Transforming growth factor (TGF)-β regulates vascular development through two type I receptors: activin receptor-like kinase (ALK) 1 and ALK5, each of which activates a different downstream Smad ...pathway. The endothelial cell (EC)-specific ALK1 increases EC proliferation and migration, whereas the ubiquitously expressed ALK5 inhibits both of these processes. As ALK1 requires the kinase activity of ALK5 for optimal activation, the lack of ALK5 in ECs results in defective phosphorylation of both Smad pathways on TGF-β stimulation. To understand why TGF-β signaling through ALK1 and ALK5 has opposing effects on ECs and whether this takes place in vivo, we carefully compared the phenotype of ALK5 knock-in (ALK5KI/KI) mice, in which the aspartic acid residue 266 in the L45 loop of ALK5 was replaced by an alanine residue, with the phenotypes of ALK5 knock-out (ALK5−/−) and wild-type mice. The ALK5KI/KI mice showed angiogenic defects with embryonic lethality at E10.5–11.5. Although the phenotype of the ALK5KI/KI mice was quite similar to that of the ALK5−/− mice, the hierarchical structure of blood vessels formed in the ALK5KI/KI embryos was more developed than that in the ALK5−/− mutants. Thus, the L45 loop mutation in ALK5 partially rescued the earliest vascular defects in the ALK5−/− embryos. This study supports our earlier observation that vascular maturation in vivo requires both TGF-β/ALK1/BMP-Smad and TGF-β/ALK5/activin-Smad pathways for normal vascular development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder in humans which has been mapped to two genes, endoglin and activin receptor-like kinase-1 (ALK-1) both of which mediate signaling by ...transforming growth factor beta ligands in vascular endothelial cells. Animal models have shown that these receptors are not only important for maintaining vascular integrity but also for angiogenesis both during embryonic development and during tumor growth. Here, we review the current status of reported mutations in the context of the clinical manifestations and the effects on the vessel wall both in patients and in animal models of the disease.
Key regulatory components of the BMP‐induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, ...Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2‐induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP‐induced osteoblast differentiation.
Introduction: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone‐related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2‐induced osteoblast differentiation.
Materials and Methods: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high‐density microarrays. The regulated genes were grouped by means of model‐based clustering, and protein functions were assigned. Real‐time quantitative RT‐PCR analysis was used to validate BMP2‐induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP‐induced osteoblast differentiation.
Results and Conclusions: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Worldwide increases in life expectancy have been paralleled by a greater prevalence of chronic and age-associated disorders, particularly of the cardiovascular, neural and metabolic systems. This has ...not been met by commensurate development of new drugs and therapies, which is in part owing to the difficulty in modelling human diseases in laboratory assays or experimental animals. Patient-specific induced pluripotent stem (iPS) cells are an emerging paradigm that may address this. Reprogrammed somatic cells from patients are already applied in disease modelling, drug testing and drug discovery, thus enabling researchers to undertake studies for treating diseases 'in a dish', which was previously inconceivable.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to ...identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.
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•T3+IGF-1+ dexamethasone improves the electrophysiology of hPSC cardiomyocytes•These factors synergistically enhance bioenergetics and contractile force generation•Cardiomyocytes with HCM-causing mutations have a contractile defect
Birket et al. identify a combination of factors that cooperatively improve the function of human pluripotent stem cell (hiPSC)-derived cardiomyocytes. Optimizing the system facilitated the identification of a contraction force defect in a model of hypertrophic cardiomyopathy (HCM), a disease affecting ∼1:500 of the population.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This ...contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal ...stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential. We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro . These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Generic methods for genetic manipulation of human embryonic stem cells (hESCs) are important for both present research and future commercial applications. To date, differences in cell derivation and ...culture have required independent optimization of transfection and transduction protocols and some lines have remained refractile to all methods. Here we describe a culture protocol that has been extensively tested in 12 different hESC lines (1, 2) and shown to support efficient gene transfer independent of the method of gene delivery or history of the cell line. The system is based on Matrigel monolayer culture and conditioned medium from mouse embryonic feeder cells (MEFs) and entails transient high-density culture followed by rapid adaptation to low density for gene transfer. Under these conditions, plasmid transfection, virus infection, and siRNA transfection are highly effective. Stable genetically modified hESC lines can be generated with plasmid transfection, viral infection, or electroporation without loss of pluripotency or differentiation potential. The majority of lines generated in this system display a normal karyotype.
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FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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