Hypoxia Preserves the Quiescent Lineage Negative Phenotype of Cardiac Stem Cells Within the Niche

In stem cell-regulated organs, a subset of niches is characterized by low oxygen tension. This metabolic adaptation offers a selective advantage to stem cells favoring the preservation of their quiescent undifferentiated phenotype. The objective of this work was to determine whether in the mouse heart cardiac niches constitute a heterogeneous compartment composed of hypoxic and normoxic niches, and whether differences in O 2 concentration affect the function of c-kit-positive cardiac stem cells (CSCs).To test this possibility, we studied first the in vivo uptake of the hypoxic marker pimonidazole (PIMO), which identifies intracellular O 2 concentration <10 mmHg. Mice were sacrificed 2 hours after intraperitoneal administration of PIMO, and PIMO-labeling was analyzed. By immunolabeling, 15% of cardiac niches were characterized by a hypoxic microenvironment and more than 20% of isolated CSCs were PIMO-positive, as measured by flow-cytometry. The cell cycle protein Ki67 was restricted to the PIMO-negative CSC class, which contained early committed cells expressing c-kit together with the myocyte specific transcription factors GATA4 and Nkx2.5. Mice were then administered tirapazamine, a compound that kills selectively hypoxic cells. One day later, the fraction of PIMO-positive CSCs was markedly decreased but, at 5 days, this compartment was partly reconstituted. This compensatory response was coupled with increased proliferation of PIMO-negative CSCs, suggesting that normoxic CSCs have the ability to replenish hypoxic niches following injury. Subsequently, the effects of hypoxia were studied in human CSCs (hCSCs) exposed in vitro to 1% O 2 . With respect to cells cultured in normoxia, 1% O 2 led to upregulation of HIF1α in hCSCs which also showed lower levels of BrdU incorporation. These cellular responses were accompanied by an increase in transcripts for the stemness genes c-kit, Oct4, Nanog and Sox2, and a decrease in mRNA for myocyte and vascular genes. Apoptosis, measured by TdT labeling, did not differ in normoxic and hypoxic hCSCs. In conclusion, our data indicate that hypoxic and normoxic niches coexist in the myocardium, and that intracellular hypoxia regulates the quiescent primitive CSC phenotype.