Expression data of quaternary clones from rat lung CD117+ endothelial cells vs. non-clonal control endothelial cells

One current concept suggests that unchecked proliferation of clonally selected precursors of endothelial cells contribute to severe pulmonary arterial hypertension (pAH). We hypothesized that clonally selected ECs expressing the progenitor marker CD117 promote severe occlusive pulmonary hypertension (PH). We used microarrays to identify the steady state gene expression profile of quaternary clones derived from CD117+ rat lung ECs vs control ECs derived from rat lung CD117- cells. Following isolation of CD117+ lin- CD31+ cells from the rat lungs, cells were clonally expanded by seeded at limiting dilution (1 cell per well) in 96 well plates. The presence of 1 cell per each well was confirmed by microscopy. After 14 days, the largest colonies with typical endothelial “cobblestone” morphology were identified and the 6 largest endothelial colonies were used for the next round of clonal expansion. This procedure was repeated until fourth generation clonal colonies were obtained. Selection of the largest clonal colonies ensured selection of the clones with the highest proliferation and self-renewal capacity. For experiments, the cells were used after 4 clonal expansions and in the first 5 post-clonal passages. CD117- lin- CD31+ ECs were isolated from rat lungs without clonal expansion and used in passage 1-5.