CRISPR-Knockout Screen Identifies Dmap1 as a Regulator of Chemically Induced Reprogramming and Differentiation of Cardiac Progenitors.

Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958-972.

How to make a cardiomyocyte.

During development, cardiogenesis is orchestrated by a family of heart progenitors that build distinct regions of the heart. Each region contains diverse cell types that assemble to form the complex structures of the individual cardiac compartments. Cardiomyocytes are the main cell type found in the heart and ensure contraction of the chambers and efficient blood flow throughout the body. Injury to the cardiac muscle often leads to heart failure due to the loss of a large number of cardiomyocytes and its limited intrinsic capacity to regenerate the damaged tissue, making it one of the leading causes of morbidity and mortality worldwide. In this Primer we discuss how insights into the molecular and cellular framework underlying cardiac development can be used to guide the in vitro specification of cardiomyocytes, whether by directed differentiation of pluripotent stem cells or via direct lineage conversion. Additional strategies to generate cardiomyocytes in situ, such as reactivation of endogenous cardiac progenitors and induction of cardiomyocyte proliferation, will also be discussed.

Domain-specific control of neurogenesis achieved through patterned regulation of Notch ligand expression.

Homeodomain (HD) transcription factors and components of the Notch pathway [Delta1 (Dll1), Jagged1 (Jag1) and the Fringe (Fng) proteins] are expressed in distinct progenitor domains along the dorsoventral (DV) axis of the developing spinal cord. However, the internal relationship between these two regulatory pathways has not been established. In this report we show that HD proteins act upstream of Notch signalling. Thus, HD proteins control the spatial distribution of Notch ligands and Fng proteins, whereas perturbation of the Notch pathway does not affect the regional expression of HD proteins. Loss of Dll1 or Jag1 leads to a domain-specific increase of neuronal differentiation but does not affect the establishment of progenitor domain boundaries. Moreover, gain-of-function experiments indicate that the ability of Dll1 and Jag1 to activate Notch is limited to progenitors endogenously expressing the respective ligand. Fng proteins enhance Dll1-activated Notch signalling and block Notch activation mediated by Jag1. This finding, combined with the overlapping expression of Fng with Dll1 but not with Jag1, is likely to explain the domain-specific activity of the Notch ligands. This outcome is opposite to the local regulation of Notch activity in most other systems, including the Drosophila wing, where Fng co-localizes with Jagged/Serrate rather than Dll/Delta, which facilitates Notch signalling at regional boundaries instead of within domains. The regulation of Notch activation in the spinal cord therefore appears to endow specific progenitor populations with a domain-wide autonomy in the control of neurogenesis and prevents any inadequate activation of Notch across progenitor domain boundaries.

Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart.

The heart depends on a functional vasculature for oxygenation and transport of nutrients, and it is of interest to learn how primary impairment of the vasculature can indirectly affect cardiac function and heart morphology. Notch3-deficiency causes vascular smooth muscle cell (VSMC) loss in the vasculature but the consequences for the heart remain largely elusive. Here, we demonstrate that Notch3-/- mice have enlarged hearts with left ventricular hypertrophy and mild fibrosis. Cardiomyocytes were hypertrophic but not hyperproliferative, and the expression of several cardiomyocyte markers, including Tnt2, Myh6, Myh7 and Actn2, was altered. Furthermore, expression of genes regulating the metabolic status of the heart was affected: both Pdk4 and Cd36 were downregulated, indicating a metabolic switch from fatty acid oxidation to glucose consumption. Notch3-/- mice furthermore showed lower liver lipid content. Notch3 was expressed in heart VSMC and pericytes but not in cardiomyocytes, suggesting that a perturbation of Notch signalling in VSMC and pericytes indirectly impairs the cardiomyocytes. In keeping with this, Pdgfbret/ret mice, characterized by reduced numbers of VSMC and pericytes, showed left ventricular and cardiomyocyte hypertrophy. In conclusion, we demonstrate that reduced Notch3 or PDGFB signalling in vascular mural cells leads to cardiomyocyte dysfunction.

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology.

Control of Notch-ligand endocytosis by ligand-receptor interaction.

In Notch signaling, cell-bound ligands activate Notch receptors on juxtaposed cells, but the relationship between ligand endocytosis, ubiquitylation and ligand-receptor interaction remains poorly understood. To study the specific role of ligand-receptor interaction, we identified a missense mutant of the Notch ligand Jagged1 (Nodder, Ndr) that failed to interact with Notch receptors, but retained a cellular distribution that was similar to wild-type Jagged1 (Jagged1(WT)) in the absence of active Notch signaling. Both Jagged1(WT) and Jagged1(Ndr) interacted with the E3 ubiquitin ligase Mind bomb, but only Jagged1(WT) showed enhanced ubiquitylation after co-culture with cells expressing Notch receptor. Cells expressing Jagged1(WT), but not Jagged1(Ndr), trans-endocytosed the Notch extracellular domain (NECD) into the ligand-expressing cell, and NECD colocalized with Jagged1(WT) in early endosomes, multivesicular bodies and lysosomes, suggesting that NECD is routed through the endocytic degradation pathway. When coexpressed in the same cell, Jagged1(Ndr) did not exert a dominant-negative effect over Jagged1(WT) in terms of receptor activation. Finally, in Jag1(Ndr/Ndr) mice, the ligand was largely accumulated at the cell surface, indicating that engagement of the Notch receptor is important for ligand internalization in vivo. In conclusion, the interaction-dead Jagged1(Ndr) ligand provides new insights into the specific role of receptor-ligand interaction in the intracellular trafficking of Notch ligands.

A single-cell transcriptomic inventory of murine smooth muscle cells.

Smooth muscle cells (SMCs) execute important physiological functions in numerous vital organ systems, including the vascular, gastrointestinal, respiratory, and urogenital tracts. SMC differ morphologically and functionally at these different anatomical locations, but the molecular underpinnings of the differences remain poorly understood. Here, using deep single-cell RNA sequencing combined with in situ gene and protein expression analysis in four murine organs-heart, aorta, lung, and colon-we identify a molecular basis for high-level differences among vascular, visceral, and airway SMC, as well as more subtle differences between, for example, SMC in elastic and muscular arteries and zonation of elastic artery SMC along the direction of blood flow. Arterial SMC exhibit extensive organotypic heterogeneity, whereas venous SMC are similar across organs. We further identify a specific SMC subtype within the pulmonary vasculature. This comparative SMC cross-organ resource offers insight into SMC subtypes and their specific functions.

The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells: Implications for COVID-19 vascular research.

Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is important to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, and in heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.

Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells.

The Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells.

Notch signaling regulates platelet-derived growth factor receptor-beta expression in vascular smooth muscle cells.

Notch signaling is critically important for proper architecture of the vascular system, and mutations in NOTCH3 are associated with CADASIL, a stroke and dementia syndrome with vascular smooth muscle cell (VSMC) dysfunction. In this report, we link Notch signaling to platelet-derived growth factor (PDGF) signaling, a key determinant of VSMC biology, and show that PDGF receptor (PDGFR)-beta is a novel immediate Notch target gene. PDGFR-beta expression was upregulated by Notch ligand induction or by activated forms of the Notch receptor. Moreover, upregulation of PDGFR-beta expression in response to Notch activation critically required the Notch signal integrator CSL. In primary VSMCs, PDGFR-beta expression was robustly upregulated by Notch signaling, leading to an augmented intracellular response to PDGF stimulation. In newborn Notch3-deficient mice, PDGFR-beta expression was strongly reduced in the VSMCs that later develop an aberrant morphology. In keeping with this, PDGFR-beta upregulation in response to Notch activation was reduced also in Notch3-deficient embryonic stem cells. Finally, in VSMCs from a CADASIL patient carrying a NOTCH3 missense mutation, upregulation of PDGFR-beta mRNA and protein in response to ligand-induced Notch activation was significantly reduced. In sum, these data reveal a hierarchy for 2 important signaling systems, Notch and PDGF, in the vasculature and provide insights into how dysregulated Notch signaling perturbs VSMC differentiation and function.

Multiplexed Optical Sensors in Arrayed Islands of Cells for multimodal recordings of cellular physiology.

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.

Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.

Publisher Correction: Single-cell analysis uncovers fibroblast heterogeneity and criteria for fibroblast and mural cell identification and discrimination.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System.

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body, including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system, provides a platform for the screening of chemical libraries that affect these processes, and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However, defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner, with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells, exhibit spontaneous electrical activity, and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months, even without astrocyte feeder layers.

Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3ß inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC(+)CD31(+)CD34(+)CD14(-)KDR(high) endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR(+) mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Hypoxia-induced arterial differentiation requires adrenomedullin and notch signaling.

Hypoxia (low oxygen) and Notch signaling are 2 important regulators of vascular development, but how they interact in controlling the choice between arterial and venous fates for endothelial cells during vasculogenesis is less well understood. In this report, we show that hypoxia and Notch signaling intersect in promotion of arterial differentiation. Hypoxia upregulated expression of the Notch ligand Dll4 and increased Notch signaling in a process requiring the vasoactive hormone adrenomedullin. Notch signaling also upregulated Dll4 expression, leading to a positive feedback loop sustaining Dll4 expression and Notch signaling. In addition, hypoxia-mediated upregulation of the arterial marker genes Depp, connexin40 (Gja5), Cxcr4, and Hey1 required Notch signaling. In conclusion, the data reveal an intricate interaction between hypoxia and Notch signaling in the control of endothelial cell differentiation, including a hypoxia/adrenomedullin/Dll4 axis that initiates Notch signaling and a requirement for Notch signaling to effectuate hypoxia-mediated induction of the arterial differentiation program.

Reprogramming a broken heart.

Fibrosis resulting from cardiac injury presents a major challenge to restoring heart function after myocardial infarction. Two recent papers in Nature report successful in vivo reprogramming of fibroblasts to cardiomyocytes in injured mouse hearts (Qian et al., 2012; Song et al., 2012), resulting in improved cardiac function and reduced scar formation.

Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.

We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on alpha6beta1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.