Bicuspid Aortic Valve-Associated Regulatory Regions Reveal GATA4 Regulation and Function During Human-Induced Pluripotent Stem Cell-Based Endothelial-Mesenchymal Transition-Brief Report.

BACKGROUND: The endothelial-mesenchymal transition (EndoMT) is a fundamental process for heart valve formation and defects in EndoMT cause aortic valve abnormalities. Our previous genome-wide association study identified multiple variants in a large chromosome 8 segment as significantly associated with bicuspid aortic valve (BAV). The objective of this study is to determine the biological effects of this large noncoding segment in human induced pluripotent stem cell (hiPSC)-based EndoMT. METHODS: A large genomic segment enriched for BAV-associated variants was deleted in hiPSCs using 2-step CRISPR/Cas9 editing. To address the effects of the variants on GATA4 expression, we generated CRISPR repression hiPSC lines (CRISPRi) as well as hiPSCs from BAV patients. The resulting hiPSCs were differentiated to mesenchymal/myofibroblast-like cells through cardiovascular-lineage endothelial cells for molecular and cellular analysis. Single-cell RNA sequencing was also performed at different stages of EndoMT induction. RESULTS: The large deletion impaired hiPSC-based EndoMT in multiple biallelic clones compared with their isogenic control. It also reduced GATA4 transcript and protein levels during EndoMT, sparing the other genes nearby the deletion segment. Single-cell trajectory analysis revealed the molecular reprogramming during EndoMT. Putative GATA-binding protein targets during EndoMT were uncovered, including genes implicated in endocardial cushion formation and EndoMT process. Differentiation of cells derived from BAV patients carrying the rs117430032 variant as well as CRISPRi repression of the rs117430032 locus resulted in lower GATA4 expression in a stage-specific manner. TWIST1 was identified as a potential regulator of GATA4 expression, showing specificity to the locus tagged by rs117430032. CONCLUSIONS: BAV-associated distal regions regulate GATA4 expression during hiPSC-based EndoMT, which in turn promotes EndoMT progression, implicating its contribution to heart valve development.

Single-Molecule Spatial Transcriptomics of Human Thoracic Aortic Aneurysms Uncovers Calcification-Related CARTPT-Expressing Smooth Muscle Cells.

BACKGROUND: Although single-cell RNA-sequencing is commonly applied to dissect the heterogeneity in human tissues, it involves the preparation of single-cell suspensions via cell dissociation, causing loss of spatial information. In this study, we employed high-resolution single-cell transcriptome imaging to reveal rare smooth muscle cell (SMC) types in human thoracic aortic aneurysm (TAA) tissue samples. METHODS: Single-molecule spatial distribution of transcripts from 140 genes was analyzed in fresh-frozen human TAA samples with region and sex-matched controls. In vitro studies and tissue staining were performed to examine human CART prepropeptide (CARTPT) regulation and function. RESULTS: We captured thousands of cells per sample including a spatially distinct CARTPT-expressing SMC subtype enriched in male TAA samples. Immunoassays confirmed human CART (cocaine- and amphetamine-regulated transcript) protein enrichment in male TAA tissue and truncated CARTPT secretion into cell culture medium. Oxidized low-density lipoprotein, a cardiovascular risk factor, induced CARTPT expression, whereas CARTPT overexpression in human aortic SMCs increased the expression of key osteochondrogenic transcription factors and reduced contractile gene expression. Recombinant human CART treatment of human SMCs further confirmed this phenotype. Alizarin red staining revealed calcium deposition in male TAA samples showing similar localization with human CART staining. CONCLUSIONS: Here, we demonstrate the feasibility of single-molecule imaging in uncovering rare SMC subtypes in the diseased human aorta, a difficult tissue to dissociate. We identified a spatially distinct CARTPT-expressing SMC subtype enriched in male human TAA samples. Our functional studies suggest that human CART promotes osteochondrogenic switch of aortic SMCs, potentially leading to medial calcification of the thoracic aorta.

Dissecting the Heterogeneity of Human Thoracic Aortic Aneurysms Using Single-Cell Transcriptomics.

Thoracic aortic aneurysm is a life-threatening condition caused by weakening of the thoracic aorta wall, often developing silently until dissection or rupture occurs. Despite substantial efforts in the past decade, there have been no significant therapeutic advances to prevent or clinically manage diverse forms of thoracic aortic aneurysm and dissection with the only effective treatment being surgical repair. There is an urgent need to understand intra- and inter-aneurysmal heterogeneity underlying thoracic aortic aneurysm and dissection pathogenesis. The human aortic wall consists of many cell types and exhibits significant regional heterogeneity. High-throughput single-cell RNA sequencing has emerged as the principal tool to reveal the complexity in human tissues and clinical specimens. Recent single-cell RNA sequencing studies of different aortic cell populations both in vivo and in vitro began to dissect this complexity and have provided valuable information. In this review, we summarize these findings and discuss the potential applications of single-cell transcriptomics and related high-content technologies in human thoracic aortic aneurysm and dissection research, as well as the challenges associated with it.

hiPSC Modeling of Lineage-Specific Smooth Muscle Cell Defects Caused by TGFBR1A230T Variant, and Its Therapeutic Implications for Loeys-Dietz Syndrome.

BACKGROUND: Loeys-Dietz syndrome (LDS) is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection. Currently, there are no medical treatments except surgical resection. Although the genetic basis of LDS is well-understood, molecular mechanisms underlying the disease remain elusive, impeding the development of a therapeutic strategy. In addition, aortic smooth muscle cells (SMCs) have heterogenous embryonic origins, depending on their spatial location, and lineage-specific effects of pathogenic variants on SMC function, likely causing regionally constrained LDS manifestations, have been unexplored. METHODS: We identified an LDS family with a dominant pathogenic variant in the TGFBR1 gene (TGFBR1A230T) causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human induced pluripotent stem cells from a subject with normal aorta to generate human induced pluripotent stem cells carrying TGFBR1A230T, and corrected the mutation in patient-derived human induced pluripotent stem cells using CRISPR-Cas9 gene editing. After their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell lineages, we used conventional molecular techniques and single-cell RNA sequencing to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments using activin A and rapamycin. RESULTS: Our results indicate the TGFBR1A230T mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in neural crest stem cell-SMC. Single-cell RNA sequencing results implicate defective differentiation even in TGFBR1A230T/+ CPC-SMC including disruption of SMC contraction and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC. TGFBR1A230T selectively disrupted SMAD3 (SMAD family member 3) and AKT (AKT serine/threonine kinase) activation in CPC-SMC, and led to increased cell proliferation. Consistently, single-cell RNA sequencing revealed molecular similarities between a loss-of-function SMAD3 mutation (SMAD3c.652delA/+) and TGFBR1A230T/+. Last, combination treatment with activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression and function, and rescued the mechanical properties of mutant CPC-SMC tissue constructs. CONCLUSIONS: This study reveals that a pathogenic TGFBR1 variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with activin A and rapamycin that can rescue the SMC defects caused by the variant.

Bioengineered vascular grafts with a pathogenic TGFBR1 variant model aneurysm formation in vivo and reveal underlying collagen defects.

Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.

Injury-induced ASCL1 expression orchestrates a transitory cell state required for repair of the neonatal cerebellum.

To understand repair processes, it is critical to identify the molecular foundations underlying progenitor diversity and plasticity. Upon injury to the neonatal cerebellum, a normally gliogenic nestin-expressing progenitor (NEP) in the Bergmann glia layer (BgL) undergoes adaptive reprograming to restore granule cell production. However, the cellular states and genes regulating the NEP fate switch are unknown. Using single-cell RNA sequencing and fate mapping, we defined molecular subtypes of NEPs and their lineages under homeostasis and repair. NEPs contain two major subtypes: Hopx+ astrogliogenic and Ascl1+ neurogenic NEPs that are further subdivided based on their location, lineage, and differentiation status. Upon injury, an Ascl1+ transitory cellular state arises from Hopx+ BgL-NEPs. Furthermore, mutational analysis revealed that induction of Ascl1 is required for adaptive reprogramming by orchestrating a glial-to-neural switch in vivo following injury. Thus, we provide molecular and cellular insights into context-dependent progenitor plasticity and repair mechanisms in the brain.

Release of stem cells from quiescence reveals gliogenic domains in the adult mouse brain.

Quiescent neural stem cells (NSCs) in the adult mouse ventricular-subventricular zone (V-SVZ) undergo activation to generate neurons and some glia. Here we show that platelet-derived growth factor receptor beta (PDGFRß) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia. Selective deletion of PDGFRß in adult V-SVZ NSCs leads to their release from quiescence, uncovering gliogenic domains for different glial cell types. These domains are also recruited upon injury. We identify an intraventricular oligodendrocyte progenitor derived from NSCs inside the brain ventricles that contacts supraependymal axons. Together, our findings reveal that the adult V-SVZ contains spatial domains for gliogenesis, in addition to those for neurogenesis. These gliogenic NSC domains tend to be quiescent under homeostasis and may contribute to brain plasticity.

Single-Cell Profiling and SCOPE-Seq Reveal Lineage Dynamics of Adult Ventricular-Subventricular Zone Neurogenesis and NOTUM as a Key Regulator.

In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.

Single-Cell Analysis of Regional Differences in Adult V-SVZ Neural Stem Cell Lineages.

The ventricular-subventricular zone (V-SVZ) harbors adult neural stem cells. V-SVZ neural stem cells exhibit features of astrocytes, have a regional identity, and depending on their location in the lateral or septal wall of the lateral ventricle, generate different types of neuronal and glial progeny. We performed large-scale single-cell RNA sequencing to provide a molecular atlas of cells from the lateral and septal adult V-SVZ of male and female mice. This revealed regional and sex differences among adult V-SVZ cells. We uncovered lineage potency bias at the single-cell level among lateral and septal wall astrocytes toward neurogenesis and oligodendrogenesis, respectively. Finally, we identified transcription factor co-expression modules marking key temporal steps in neurogenic and oligodendrocyte lineage progression. Our data suggest functionally important spatial diversity in neurogenesis and oligodendrogenesis in the adult brain and reveal molecular correlates of adult NSC dormancy and lineage specialization.

Age-Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells.

Specialized niches support the lifelong maintenance and function of tissue-specific stem cells. Adult neural stem cells in the ventricular-subventricular zone (V-SVZ) contact the cerebrospinal fluid (CSF), which flows through the lateral ventricles. A largely ignored component of the V-SVZ stem cell niche is the lateral ventricle choroid plexus (LVCP), a primary producer of CSF. Here we show that the LVCP, in addition to performing important homeostatic support functions, secretes factors that promote colony formation and proliferation of purified quiescent and activated V-SVZ stem cells and transit-amplifying cells. The functional effect of the LVCP secretome changes throughout the lifespan, with activated neural stem cells being especially sensitive to age-related changes. Transcriptome analysis identified multiple factors that recruit colony formation and highlights novel facets of LVCP function. Thus, the LVCP is a key niche compartment that translates physiological changes into molecular signals directly affecting neural stem cell behavior.

A mechanism for circadian control of pacemaker neuron excitability.

Although the intracellular molecular clocks that regulate circadian (~24 h) behavioral rhythms are well understood, it remains unclear how molecular clock information is transduced into rhythmic neuronal activity that in turn drives behavioral rhythms. To identify potential clock outputs, the authors generated expression profiles from a homogeneous population of purified pacemaker neurons (LN(v)s) from wild-type and clock mutant Drosophila. They identified a group of genes with enriched expression in LN(v)s and a second group of genes rhythmically expressed in LN(v)s in a clock-dependent manner. Only 10 genes fell into both groups: 4 core clock genes, including period (per) and timeless (tim), and 6 genes previously unstudied in circadian rhythms. The authors focused on one of these 6 genes, Ir, which encodes an inward rectifier K(+) channel likely to regulate resting membrane potential, whose expression peaks around dusk. Reducing Ir expression in LN(v)s increased larval light avoidance and lengthened the period of adult locomotor rhythms, consistent with increased LN(v) excitability. In contrast, increased Ir expression made many adult flies arrhythmic and dampened PER protein oscillations. The authors propose that rhythmic Ir expression contributes to daily rhythms in LN(v) neuronal activity, which in turn feed back to regulate molecular clock oscillations.

Electrical activity can impose time of day on the circadian transcriptome of pacemaker neurons.

BACKGROUND: Circadian (∼24 hr) rhythms offer one of the best examples of how gene expression is tied to behavior. Circadian pacemaker neurons contain molecular clocks that control 24 hr rhythms in gene expression that in turn regulate electrical activity rhythms to control behavior. RESULTS: Here we demonstrate the inverse relationship: there are broad transcriptional changes in Drosophila clock neurons (LN(v)s) in response to altered electrical activity, including a large set of circadian genes. Hyperexciting LN(v)s creates a morning-like expression profile for many circadian genes while hyperpolarization leads to an evening-like transcriptional state. The electrical effects robustly persist in per(0) mutant LN(v)s but not in cyc(0) mutant LN(v)s, suggesting that neuronal activity interacts with the transcriptional activators of the core circadian clock. Bioinformatic and immunocytochemical analyses suggest that CREB family transcription factors link LN(v) electrical state to circadian gene expression. CONCLUSIONS: The electrical state of a clock neuron can impose time of day to its transcriptional program. We propose that this acts as an internal zeitgeber to add robustness and precision to circadian behavioral rhythms.