primates2. By contrast, the mouse orthologues have large receptive fields and comprise around 2% of mouse RGCs. Projections of both primate and mouse orthologous types are overrepresented in the thalamus, which supplies the primary visual cortex. We suggest that midget RGCs are not primate innovations, but are descendants of evolutionarily ancient types that decreased in size and increased in number as primates evolved, thereby facilitating high visual acuity and increased cortical processing of visual information.

A longstanding goal in biomedical research has been to create organotypic cocultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized three-dimensional (3D) coculture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D coculture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells (SMCs), fibroblasts, and epithelial cells (EpiCs). This study represents the first effort to combine these particular cell types into an organized bronchiole coculture. These cell layers were first cultured in 3D by magnetic levitation, and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin and eosin staining of the resulting coculture showed four distinct layers within the 3D coculture. Immunohistochemistry confirmed the phenotype of each of the four cell types and showed organized extracellular matrix formation, particularly, with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle α-actin, vimentin, and fibronectin demonstrate the maintenance of the phenotype for endothelial cells, SMCs, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% fetal bovine serum showed that EpiCs maintained the phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D cocultures that maintain the phenotype and induce extracellular matrix formation.

Responses to social cues, such as pheromones, can be modified by genotype, physiology, or environmental context. Honey bee queens produce a pheromone (queen mandibular pheromone; QMP) which regulates aspects of worker bee behavior and physiology. Forager bees are less responsive to QMP than young bees engaged in brood care, suggesting that physiological changes associated with behavioral maturation modulate response to this pheromone. Since 3',5'-cyclic guanosine monophosphate (cGMP) is a major regulator of behavioral maturation in workers, we examined its role in modulating worker responses to QMP. Treatment with a cGMP analog resulted in significant reductions in both behavioral and physiological responses to QMP in young caged workers. Treatment significantly reduced attraction to QMP and inhibited the QMP-mediated increase in vitellogenin RNA levels in the fat bodies of worker bees. Genome-wide analysis of brain gene expression patterns demonstrated that cGMP has a larger effect on expression levels than QMP, and that QMP has specific effects in the presence of cGMP, suggesting that some responses to QMP may be dependent on an individual bees' physiological state. Our data suggest that cGMP-mediated processes play a role in modulating responses to QMP in honey bees at the behavioral, physiological, and molecular levels.

This study explores the theoretical framework for constructing personalized pathways in receptive music therapy by integrating the Traditional Chinese Medicine (TCM) principle of “Three Categories of Etiological Factors System” with artificial intelligence (AI) technology. The three dimensions—temporal appropriateness, geographical appropriateness, and individual appropriateness - offers a holistic approach to personalized healthcare that aligns with modern precision medicine. Through systematic analysis of how these three dimensions apply to music therapy practice, we propose a comprehensive AI-based multidimensional assessment model and dynamic adjustment mechanism. The research establishes a theoretical system encompassing three core modules: individual characteristic identification, environmental factor analysis, and temporal pattern recognition. This framework enables the intelligent selection and dynamic optimization of therapeutic music based on patients' physiological states, psychological profiles, cultural backgrounds, circadian rhythms, seasonal variations, and environmental contexts. By leveraging machine learning algorithms and deep learning technologies, the system can process multimodal data to generate highly personalized treatment protocols and adjust them in real-time according to therapeutic responses. This integration of ancient medical wisdom with cutting-edge AI technology provides novel theoretical perspectives and practical pathways for achieving precision-oriented and intelligentized music therapy, potentially enhancing treatment efficacy and accessibility across diverse clinical settings including mental health, neurological rehabilitation, pain management, and pediatric developmental disorders.

The external globus pallidus (GPe) is traditionally viewed as a homogeneous relay in the indirect basal ganglia pathway that broadly suppresses movement. Using whole-brain anterograde axon mapping, rabies tracing, single-neuron reconstruction, and single-nucleus RNA sequencing, we reveal that the GPe is a major basal ganglia output nucleus, composed of anatomically and molecularly distinct populations. Besides the neurons with canonical projections, and projections to striatum and cortex, the GPe contains specific neuronal populations that target the thalamus and brainstem directly, including the parafascicular thalamus (GPePf) and the pedunculopontine nucleus (GPePPN). These projection-defined subpopulations exhibit distinct behavioral functions. GPe-PPN neurons are selectively suppressed at locomotor onset, and bidirectional manipulation of this pathway is sufficient to promote or suppress locomotion. Notably, D2-MSN stimulation upstream of these neurons evokes locomotion, and co-activation of GPePPN pathway blocks this D2-MSN-evoked locomotion, demonstrating a disinhibitory circuit for action stemming from D2-MSNs. In contrast, GPePf neurons are not engaged during locomotion but are selectively recruited during skilled forelimb actions, and their activation disrupts forelimb movements without affecting locomotion. Together, these findings establish the GPe as a basal ganglia output hub composed of modules that mediate distinct behaviors. This organization revises canonical models of the indirect pathway by demonstrating that D2-MSNs can also facilitate, rather than only suppress, movement depending on the downstream GPe output channels they engage.