Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause.

Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state.

Bone marrow-derived myeloid progenitors in the leptomeninges of adult mice.

Although the bone marrow contains most hematopoietic activity during adulthood, hematopoietic stem and progenitor cells can be recovered from various extramedullary sites. Cells with hematopoietic progenitor properties have even been reported in the adult brain under steady-state conditions, but their nature and localization remain insufficiently defined. Here, we describe a heterogeneous population of myeloid progenitors in the leptomeninges of adult C57BL/6 mice. This cell pool included common myeloid, granulocyte/macrophage, and megakaryocyte/erythrocyte progenitors. Accordingly, it gave rise to all major myelo-erythroid lineages in clonogenic culture assays. Brain-associated progenitors persisted after tissue perfusion and were partially inaccessible to intravenous antibodies, suggesting their localization behind continuous blood vessel endothelium such as the blood-arachnoid barrier. Flt3Cre lineage tracing and bone marrow transplantation showed that the precursors were derived from adult hematopoietic stem cells and were most likely continuously replaced via cell trafficking. Importantly, their occurrence was tied to the immunologic state of the central nervous system (CNS) and was diminished in the context of neuroinflammation and ischemic stroke. Our findings confirm the presence of myeloid progenitors at the meningeal border of the brain and lay the foundation to unravel their possible functions in CNS surveillance and local immune cell production.

Resident CD34-positive cells contribute to peri-endothelial cells and vascular morphogenesis in salivary gland after irradiation.

Salivary gland (SG) hypofunction is a common post-radiotherapy complication. Besides the parenchymal damage after irradiation (IR), there are also effects on mesenchymal stem cells (MSCs) which were shown to contribute to regeneration and repair of damaged tissues by differentiating into stromal cell types or releasing vesicles and soluble factors supporting the healing processes. However, there are no adequate reports about their roles during SG damage and regeneration so far. Using an irradiated SG mouse model, we performed certain immunostainings on tissue sections of submandibular glands at different time points after IR. Immunostaining for CD31 revealed that already one day after IR, vascular impairment was induced at the level of capillaries. In addition, the expression of CD44-a marker of acinar cells-diminished gradually after IR and, by 20 weeks, almost disappeared. In contrast, the number of CD34-positive cells significantly increased 4 weeks after IR and some of the CD34-positive cells were found to reside within the adventitia of arteries and veins. Laser confocal microscopic analyses revealed an accumulation of CD34-positive cells within the area of damaged capillaries where they were in close contact to the CD31-positive endothelial cells. At 4 weeks after IR, a fraction of the CD34-positive cells underwent differentiation into α-SMA-positive cells, which suggests that they may contribute to regeneration of smooth muscle cells and/or pericytes covering the small vessels from the outside. In conclusion, SG-resident CD34-positive cells represent a population of progenitors that could contribute to new vessel formation and/or remodeling of the pre-existing vessels after IR and thus, might be an important player during SG tissue healing.

Do not keep it simple: recent advances in the generation of complex organoids.

3D cell culture models which closely resemble real human tissues are of high interest for disease modelling, drug screening as well as a deeper understanding of human developmental biology. Such structures are termed organoids. Within the last years, several human organoid models were described. These are usually stem cell derived, arise by self-organization, mimic mechanisms of normal tissue development, show typical organ morphogenesis and recapitulate at least some organ specific functions. Many tissues have been reproduced in vitro such as gut, liver, lung, kidney and brain. The resulting entities can be either derived from an adult stem cell population, or generated from pluripotent stem cells using a specific differentiation protocol. However, many organoid models only recapitulate the organs parenchyma but are devoid of stromal components such as blood vessels, connective tissue and inflammatory cells. Recent studies show that the incorporation of endothelial and mesenchymal cells into organoids improved their maturation and might be required to create fully functional micro-tissues, which will allow deeper insights into human embryogenesis as well as disease development and progression. In this review article, we will summarize and discuss recent works trying to incorporate stromal components into organoids, with a special focus on neural organoid models.

Generation of Cardiomyocytes From Vascular Adventitia-Resident Stem Cells.

RATIONALE: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. OBJECTIVE: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. METHODS AND RESULTS: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1+ AoCs from GFP (green fluorescent protein) mice, we identified Flk1+CD (cluster of differentiation) 34+Sca-1 (stem cell antigen-1)-CD44- AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1+ and CD34+ cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1+ (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. CONCLUSIONS: Our data suggest Flk1+CD34+ vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.

The role of connexins during early embryonic development: pluripotent stem cells, gene editing, and artificial embryonic tissues as tools to close the knowledge gap.

Since almost 4 decades, connexins have been discussed as important regulators of embryogenesis. Several different members of the gene family can be detected in the preimplantation embryo and during gastrulation. However, genetically engineered mice deficient for every connexin expressed during early development are available and even double-deficient mice were generated. Interestingly, all of these mice complete gastrulation without any abnormalities. This raises the question if the role of connexins has been overrated or if other gene family members compensate and mask their importance. To answer this question, embryos completely devoid of any gap junctional communication need to be investigated. This is challenging because a variety of connexin genes are co-expressed and some null mutations lead to a lethal phenotype. In addition, maternal connexin transcripts were described to persist until the blastocyst stage. In this review, we summarize the current knowledge about the role of connexins during preimplantation development and in embryonic stem cells. We propose that the use of pluripotent stem cells, trophoblast stem cells, as well as artificial embryo-like structures and organoid cultures in combination with multiplex CRISPR/Cas9-based genome editing provides a powerful platform to comprehensively readdress this issue and decipher the role of connexins during lineage decision, differentiation, and morphogenesis in a cell culture model for mouse and human development.

Abrogation of Gap Junctional Communication in ES Cells Results in a Disruption of Primitive Endoderm Formation in Embryoid Bodies.

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells. Stem Cells 2017;35:859-871.

A LewisX glycoprotein screen identifies the low density lipoprotein receptor-related protein 1 (LRP1) as a modulator of oligodendrogenesis in mice.

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.

Mutations in DNAJC19 cause altered mitochondrial structure and increased mitochondrial respiration in human iPSC-derived cardiomyocytes.

BACKGROUND: Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy. METHODS: We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca2+ kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tvHeLa). RESULTS: Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca2+ concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to ß-adrenergic stimulation. CONCLUSIONS: Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca2+ kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.

Neuro-mesodermal assembloids recapitulate aspects of peripheral nervous system development in vitro.

Here we describe a novel neuro-mesodermal assembloid model that recapitulates aspects of peripheral nervous system (PNS) development such as neural crest cell (NCC) induction, migration, and sensory as well as sympathetic ganglion formation. The ganglia send projections to the mesodermal as well as neural compartment. Axons in the mesodermal part are associated with Schwann cells. In addition, peripheral ganglia and nerve fibers interact with a co-developing vascular plexus, forming a neurovascular niche. Finally, developing sensory ganglia show response to capsaicin indicating their functionality. The presented assembloid model could help to uncover mechanisms of human NCC induction, delamination, migration, and PNS development. Moreover, the model could be used for toxicity screenings or drug testing. The co-development of mesodermal and neuroectodermal tissues and a vascular plexus along with a PNS allows us to investigate the crosstalk between neuroectoderm and mesoderm and between peripheral neurons/neuroblasts and endothelial cells.

Loss of connexin43-mediated gap junctional coupling in the mesenchyme of limb buds leads to altered expression of morphogens in mice.

Mutations in the GJA1 gene coding for connexin43 (Cx43) cause oculodentodigital dysplasia (ODDD), a pleiotropic human disorder with characteristic morphologic anomalies of face, teeth, bones and digits. Interdigital webbings, also called syndactylies, are a characteristic phenotype of this disease showing high intra- and interfamilial penetrance. Therefore, we decided to study the molecular basis of syndactylies caused by Cx43 mutations. In order to reveal the impact of Cx43-mediated gap junctional coupling, we used mice expressing the human point mutation Cx43G138R and, in addition, 'knock-out' mice lacking Cx43. Both conditional mouse models developed syndactylies as a consequence of disturbed interdigital apoptosis, which we show to be due to reduced expression of two key morphogens: sonic hedgehog (Shh) and bone morphogenic protein 2 (Bmp2). Diminished levels of Bmp2 and subsequent up-regulation of fibroblast growth factors (Fgfs) lead to an insufficient induction of interdigital apoptosis. Interestingly, the reduction of Shh expression in Cx43 mutants begins on embryonic day 10.5 indicating a disturbance of the Fgf/Shh regulatory feedback loop, and confirming the recently published observation that gap junctions can relay Fgf signals to neighboring cells. Thus, Cx43-mediated gap junctional coupling in the mesenchyme of limb buds after ED11 is essential to maintain Shh expression, which regulates the downstream signaling of Bmp2. Besides diminished interdigital apoptosis, the decreased expression of Bmp2 in Cx43 mutants may also be involved in other morphological alterations in patients suffering from ODDD.

The Impact of Oxygen Availability and Multilineage Communication on Organoid Maturation.

Significance: An optimal supply with oxygen is of high importance during embryogenesis and a prerequisite for proper organ development. Different tissues require varying amounts of oxygen, and even within single organs, different phases of development go alongside with either physiological hypoxia or the need for sufficient oxygen supply. Recent Advances: Human induced pluripotent stem cell-derived organoid models are state of the art cell culture platforms for the investigation of developmental processes, disease modeling, and drug testing. Organoids modeling the development of multiple tissues were developed within the past years. Critical Issues: Until now, optimization of oxygen supply and its role during organoid growth, differentiation, and maturation have only rarely been addressed. Recent publications indicate that hypoxia-induced processes play an important role in three-dimensional tissue cultures, triggering multilineage communication between mesenchymal cells, the endothelium, as well as organotypic cells. Later in culture, a sufficient supply with oxygen is of high importance to allow larger organoid sizes. Moreover, cellular stress is reduced and tissue maturation is improved. Therefore, a functional blood vessel network is required. Future Directions: In this review, we will briefly summarize aspects of the role of oxygen during embryonic development and organogenesis, present an update on novel organoid models with a special focus on organoid vascularization, and discuss the importance of complex organoids involving parenchymal cells, mesenchymal cells, inflammatory cells, and functional blood vessels for the generation of mature and fully functional tissues in vitro. Antioxid. Redox Signal. 35, 217-233.

Human iPSC-derived mesodermal progenitor cells preserve their vasculogenesis potential after extrusion and form hierarchically organized blood vessels.

Post-fabrication formation of a proper vasculature remains an unresolved challenge in bioprinting. Established strategies focus on the supply of the fabricated structure with nutrients and oxygen and either rely on the mere formation of a channel system using fugitive inks or additionally use mature endothelial cells and/or peri-endothelial cells such as smooth muscle cells for the formation of blood vesselsin vitro.Functional vessels, however, exhibit a hierarchical organization and multilayered wall structure that is important for their function. Human induced pluripotent stem cell-derived mesodermal progenitor cells (hiMPCs) have been shown to possess the capacity to form blood vesselsin vitro, but have so far not been assessed for their applicability in bioprinting processes. Here, we demonstrate that hiMPCs, after formulation into an alginate/collagen type I bioink and subsequent extrusion, retain their ability to give rise to the formation of complex vessels that display a hierarchical network in a process that mimics the embryonic steps of vessel formation during vasculogenesis. Histological evaluations at different time points of extrusion revealed the initial formation of spheres, followed by lumen formation and further structural maturation as evidenced by building a multilayered vessel wall and a vascular network. These findings are supported by immunostainings for endothelial and peri-endothelial cell markers as well as electron microscopic analyses at the ultrastructural level. Moreover, endothelial cells in capillary-like vessel structures deposited a basement membrane-like matrix at the basal side between the vessel wall and the alginate-collagen matrix. After transplantation of the printed constructs into the chicken chorioallantoic membrane (CAM) the printed vessels connected to the CAM blood vessels and get perfusedin vivo. These results evidence the applicability and great potential of hiMPCs for the bioprinting of vascular structures mimicking the basic morphogenetic steps ofde novovessel formation during embryogenesis.

SARS-CoV-2 Infects Endothelial Cells In Vivo and In Vitro.

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.

Generation of Vascularized Neural Organoids by Co-culturing with Mesodermal Progenitor Cells.

Organoids are three-dimensional (3D) constructs generated in stem cell cultures and are thought to mimic tissue and organ development in situ. However, until recently, they often exclusively recapitulated the development of the organ`s parenchyma without the major components of the organ stroma. Here, we describe a protocol to incorporate stromal components, first of all blood vessels, by co-culturing with induced pluripotent stem cell-derived mesodermal progenitor cells. For complete details on the use and execution of this protocol, please refer to Wörsdörfer et al. (2019).

Connexin expression and functional analysis of gap junctional communication in mouse embryonic stem cells.

Gap junctional intercellular communication (GJIC) has been suggested to be necessary for cellular proliferation and differentiation. We wanted to investigate the function of GJIC in mouse embryonic stem (ES) cells using pharmacological inhibitors or a genetic approach to inhibit the expression of connexins, that is, the subunit proteins of gap junction channels. For this purpose, we have analyzed all known connexin genes in mouse ES cells but found only three of them, Cx31, Cx43, and Cx45, to be expressed as proteins. We have demonstrated by coimmunoprecipitation that Cx31 and Cx43, as well as Cx43 and Cx45, probably form heteromeric gap junction channels, whereas Cx31 and Cx45 do not. The pharmacological inhibitors reduced GJIC between ES cells to approximately 3% and initiated apoptosis, suggesting an antiapoptotic effect of GJIC. In contrast to these results, reduction of GJIC to approximately 5% by decreased expression of Cx31 or Cx45 via RNA interference in homozygous Cx43-deficient ES cells did not lead to apoptosis. Additional studies suggested that apoptotic death of ES cells and adult stem cells reported in the literature is likely due to a cytotoxic side effect of the inhibitors and not due to a decrease of GJIC. Using the connexin expression pattern in mouse ES cells, as determined in this study, multiple connexin-deficient ES cells can now be genetically engineered in which the level of GJIC is further decreased, to clarify whether the differentiation of ES cells is qualitatively or quantitatively compromised.

Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells.

Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and disease. We propose, that the directed incorporation of mesodermal progenitor cells (MPCs) into organoids will overcome the aforementioned limitations. In order to demonstrate the feasibility of the method, we generated complex human tumor as well as neural organoids. We show that the formed blood vessels display a hierarchic organization and mural cells are assembled into the vessel wall. Moreover, we demonstrate a typical blood vessel ultrastructure including endothelial cell-cell junctions, a basement membrane as well as luminal caveolae and microvesicles. We observe a high plasticity in the endothelial network, which expands, while the organoids grow and is responsive to anti-angiogenic compounds and pro-angiogenic conditions such as hypoxia. We show that vessels within tumor organoids connect to host vessels following transplantation. Remarkably, MPCs also deliver Iba1+ cells that infiltrate the neural tissue in a microglia-like manner.

Aging-related carcinoembryonic antigen-related cell adhesion molecule 1 signaling promotes vascular dysfunction.

Aging is an independent risk factor for cardiovascular diseases and therefore of particular interest for the prevention of cardiovascular events. However, the mechanisms underlying vascular aging are not well understood. Since carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is crucially involved in vascular homeostasis, we sought to identify the role of CEACAM1 in vascular aging. Using human internal thoracic artery and murine aorta, we show that CEACAM1 is upregulated in the course of vascular aging. Further analyses demonstrated that TNF-α is CEACAM1-dependently upregulated in the aging vasculature. Vice versa, TNF-α induces CEACAM1 expression. This results in a feed-forward loop in the aging vasculature that maintains a chronic pro-inflammatory milieu. Furthermore, we demonstrate that age-associated vascular alterations, that is, increased oxidative stress and vascular fibrosis, due to increased medial collagen deposition crucially depend on the presence of CEACAM1. Additionally, age-dependent upregulation of vascular CEACAM1 expression contributes to endothelial barrier impairment, putatively via increased VEGF/VEGFR-2 signaling. Consequently, aging-related upregulation of vascular CEACAM1 expression results in endothelial dysfunction that may promote atherosclerotic plaque formation in the presence of additional risk factors. Our data suggest that CEACAM1 might represent an attractive target in order to delay physiological aging and therefore the transition to vascular disorders such as atherosclerosis.

Identification of Embryonic Neural Plate Border Stem Cells and Their Generation by Direct Reprogramming from Adult Human Blood Cells.

We report the direct reprogramming of both adult human fibroblasts and blood cells into induced neural plate border stem cells (iNBSCs) by ectopic expression of four neural transcription factors. Self-renewing, clonal iNBSCs can be robustly expanded in defined media while retaining multilineage differentiation potential. They generate functional cell types of neural crest and CNS lineages and could be used to model a human pain syndrome via gene editing of SCN9A in iNBSCs. NBSCs can also be derived from human pluripotent stem cells and share functional and molecular features with NBSCs isolated from embryonic day 8.5 (E8.5) mouse neural folds. Single-cell RNA sequencing identified the anterior hindbrain as the origin of mouse NBSCs, with human iNBSCs sharing a similar regional identity. In summary, we identify embryonic NBSCs and report their generation by direct reprogramming in human, which may facilitate insights into neural development and provide a neural stem cell source for applications in regenerative medicine.

Functional Analysis of Connexin Channels in Cultured Cells by Neurobiotin Injection and Visualization.

Functional gap junction channels between neighboring cells can be assessed by microinjection of low molecular weight tracer substances into cultured cells. The extent of direct intercellular communication can be precisely quantified by this method. This protocol describes the iontophoretic injection and visualisation of Neurobiotin into cultured cells.