ABSTRACT
Electrical stimulation is the standard technique for exploring electrical behavior of heart muscle, but this approach has considerable technical limitations. Here we report expression of the light-activated cation channel channelrhodopsin-2 for light-induced stimulation of heart muscle in vitro and in mice. This method enabled precise localized stimulation and constant prolonged depolarization of cardiomyocytes and cardiac tissue resulting in alterations of pacemaking, Ca(2+) homeostasis, electrical coupling and arrhythmogenic spontaneous extrabeats.
Subject(s)
Myocytes, Cardiac/physiology , Animals , Calcium/metabolism , Channelrhodopsins , Electric Stimulation , Electrocardiography , Mice , Pacemaker, ArtificialABSTRACT
Vessel formation is of critical importance for organ function in the normal and diseased state. In particular, the labeling and quantitation of small vessels prove to be technically challenging using current approaches. We have, therefore, established a transgenic embryonic stem (ES) cell line and a transgenic mouse model where the vascular endothelial growth factor receptor VEGFR-1 (flt-1) promoter drives the expression of the live reporter eGFP. Fluorescence microscopy and immunostainings revealed endothelial-specific eGFP labeling of vascular networks. The expression pattern recapitulates that of the endogenous flt-1 gene, because small and large vessels are labeled by eGFP during embryonic development; after birth, the expression becomes more restricted to small vessels. We have explored this in the cardiovascular system more in detail and found that all small vessels and capillaries within the heart are strongly eGFP+. In addition, myocardial injuries have been induced in transgenic mice and prominent vascular remodeling, and an increase in endothelial cell area within the peri-infarct area could be observed underscoring the utility of this mouse model. Thus, the transgenic flt-1/eGFP models are powerful tools to investigate and quantify vascularization in vivo and to probe the effect of different compounds on vessel formation in vitro.
Subject(s)
Endothelium, Vascular/cytology , Mice, Transgenic , Myocardial Infarction/physiopathology , Neovascularization, Physiologic/physiology , Promoter Regions, Genetic , Vascular Endothelial Growth Factor Receptor-1/genetics , Animals , Blotting, Western , Disease Models, Animal , Green Fluorescent Proteins , Immunohistochemistry , Mice , Microscopy, Fluorescence , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.
Subject(s)
5'-Nucleotidase/metabolism , Bone Marrow Cells/metabolism , Endothelial Cells/metabolism , Multipotent Stem Cells/metabolism , Staining and Labeling , Stem Cell Niche , Animals , Bone Marrow/metabolism , Bone Marrow Cells/cytology , Chondrogenesis , Endothelial Cells/cytology , Female , Genes, Reporter , Green Fluorescent Proteins/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Multipotent Stem Cells/cytology , Organ Specificity , Stromal Cells/cytology , Stromal Cells/metabolismABSTRACT
Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes.