The Cell Biology Core will be a critical resource to this PPG. It will provide the four Projects with access to a wide array of reagents, procedures, and analyses, including fully characterized cardiac mesenchymal cells (CMCs) and CMC-derived extracellular vesicles (CMC-EVs, hereafter referred to as EVs) isolated from mice and pigs, assays of CMC function in vitro, and characterization of EVs (e.g., arrays of EV-related non-coding RNAs, particle size analysis, protein assays). Specifically, the Core will i) isolate CMCs from mouse and pig hearts; ii) perform comprehensive CMC characterization, including proliferation, population doubling time, morphometric analysis (cell length and area), clonogenicity, analysis of marker expression, secretome analysis, and gene profiling; iii) isolate EVs from mouse and pig CMCs; iv) perform characterization of EVs, including particle size analysis, validation of markers expression, and RNA and protein profiling; v) isolate cells utilizing multiple markers; vi) assess cell viability; vii) determine changes in cell signaling; and viii) quantify cells at each phase of the cell cycle. The individual Projects do not have the facilities or expertise needed to isolate and characterize CMCs consistently or to perform the extensive EV characterizations. Consolidation of all CMC/EV work into a Core facility will decrease the costs of supplies and equipment because the Core will make bulk purchases of supplies (thereby reducing expenses) and because waste and unnecessary duplication of supplies, reagents, and equipment will be eliminated with the maintenance of centralized stocks and inventories. Consolidating CMC/EV work into a Core facility is also time-effective because the techniques involved in this work are very labor intensive and require dedicated, skilled personnel. Core C will make available to the four Projects the tremendous experience and expertise of the Core personnel in the preparation and analysis of CMCs and EVs. The Core staff, a strong team of dedicated individuals under the immediate supervision of the Core Leader, will provide consistency and reproducibility of analysis. This is crucial, because it will ensure that all four Projects will use CMCs isolated and expanded the same way, thereby making the results comparable. A single source of CMCs and EVs with rigorous standardization to ensure consistency will result in transplantation of uniform CMC and EV populations in all four Projects and thus is extremely important for the integration and comparison of results from each of the Projects. This will make it possible to compare results of different experiments within the same Project and among different Projects. In summary, the Core will ensure quality control and, by eliminating duplication of effort and maximizing the use of personnel and supplies, will enable an efficient use of resources.
The Cell Biology Core will provide the four Projects with a uniform source of cardiac mesenchymal cells (CMCs) and CMC-derived extracellular vesicles. It will provide uniform assays of CMC function in vitro and characterization of EVs.
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