Oxygen Sensing of Mesenchymal Stem and Progenitor Cells Facilitates Neo-Vasculogenesis In Vivo

Vascular homeostasis and regeneration are maintained by proliferating vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs). Despite promising experimental data, regenerative stem cell therapy approaches employing ECFCs have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. We and others have recently shown that ECFC function in vivo requires a stringent interaction with mesenchymal stem and progenitor cells (MSPCs) * [Blood 2009; 113 (26):6716-25]. Co-transplantation of ECFCs and MSPCs is considered to be an advantageous strategy for vascular regenerative medicine.

Hypoxia in ischemic tissue is considered to be a key factor influencing pro- and anti-angiogenic treatment by driving the revascularization machinery. In vivo most cells exist under an O₂ pressure considerably below air oxygen. In vitro cells are usually expanded under air oxygen and suddenly encounter reduced O₂ conditions when re-injected for therapy. Preliminary data suggests that low oxygen conditions differentially regulate stem cell function. We hypothesized that MSPCs act as hypoxia sensors and drive ECFCs to form functioning vessels in vivo.

Adult human ECFCs were isolated and propagated directly from whole venous blood using a novel recovery strategy **[J Vis Exp. 2009;(32) pii: 1524]. MSPCs were isolated from human bone marrow aspirates. During cell culture, pooled human platelet lysate (pHPL) entirely replaced fetal bovine serum. Throughout this study we designated the oxygen level present in vivo in the venous environment as euoxia (41.5±3.4 mmHg). Oxygen levels below euoxia are defined as hypoxia (27.4±7.3 mmHg). Air-oxygen commonly used in standard laboratory practice is above euoxia and is therefore referred to as hyperoxia (139.8±2.9 mmHg). Progenitor cell phenotype, hierarchy, long-term proliferation, wound repair as well as migratory and vasculogenic functions were monitored under euoxia as compared to hypoxic or hyperoxic conditions. Molecular regulation of cellular responses to different oxygen levels was assessed by flow cytometry, immune cytochemistry and proteomic profiling. ECFC and MSPC interactions in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) after sub-cutaneous co-implantation in matrigel plugs. Immune histochemistry and TUNEL assays were performed on plugs at day 1, 7 and 14 after transplantation.

Compared to hyperoxic standard laboratory conditions in vitro, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced under euoxia, and even more under hypoxic conditions. Hyperoxic conditioning resulted in a shift in progenitor hierarchy with an augmented number of ECFC high proliferative potential (HPP) colonies (60±18% of total colonies) as compared to euoxia (9±6%) and a complete loss of HPP colonies under hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair in scratch assays and matrigel vascular-like network formation in vitro were improved with escalating oxygen supply. The reoxygenation of hypoxic and euoxic ECFCs led to enhanced proliferation and function. Furthermore, MSPCs stabilized hypoxia inducible factor-1α (HIF-1α) under hypoxic as well as euoxic conditions, whereas ECFCs only stabilized HIF-1α when confronted with hypoxia in vitro. In a mouse model, subcutaneously injected ECFCs in matrigel underwent apoptosis after 1 day and attracted mouse leucocytes which infiltrated the matrigel plug. Co-implantation of ECFCs and MSPCs in these matrigel plugs resulted in reduced apoptosis and formation of perfused human vessels as soon as 7 days after transplantation. In this in vivo setting, perivascular cells but not endothelial cells were positive for HIF-1α in immune histochemistry.

These data indicate that oxygen levels differentially regulate ECFC and MSPC function during vascular homeostasis and regeneration. While hypoxic ECFCs alone are not able to function in vitro and form patent vessels in vivo, MSPCs react to the low oxygen environment and support ECFCs to perform vessel formation in vivo at least in part by rescuing ECFCs from hypoxia-induced apoptosis. This suggests that oxygen appears to be a key factor in stem cell transplantation and regenerative medicine.

No relevant conflicts of interest to declare.