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Nature Communications May 2024Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of...
Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.
Topics: Animals; Forkhead Transcription Factors; Retinal Neovascularization; Mice; Endothelial Cells; Blood-Retinal Barrier; TOR Serine-Threonine Kinases; Pericytes; Fusion Regulatory Protein 1, Heavy Chain; Retinal Vessels; Humans; Large Neutral Amino Acid-Transporter 1; Mice, Knockout; Mice, Inbred C57BL; Retina; Male; Angiogenesis
PubMed: 38755144
DOI: 10.1038/s41467-024-48134-2 -
Microbiology Spectrum Jun 2024Enterovirus A71 (EV-A71) is associated with neurological conditions such as acute meningitis and encephalitis. The virus is detected in the bloodstream, and high blood...
Enterovirus A71 (EV-A71) is associated with neurological conditions such as acute meningitis and encephalitis. The virus is detected in the bloodstream, and high blood viral loads are associated with central nervous system (CNS) manifestations. We used an blood-brain barrier (BBB) model made up of human brain-like endothelial cells (hBLECs) and brain pericytes grown in transwell systems to investigate whether three genetically distinct EV-A71 strains (subgenogroups C1, C1-like, and C4) can cross the human BBB. EV-A71 poorly replicated in hBLECs, which released moderate amounts of infectious viruses from their luminal side and trace amounts of infectious viruses from their basolateral side. The barrier properties of hBLECs were not impaired by EV-A71 infection. We investigated the passage through hBLECs of EV-A71-infected white blood cells. EV-A71 strains efficiently replicated in immune cells, including monocytes, neutrophils, and NK/T cells. Attachment to hBLECs of immune cells infected with the C1-like virus was higher than attachment of cells infected with C1-06. EV-A71 infection did not impair the transmigration of immune cells through hBLECs. Overall, EV-A71 targets different white blood cell populations that have the potential to be used as a Trojan horse to cross hBLECs more efficiently than cell-free EV-A71 particles.IMPORTANCEEnterovirus A71 (EV-A71) was first reported in the USA, and numerous outbreaks have since occurred in Asia and Europe. EV-A71 re-emerged as a new multirecombinant strain in 2015 in Europe and is now widespread. The virus causes hand-foot-and-mouth disease in young children and is involved in nervous system infections. How the virus spreads to the nervous system is unclear. We investigated whether white blood cells could be infected by EV-A71 and transmit it across human endothelial cells mimicking the blood-brain barrier protecting the brain from adverse effects. We found that endothelial cells provide a strong roadblock to prevent the passage of free virus particles but allow the migration of infected immune cells, including monocytes, neutrophils, and NK/T cells. Our data are consistent with the potential role of immune cells in the pathogenesis of EV-A71 infections by spreading the virus in the blood and across the human blood-brain barrier.
Topics: Blood-Brain Barrier; Humans; Enterovirus A, Human; Enterovirus Infections; Endothelial Cells; Virus Replication; Monocytes; Pericytes; Leukocytes; Brain; Killer Cells, Natural; Neutrophils
PubMed: 38752731
DOI: 10.1128/spectrum.00690-24 -
Alzheimer Disease and Associated...Blood-brain barrier (BBB) dysfunction is emerging as an important pathophysiologic factor in Alzheimer disease (AD). Cerebrospinal fluid (CSF) platelet-derived growth...
BACKGROUND
Blood-brain barrier (BBB) dysfunction is emerging as an important pathophysiologic factor in Alzheimer disease (AD). Cerebrospinal fluid (CSF) platelet-derived growth factor receptor-β (PDGFRβ) is a biomarker of BBB pericyte injury and has been implicated in cognitive impairment and AD.
METHODS
We aimed to study CSF PDGFRβ protein levels, along with CSF biomarkers of brain amyloidosis and tau pathology in a well-characterized population of cognitively unimpaired individuals and correlated CSF findings with amyloid-PET positivity. We performed an institutional review board (IRB)-approved cross-sectional analysis of a prospectively enrolled cohort of 36 cognitively normal volunteers with available CSF, Pittsburgh compound B PET/CT, Mini-Mental State Exam score, Global Deterioration Scale, and known apolipoprotein E ( APOE ) ε4 status.
RESULTS
Thirty-six subjects were included. Mean age was 63.3 years; 31 of 36 were female, 6 of 36 were amyloid-PET-positive and 12 of 36 were APOE ε4 carriers. We found a moderate positive correlation between CSF PDGFRβ and both total Tau (r=0.45, P =0.006) and phosphorylated Tau 181 (r=0.51, P =0.002). CSF PDGFRβ levels were not associated with either the CSF Aβ42 or the amyloid-PET.
CONCLUSIONS
We demonstrated a moderate positive correlation between PDGFRβ and both total Tau and phosphorylated Tau 181 in cognitively normal individuals. Our data support the hypothesis that BBB dysfunction represents an important early pathophysiologic step in AD, warranting larger prospective studies.
TRIAL REGISTRATION
ClinicalTrials.gov Identifier: NCT00094939.
Topics: Humans; Female; Alzheimer Disease; Male; Biomarkers; Middle Aged; Cross-Sectional Studies; Aged; tau Proteins; Pericytes; Positron-Emission Tomography; Amyloid beta-Peptides; Blood-Brain Barrier; Receptor, Platelet-Derived Growth Factor beta; Prospective Studies; Cohort Studies
PubMed: 38752577
DOI: 10.1097/WAD.0000000000000623 -
Circulation Jun 2024Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic...
BACKGROUND
Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear.
METHODS
We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 ()-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays.
RESULTS
Transcriptional regulation and trajectory inference from -null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor and upregulation of late anticardiac regulators and in knockout cells reflect a necessary role for in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation.
CONCLUSIONS
These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.
Topics: Humans; Myocytes, Cardiac; Cell Differentiation; Cell Lineage; Neurofibromin 2; CRISPR-Cas Systems; Human Embryonic Stem Cells
PubMed: 38752370
DOI: 10.1161/CIRCULATIONAHA.122.061335 -
Frontiers in Cell and Developmental... 2024Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC...
Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor -GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes -GlcNAcase (OGA) and -GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of -depleted (OGAi) and -depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34 hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular -GlcNAcylation concomitant with their loss of OGA and OGT, respectively, appeared normal in phenotype and karyotype, and retained pluripotency, although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition, we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of -GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors, thus confirming their functional characteristics. Altogether, the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of -GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of and in these hiPSCs makes them susceptible to additional manipulation, i.e., by small molecules, allowing the molecular dynamics studies of -GlcNAcylation.
PubMed: 38752196
DOI: 10.3389/fcell.2024.1361943 -
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 -
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