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Journal of Medical Virology May 2024The coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although severe acute respiratory...
The coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus mainly infects the respiratory system, neurological complications are widely reported in both acute infection and long-COVID cases. Despite the success of vaccines and antiviral treatments, neuroinvasiveness of SARS-CoV-2 remains an important question, which is also centered on the mystery of whether the virus is capable of breaching the barriers into the central nervous system. By studying the K18-hACE2 infection model, we observed clear evidence of microvascular damage and breakdown of the blood-brain barrier (BBB). Mechanistically, SARS-CoV-2 infection caused pericyte damage, tight junction loss, endothelial activation and vascular inflammation, which together drive microvascular injury and BBB impairment. In addition, the blood-cerebrospinal fluid barrier at the choroid plexus was also impaired after infection. Therefore, cerebrovascular and choroid plexus dysfunctions are important aspects of COVID-19 and may contribute to neurological complications both acutely and in long COVID.
Topics: Blood-Brain Barrier; Animals; Choroid Plexus; COVID-19; Mice; SARS-CoV-2; Tight Junctions; Disease Models, Animal; Angiotensin-Converting Enzyme 2; Inflammation; Humans; Pericytes
PubMed: 38747003
DOI: 10.1002/jmv.29671 -
BioRxiv : the Preprint Server For... May 2024Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples....
UNLABELLED
Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.
SUMMARY
We establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.
PubMed: 38746120
DOI: 10.1101/2024.04.29.591777 -
Methods in Molecular Biology (Clifton,... 2024The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions...
The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.
Topics: Blood-Brain Barrier; Humans; Astrocytes; Endothelial Cells; HIV-1; HIV Infections; Pericytes; Neuroinflammatory Diseases; Coculture Techniques; Cells, Cultured; Brain
PubMed: 38743235
DOI: 10.1007/978-1-0716-3862-0_19 -
Human Cell May 2024Mesenchymal stem/stromal cells (MSCs), originating from the mesoderm, represent a multifunctional stem cell population capable of differentiating into diverse cell types... (Review)
Review
Mesenchymal stem/stromal cells (MSCs), originating from the mesoderm, represent a multifunctional stem cell population capable of differentiating into diverse cell types and exhibiting a wide range of biological functions. Despite more than half a century of research, MSCs continue to be among the most extensively studied cell types in clinical research projects globally. However, their significant heterogeneity and phenotypic instability have significantly hindered their exploration and application. Single-cell sequencing technology emerges as a powerful tool to address these challenges, offering precise dissection of complex cellular samples. It uncovers the genetic structure and gene expression status of individual contained cells on a massive scale and reveals the heterogeneity among these cells. It links the molecular characteristics of MSCs with their clinical applications, contributing to the advancement of regenerative medicine. With the development and cost reduction of single-cell analysis techniques, sequencing technology is now widely applied in fundamental research and clinical trials. This study aimed to review the application of single-cell sequencing in MSC research and assess its prospects.
PubMed: 38743204
DOI: 10.1007/s13577-024-01076-9 -
Development (Cambridge, England) May 2024During mouse development, presomitic mesoderm cells synchronize Wnt and Notch oscillations, creating sequential phase waves that pattern somites. Traditional...
During mouse development, presomitic mesoderm cells synchronize Wnt and Notch oscillations, creating sequential phase waves that pattern somites. Traditional somitogenesis models attribute phase waves to a global modulation of the oscillation frequency. However, increasing evidence suggests that they could arise in a self-organizing manner. Here, we introduce the Sevilletor, a novel reaction-diffusion system that serves as a framework to compare different somitogenesis patterning hypotheses. Using this framework, we propose the Clock and Wavefront Self-Organizing model that considers an excitable self-organizing region where phase waves form independent of global frequency gradients. The model recapitulates the change in relative phase of Wnt and Notch observed during mouse somitogenesis and provides a theoretical basis for understanding the excitability of mouse presomitic mesoderm cells in vitro.
Topics: Animals; Mice; Somites; Receptors, Notch; Mesoderm; Models, Biological; Body Patterning; Wnt Proteins; Embryonic Development; Biological Clocks
PubMed: 38742434
DOI: 10.1242/dev.202606 -
Gastro Hep Advances 2024
The IncRNA, Cardiac Mesoderm Enhancer-Associated Noncoding RNA Is Indispensable for Intestinal Smooth Muscle Homeostasis in Female Mice as Revealed by a Novel Endogenous -Encoded Inducible Cre Model.
PubMed: 38737599
DOI: 10.1016/j.gastha.2023.12.012 -
Bio-protocol May 2024Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study...
Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.
PubMed: 38737507
DOI: 10.21769/BioProtoc.4984 -
Stem Cell Research Jun 2024Y chromosome deletion and karyotype abnormalities are commonly associated with congenital non-obstructive azoospermia, impairing spermatogenesis. Specifically, the...
Y chromosome deletion and karyotype abnormalities are commonly associated with congenital non-obstructive azoospermia, impairing spermatogenesis. Specifically, the deletion of the Y chromosome Azoospermia factor a (AZFa) has been identified in infertile males with severely impaired spermatogenesis. AZFa, encompassing megabase-scale of the Y chromosome region, poses challenges in modeling AZFa deletion-related male infertility using gene editing tools. Here, we successfully created an AZFa-deleted human embryonic stem cell line utilizing the CRISPR/Cas9 gene editing tool. Our analysis indicates the AZFa-deleted stem cell line holds promise for differentiation into ectoderm, mesoderm, and endoderm, highlighting its potential for further comprehensive study.
Topics: Humans; Human Embryonic Stem Cells; Male; Cell Line; Chromosomes, Human, Y; Cell Differentiation; CRISPR-Cas Systems; Chromosome Deletion; Gene Editing
PubMed: 38733811
DOI: 10.1016/j.scr.2024.103436 -
International Journal of Molecular... Apr 2024Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The...
Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.
Topics: Melatonin; Mesenchymal Stem Cells; Humans; Cell Differentiation; Cells, Cultured; Adipose Tissue; Neurons; Culture Media, Conditioned; Schwann Cells; Neurogenesis; Adult; Nestin; Glial Fibrillary Acidic Protein; Neuroglia; Synapsins
PubMed: 38732109
DOI: 10.3390/ijms25094891 -
EvoDevo May 2024Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using...
Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5 k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. We found differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We identified many Hox genes as markers of cell clusters, and Hox gene ohnologs were often present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in an arachnopulmonate ancestor (spiders, scorpions, and related orders). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.
PubMed: 38730509
DOI: 10.1186/s13227-024-00224-4