Single cell cloning generates lung endothelial colonies with conserved growth, angiogenic, and bioenergetic characteristics

Pulmonary artery, capillary, and vein endothelial cells possess distinctive structures and functions, which represent a form of vascular segment specific macroheterogeneity. However, within each of these segmental populations, individual cell functional variability represents a poorly characterized microheterogeneity. Here, we hypothesized that single cell clonogenic assays would reveal microheterogeneity among the parent cell population and enable isolation of highly representative cells with committed parental characteristics. To test this hypothesis, pulmonary microvascular endothelial cells (PMVECs) and pulmonary arterial endothelial cells (PAECs) were isolated from different Sprague Dawley rats. Serum stimulated proliferation of endothelial populations and single cell clonogenic potential were evaluated. In vitro Matrigel assays were utilized to analyze angiogenic potential and the Seahorse assay was used to evaluate bioenergetic profiles. PMVEC populations grew faster and had a higher proliferative potential than PAEC populations. Fewer PMVECs were needed to form networks on Matrigel when compared with PAECs. PMVECs primarily utilized aerobic glycolysis, while PAECs relied more heavily on oxidative phosphorylation, to support bioenergetic demands. Repeated single cell cloning and expansion of PAEC colonies generated homogeneous first-generation clones that were highly reflective of the parental population in terms of growth, angiogenic potential, and bioenergetic profiles. Repeated single cell cloning of the first-generation clones generated second-generation clones with increased proliferative potential while maintaining other parental characteristics. Second-generation clones were highly homogeneous populations. Thus, single cell cloning reveals microheterogeneity among the parent cell population and enables isolation of highly representative cells with parental characteristics.