Biology subjects

Grubb, S.

Publications and source records attributed to Grubb, S..

10 recordsLinked to original sources

bioRxiv2026

The Genetic Discovery (GeDi) resource: Deep characterization of a multi-ancestry panel of human reference iPSCs for functional genomic studies

Human induced pluripotent stem cells (iPSCs) are essential tools for disease modelling, drug discovery, and functional genomics, yet the field remains anchored to a small number of reference lines, limiting our understanding of how genetic background shapes cellular phenotypes across human populations. Here we describe the Genetic Discovery iPSC panel (GeDi), eight clonal iPSC lines reprogrammed from male and female donors that broadly span global human population diversity. Ancestry analysis confirmed that each line's genetic profile is concordant with donor self-reported ancestry. All GeDi lines are pluripotent and passed comprehensive quality control, demonstrating Sendai virus clearance, intact p53 pathway integrity, reproducible proliferative capacity, and compatibility with standardized genome editing workflows. Transcriptome profiling across five differentiated lineages covered 80% of annotated protein-coding genes and revealed stable donor-intrinsic variability in gene expression within each lineage. Haplotype-resolved genome assemblies were used to catalog structural variants and regions of absence of heterozygosity invisible to short-read sequencing. Whole-genome methylation analysis confirmed successful epigenetic reprogramming, catalogued the status of imprinted loci and captured the early dynamics of erosion of X chromosome inactivation (XCI) in female lines. The GeDi panel is available from The Jackson Laboratory as a quality-controlled resource designed to support functional genomic studies across varied genetic backgrounds.

cell biology↗

bioRxiv2025

Pericyte and Endothelial Primary Cilia and Centrioles have Disparate Organization Across the Brain Microvasculature

The brain microvascular functions are strongly influenced by the local microenvironment and cellular organization. Intracellular organelles, including primary cilia and centrioles, play critical roles in sensing and transmitting environmental cues and maintaining vascular integrity. However, their distribution across the brain vasculature remains poorly understood. In this study, we utilized publicly available large-volume electron microscopy datasets encompassing the cerebral vasculature from pial arterioles through parenchymal capillaries to pial venules. We systematically analyzed the cellular organization and characterized the distribution of primary cilia and centrioles in the mouse and human brain microvasculature. We found primary cilia exclusively on human cortical endothelial cells (ECs), indicating inter-species differences between mouse and human. Primary cilia were frequently present on mural cells (MCs, smooth muscle cells or pericytes) surrounding venules and capillaries but rarely observed on arterioles in both mouse and human brains. These MC primary cilia exhibited heterogeneity in ciliogenesis, including cells with ciliary pockets, surface cilia, and a hybrid configuration we refer as a partial pocket. In the mouse brain, many MC primary cilia were closely ensheathed by astrocytic endfeet and occasionally extended between them to establish proximity to synapses, whereas all primary cilia in the human brain were confined within the basal lamina. Our analysis of cellular density revealed similar EC densities between arterioles and venules in mice, but not in human. EC centrioles were consistently positioned against the direction of blood flow relative to the nuclei, suggesting that they may serve as a structural marker for flow direction. Collectively, these findings provide a comprehensive characterization of primary cilia and centrioles, highlighting distinct interspecies differences between mouse and human brain microvasculature. The proximity to neural cells and gradient distribution of these subcellular structures suggest that they may act as antennae for sensing mechanical and chemical signals within the brain microvascular environment.

neuroscience↗

bioRxiv2025

Distinct Ca2+ signatures of leptomeningeal fibroblast subgroups in awake mouse brains in health and inflammation

The leptomeninges, composed of the arachnoid mater, and pia mater, contains distinct subgroups of fibroblasts that differ in location and transcriptomic profiles. These fibroblasts contribute to the blood-cerebrospinal fluid barrier under physiological conditions, participate in fibrosis, and support blood-brain barrier integrity during injury and disease. However, their Ca2+ signaling profiles and underlying mechanisms in health and disease remain poorly understood. In this study, we divided leptomeningeal fibroblasts into three subgroups based on their locations: arachnoid fibroblasts, pia mater fibroblasts and perivascular fibroblasts. We employed two-photon microscopy in awake transgenic mice expressing Ca{superscript 2} indicators in leptomeningeal fibroblasts to investigate spontaneous and behaviorally evoked Ca{superscript 2} transients across different fibroblast subgroups. We found that each subgroup exhibits a distinct Ca{superscript 2} activity profile, with pia mater fibroblasts showing the highest-amplitude Ca{superscript 2} transients. Moreover, these fibroblasts displayed unique responses to both whisker air-puff stimulation and locomotion. We further demonstrated, using a chronically implanted cannula beneath the cranial window, that locomotion-associated vasodilation is followed by TRPV4 channel-mediated fibroblast Ca{superscript 2} elevations. Finally, systemic inflammation induced by lipopolysaccharide (LPS) reduced spontaneous Ca{superscript 2} transients in pia mater fibroblasts, likely due to macrophage infiltration following the inflammatory response. For the first time, this study characterizes spontaneous and behaviorally evoked Ca{superscript 2} dynamics in distinct leptomeningeal fibroblast subgroups in awake animals, providing novel insights into the functional roles of leptomeningeal fibroblasts in the healthy and diseased brain.

neuroscience↗

bioRxiv2025

Brain precapillary sphincters modulate myogenic tone in adult and aged mice

Brain precapillary sphincters, which are surrounded by contractile pericytes and are located at the junction of penetrating arterioles and first-order capillaries, can increase their diameter by [~]30% in a few seconds during sensory stimulation, allowing for rapid control of capillary blood flow over a wide dynamic range. We hypothesized that these properties could help precapillary sphincters maintain the capillary blood flow and shield the downstream capillaries during surges in blood pressure. To test this, we visualized microvessels in adult and old anaesthetized mice using in vivo two-photon microscopy. We showed that a blood pressure surge disrupts both microvascular myogenic response and neurovascular coupling in both adult and old mice, with old mice exhibiting a more diminished myogenic response. Similarly, laser ablation of contractile pericytes encircling precapillary sphincters disrupted neurovascular coupling and myogenic response. The resistance provided by precapillary sphincters may be increasingly important in old mice, where we found changes in the topology of microvessels, potentially affecting microvascular blood flow. Old mice displayed more tortuous penetrating arterioles, reduced pial collateral arteriolar density and altered capillary densities: reduced in the arterial end and increased in the venous end. Our results illustrate how blood pressure surges affect brain microvascular function, underscore the protective role of precapillary sphincters during cerebrovascular autoregulation in response to blood pressure surges and compare vascular topology in adult and old mice in vivo.

neuroscience↗

bioRxiv2024

Microglia contact cerebral vasculature through gaps between astrocyte endfeet

The close spatial relationship between microglia and cerebral blood vessels implicates microglia in vascular development, homeostasis and disease. In this study we used the publicly available Cortical MM^3 electron microscopy dataset to systematically investigate microglial interactions with the vasculature. Our analysis revealed that approximately 20% of microglia formed direct contacts with blood vessels through gaps between adjacent astrocyte endfeet. We termed these contact points "plugs". Plug-forming microglia exhibited closer proximity to blood vessels than non-plug forming microglia and formed multiple plugs, predominantly near the soma, ranging in surface area from [~]0.01 m2 to [~]15 m2. Plugs were enriched at the venule end of the vascular tree and displayed a preference for contacting endothelial cells over pericytes at a ratio of 3:1. In summary, we provide novel insights into the ultrastructural relationship between microglia and the vasculature, laying a foundation for understanding how these contacts contribute to the functional cross-talk between microglia and cells of the vasculature in health and disease.

neuroscience↗

bioRxiv2023

Ultrastructure of the brain waste-clearance pathway

Efficient metabolic waste clearance is essential for optimal brain function. Waste accumulation can cause inflammation, tissue damage, and potentially lead to dementia and other neurological disorders. The primary waste exit route is debated, with evidence suggesting the lymphatic system or arachnoid villi/granulations as possible pathways. This study uses an ultrastructural dataset to emphasize the potential underestimation of macrophages and arachnoid villi/granulations in waste removal. Based on brain surface ultrastructure analysis of the MICrONS dataset, waste clearance may occur through a transcellular route involving astrocytes to the perivascular space. Waste is then either phagocytosed by perivascular macrophages or directed to the subarachnoid space for meningeal macrophage phagocytosis. Evidence is provided that macrophages transport non-degradable waste in lysosomes to arachnoid villi for exocytosis into the villus lumen and venous system routing. Additionally, the study investigates fibroblast-macrophage interactions via primary cilia, potentially optimizing waste clearance. The role of astrocytes in brain water homeostasis and GFAPs potential significance in water filtration are also examined. This study offers valuable insights into waste clearance mechanisms in the brain, potentially aiding dementia understanding and treatment.

neuroscience↗

bioRxiv2022

Ultrastructure of Precapillary Sphincters and the Neurovascular Unit

Neurons communicate with vasculature to regulate blood flow in the brain. The cells that maintain this are collectively named the neurovascular unit (NVU). This communication, known as neurovascular coupling, is thought to involve astrocytes or molecules that can pass through the astrocytic endfeet. However, the exact mechanism is still unclear. Using large 3D electron microscopy datasets, we can now study the entire NVU in context. In this study, I provide evidence for the role of precapillary sphincters as a hub for neurovascular coupling and endothelial transcytosis, as well as the role of collagen synthesized by fibroblasts in strengthening first-order capillaries. I also show how astrocytic endfeet form a barrier for fluid flow and how the microvasculature of the cortex is not innervated but is surrounded by a surprising organization of parenchymal neuronal processes around penetrating arterioles and arterial-end capillaries in both mouse and human brains. Significance statementThe neurovascular unit (NVU) is made up of various types of cells, including neurons, astrocytes, and endothelial cells, which work together to regulate blood flow in response to changes in neural activity. This process, known as neurovascular coupling, is crucial for ensuring that the brain receives an adequate supply of oxygen and nutrients. This study suggests a novel organization of the NVU and neurovascular coupling. Through ultrastructural analysis, I was able to identify previously unknown relationships between the different types of cells in the NVU. These findings provide new insights into the structure of the NVU and how it functions, which may help researchers develop new strategies for preserving cognitive function and promoting healthy aging.

neuroscience↗

bioRxiv2021

Impaired dynamics of brain precapillary sphincters and first order capillaries explains reduced neurovascular functions in aging

The microvascular inflow tract (MIT), comprising the penetrating arterioles, precapillary sphincters, and first order capillaries, is the bottleneck for brain blood flow and energy supply. However, the exact structural and functional alterations of the MIT during aging remain elusive. In vivo 4-dimensional two-photon imaging showed an age-dependent decrease in vaso-responsivity, with reduced sensitivity of the MIT to pinacidil and papaverine, vasoconstrictor endothelin-1, and nitric oxide synthase inhibitor L-NAME. This was accompanied by an age-dependent decrease in capillary density close to the arterioles and loss of pericyte processes, though the number of pericyte somas and pericyte SMA density were preserved. The age-related reduction in vascular reactivity was most pronounced at precapillary sphincters, highlighting their crucial role in capillary blood flow regulation. Mathematical modeling revealed dysregulated but preserved pressure and flow in aged mice during vasoconstriction. Preventing reduced responsivity of the MIT may ameliorate the blood flow decrease associated with aging-related brain frailty.

neuroscience↗

bioRxiv2019

Precapillary sphincters control cerebral blood flow

Active nerve cells produce and release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling, which is the basis of the BOLD (blood-oxygen-level-dependent) functional neuroimaging signals. We here reveal a unique mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and the first capillary that links blood flow in capillaries to the arteriolar inflow. Large NG2-positive cells, containing smooth muscle actin, encircle the sphincters and rises in nerve cell activity cause astrocyte and neuronal Ca2+ rises that correlate to dilation and shortening of the sphincter concomitant with substantial increases in the RBC flux. Global ischemia and cortical spreading depolarization constrict sphincters and cause vascular trapping of blood cells. These results reveal precapillary sphincters as bottlenecks for brain capillary blood flow.

neuroscience↗

bioRxiv2018

Bioinformatics workflows for genomic analysis of tumors from Patient Derived Xenografts (PDX): challenges and guidelines

Bioinformatics workflows for analyzing genomic data obtained from xenografted tumor (e.g., human tumors engrafted in a mouse host) must address several challenges, including separating mouse and human sequence reads and accurate identification of somatic mutations and copy number aberrations when paired normal DNA from the patient is not available. We report here data analysis workflows that address these challenges and result in reliable identification of somatic mutations, copy number alterations, and transcriptomic profiles of tumors from patient derived xenograft models. We validated our analytical approaches using simulated data and by assessing concordance of the genomic properties of xenograft tumors with data from primary human tumors in The Cancer Genome Atlas (TCGA). The commands and parameters for the workflows are available at https://github.com/TheJacksonLaboratory/PDX-Analysis-Workflows.

bioinformatics↗