Abstract 2987 Mesenchymal stem/progenitor cells (MSPCs) from numerous tissues are currently tested in clinical trials despite a limited understanding of their in vivo behavior. In this study we used MSPCs from adult and fetal tissues to select the appropriate source for clinical application. We asked whether MSPCs derived from human bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC), compared to skin fibroblasts, bear an equivalent bone and marrow niche formation potential with of in vivo . Furthermore we evaluated attraction and engraftment of murine as well as human hematopoietic stem/progenitor cells (HSPCs) into newly formed MSPC-derived niches. To elucidate potential mechanisms responsible for a tissue-specific MSPC potential after transplantation gene expression profiling and DNA methylation analysis on a novel high resolution 450K-CpG methylation array were employed.MSPCs were transplanted subcutaneously to test for their spontaneous bone and marrow niche formation potential in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). BM-derived MSPC transplantation reproducibly led to the development of mature bone (17/17 donors) through an endochondral ossification process leading to subsequent marrow niche formation. Additionally, these newly formed hematopoietic microenvironments attracted complete mouse hematopoiesis including immature lineage negative, Sca-1 positive, c-kit positive (LSK) HSPCs. Non-BM derived MSPCs completely lacked bone and marrow niche-forming potential and did not attract hematopoietic cells (0/9 donors). Induction of human hematopoietic chimerism through transplantation of umbilical cord blood (UCB)-derived human CD34+ HSPCs in advance of subcutaneous ectopic bone and marrow development resulted in immigration of re-transplantable human hematopoiesis into extra-medullary ossicles. Comparative 450K-CpG methylation array profiling of MSPCs revealed a tissue-specific epigenetic signature virtually corresponding to the in vivo differentiation. MSPCs from BM but neither fibroblasts nor MSPCs from WAT or UC showed epigenetically imprinted human bone and marrow niche (HuNiche) formation capacity favoring BM-MSPCs for skeletal regeneration. This novel HuNiche model should be ideally suited for studying normal and malignant hematopoiesis regulation in an ectopic human marrow with subsequent human hematopoietic engraftment that mimics clinical BM transplantation reality.Disclosures: No relevant conflicts of interest to declare.

Despite insights into the molecular pathways regulating hypoxia-induced gene expression, it is not known which cell types accomplish oxygen sensing during neo-vasculogenesis. We have developed a humanized mouse model of endothelial and mesenchymal progenitor co-transplantation to delineate the cellular compartments responsible for hypoxia response during vasculogenesis. Mesenchymal stem/progenitor cells (MSPCs) accumulated nuclear hypoxia-inducible transcription factor (HIF)-1α earlier and more sensitively than endothelial colony forming progenitor cells (ECFCs) in vitro and in vivo. Hypoxic ECFCs showed reduced function in vitro and underwent apoptosis within 24h in vivo when used without MSPCs. Surprisingly, only in MSPCs did pharmacologic or genetic inhibition of HIF-1α abrogate neo-vasculogenesis. HIF deletion in ECFCs caused no effect. ECFCs could be rescued from hypoxia-induced apoptosis by HIF-competent MSPCs resulting in the formation of patent perfused human vessels. Several angiogenic factors need to act in concert to partially substitute mesenchymal HIF-deficiency. Results demonstrate that ECFCs require HIF-competent vessel wall progenitors to initiate vasculogenesis in vivo and to bypass hypoxia-induced apoptosis. We describe a novel mechanistic role of MSPCs as oxygen sensors promoting vasculogenesis thus underscoring their importance for the development of advanced cellular therapies.

Abstract 613 Background: Hypoxia is a major stimulus of neo-vasculogenesis. Under hypoxic conditions endothelial colony-forming progenitor cells (ECFCs) arrange tubular structures, which can connect to the pre-existing vasculature forming functional perfused vessels. The current view is that mesenchymal stem and progenitor cells (MSPCs) or their pericyte progeny are recruited subsequently to stabilize vessels. So far, clinical applications of endothelial progenitors to restore tissue oxygenation after ischemia, cardiovascular disease or stroke largely failed to meet medical needs. Based on previous work demonstrating patent vessel formation after MSPC/ECFC co-transplantation in vivo (Blood 2009), we hypothesized that MSPCs have a decisive role in the vasculogenic response to hypoxia. Here we show for the first time that ECFCs in hypoxic conditions in vivo strictly require the presence of functional MSPCs not only to stabilize but primarily to initiate neo-vasculogenesis by a hypoxia-inducible transcription factor (HIF)-dependent mechanism. Methods: Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. Progenitor cell phenotype, long-term proliferation, HIF stabilization, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O 2 ), venous oxygen (5% O 2 ) and standard ambient air culture conditions (20% O 2 ). ECFC and MSPC crosstalk in vivo was studied in immune-deficient NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelet lysate gel). Cell type-specific chemical and genetic inhibition of HIF (YC-1, shRNA) was used to delineate the role of hypoxia sensing in MSPCs and ECFCs, respectively, during vasculogenesis in vivo . To determine if downstream target proteins of HIF-1α could substitute for MSPC presence during vasculogenesis, selected growth factors and cytokines were tested. Results: Progenitor proliferation and function in vitro were reduced with declining oxygen levels. ECFCs stabilized hypoxia-inducible factor-1α (HIF-1α) only at 1% O 2 , while MSPCs stabilized HIF-1α already at 5% O 2 . In an NSG mouse model, ECFCs transplanted into a hypoxic environment did not stabilize HIF-1α, while transplanted sole MSPCs or MSPCs in co-transplants showed strong nuclear HIF-1α stabilization 1 day after transplantation preceding any vessel formation or perfusion. In the absence of MSPCs, the majority of ECFCs underwent apoptosis within 24h in vivo. Inhibition of HIF-1α stabilization in MSPCs but not in ECFCs significantly abrogated vessel formation in vivo . Blocking the prominent HIF-1α down-stream target vascular endothelial growth factor (VEGF) resulted in the expected inhibition of neo-vasculogenesis. Interestingly, substitution of VEGF alone could not restore vessel formation, neither when injected together with sole ECFCs nor in a model where ECFCs were co-transplanted with HIF-depleted MSPCs. Substitution of a complex mixture of platelet-derived factors in vivo partly restored the vasculogenic function of HIF-depleted MSPCs. Conclusions: MSPCs react to a low oxygen environment by stabilizing HIF-1α earlier and more sensitively than ECFCs. MSPCs promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis in the initial phase of vasculogenesis by a HIF-dependent trophic mechanism. Surprisingly, therapeutic vasculogenesis can occur independently of endothelial HIF stabilization. These results argue in favor of MSPC/ECFC co-transplantation as a promising strategy for vascular regenerative therapy. The observation that VEGF alone could not compensate for the vasculogenic competence of pericyte precursors in vivo underlines the complexity of the hypoxia-induced cytokine network. The fact that hypoxia sensing in MSPCs but not in ECFCs is crucial to initiate vascular regeneration supports a shift of focus from endothelial cells to perivascular mesenchymal cells as a therapeutic target in anti-angiogenic therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1916 Clinical trials are underway to test the safety and efficacy of mesenchymal stem/progenitor cells (MSPCs) in various diseases. Due to their low frequency in situ, MSPC expansion is the prerequisite for dose finding studies as well as for most applications in adult patients. Notably, cultured MSPCs are a mixture of heterogeneous cells in various stages of cell cycle, proliferation and differentiation activity. A major safety concern for MSPC propagation is the risk of malignant transformation or premature senescence hampering MSPC function. The in vitro and consequently in vivo cellular characteristics may be influenced by factors as tissue source, age of the donor, materials and media, growth factors and oxygen pressure, arguing for standardized culture procedures at least in clinical trials. Defining the optimal conditions for efficient expansion of clinical grade cell therapeutics is still a challenge. We have previously shown that long-term expanded human bone marrow-derived MSPCs acquired senescence-related gene expression changes independent of culture conditions (Haematologica 2010). It has been speculated that elevated oxygen (20% air O2) contributes to genomic instability and malignant transformation in vitro. We therefore analyzed the influence of different oxygen conditions during long-term expansion on MSPC behavior focussing osteogenic differentiation. A gene panel previously defined as senescence markers was tested for differential expression after varying culture conditions. Bone marrow-derived MSPCs were expanded in α-MEM supplemented with 10% human platelet lysate replacing fetal bovine serum under physiologic conditions (5% O2) or air oxygen (20% O2) until spontaneous cessation of proliferation. Osteogenic induction was analyzed by Alizarin red. RNA was isolated from corresponding early and late passages and analyzed by qRT-PCR for p16ink4a, PARG1, CDKN2B, PTN and MCM6. In total, MSPCs could be cultured for 5 passages at 30 cells/cm2 and for 10 passages at 3,000 cells/cm2 for up to 85 days resulting in more cumulative population doublings (PDs) of MSPCs at air O2 compared to 5% O2 and in cultures with low compared to standard seeding density. Long-term cultured MSPCs after 40 PDs (air O2) and 35 PDs (5% O2) retained their osteogenic differentiation capacity. Compared to early passages, RT-PCR in late passages revealed an up-regulation of p16ink4a, PARG1 and CDKN2B without specific influence of culture conditions. PTN and MCM6 were significantly down-regulated, mainly in air O2 cultures with high seeding density correlating with diminished cell proliferation compared to low density cultures. There was no evidence of immortalization or malignant transformation. The capacity for in vivo bone formation of long term cultured MSPCs is currently tested in a novel humanized mouse model for bone and marrow niche formation (Blood 2012). Long term expansion of MSPCs under animal serum-free air oxygen conditions was safe and most efficient at low seeding density. Even in late passages (>30 PDs) MSPCs preserved their potential for osteogenic differentiation in vitro. At air oxygen delayed replicative senescence was observed, mainly at low seeding density. There was no evidence for immortalization or transformation indicating applicability of standardized ambient air culture conditions for pre-clinical cell expansion. Disclosures: No relevant conflicts of interest to declare.

Abstract 2386 Human mesenchymal stem and progenitor cells (MSPCs) from various tissues are currently evaluated in clinical trials for bone and marrow regeneration and their immune modulation potential. MSPCs from virtually all tissues appear indistinguishable regarding immune phenotype and their multipotent differentiation capacity in vitro. Improvement of so far limited clinical efficiency is hampered by a lack of understanding MSPC functionality in vivo. Here we demonstrate that the capacity of in vivo endochondral bone formation followed by establishment of a hematopoietic niche through infiltration of blood producing hematopoietic components function as a surrogate to determine in vivo multipotentiality (differentiation into more than 3 mesodermal lineages) of isolated MSPCs. MSPCs from bone marrow (BM), adipose tissue (AT) and umbilical cord (UC) have been isolated by plastic adherence and were propagated under humanized culture conditions using pooled human platelet lysate (pHPL) as previously described. Human skin fibroblast (Fibs) derived under identical culture conditions where used as control throughout the study. Comparative analyses of surface immune phenotype, adipo-, chondro- and osteogenic differentiation potential in vitro as well as expression analysis of key mesenchymal lineage genes were performed. Epigenetic profiling of MSPCs from different tissues was done using a methylation array including CpG-islands in- and outside of coding regions, CpG-shores and non CpG sides (450K array; Illumina). In vivo differentiation capacity was tested by using two million of MSPCs transplanted subcutaneously into immune-deficient NSG mice. The developmental sequence of chondro- and osteogenic as compared to perivascular mesenchymal tissue formation was analyzed using histology and immune histochemistry. In vivo near infrared (NIR) fluorescence imaging and micro computed tomography (microCT) was used to study bone development. Formation of a human MSPC-derived marrow niche with establishment of the complete host hematopoiesis was studied in situ and by polychromatic flow cytometry. Secondary transplants of MSPCs isolated from primary marrow organs were performed and analyzed equally. MSPCs from all tissues analyzed and control Fibs show an almost identical immune phenotype using a classical MSPC marker profile. Osteo- and adipogenic differentiation potential in vitro as well as gene expression did not distinguish tissue-specific MSPCs. Using a stringent 3D chondrogenesis assay and appropriate histological stainings of synthesized chondrogenic matrix proteins (Safranin O, Alcian Blue, Toluidin Blue) MSPCs from all tissues except BM failed to form cartilage in vitro. In vivo mouse studies could further strengthen these findings, because BM-derived MSPCs were the only cell type capable of generating ectopic bone through an endochondral ossification process. Bone formation was followed by mouse marrow infiltration including megakaryocytes as well as lineage negative, sca-1 positive, c-kit positive (LSK) hematopoietic stem and progenitor cells (HSPCs). These results correlated with the epigenetic status of the cells. Comparing their methylation profile using principal component analysis (PCA), BM-MSPCs cluster separately, whereas MSPCs from all other tissues cluster together. In this study BM was the only tissue containing MSPCs with multipotent differentiation capacity including chondrogenesis, osteogenesis and hematopoietic niche formation. This is reflected by a BM specific epigenetic profile that differs from that of all other tissues analyzed. Since cartilage formation is one initial developmental process important for bone generation and hematopoiesis attraction, an epigenetic predisposition of BM MSPCs to undergo endochondral ossification seems to make these cells unique for bone and marrow regeneration purposes. More stringent test systems including consequent in vivo potency assays should be mandatory in context of clinical studies. Our data may argue against the application of non BM-MSPCs for bone and marrow regeneration in patients in the absence of experimental in vivo evidence. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 699 Rationale: Vascular repair after hypoxic tissue damage requires a stringent interaction between somatic endothelial colony-forming progenitor cells (ECFCs) and mesenchymal stem and progenitor cells (MSPCs). Stem cell therapy to re-vascularize ischemic tissue has been a promising tool for various therapeutic targets including stroke, myocardial infarction and peripheral artery disease. Despite promising experimental data, therapeutic approaches employing endothelial progenitor cells have been of rather limited efficiency in clinical trials for both therapeutic vasculogenesis as well as anti-angiogenic therapy. Hypoxia in ischemic tissue is an extensively studied key factor that influences pro- and anti-angiogenic treatment by driving the revascularization machinery. We and others have shown that despite hypoxic stimulation, ECFCs in vivo only form patent vessels in the presence of MSPCs. Here we show that MSPCs but not ECFCs are the oxygen sensors enabling vasculogenesis in vivo. Methods: Adult human ECFCs were isolated from blood and MSPCs from bone marrow aspirates and expanded under humanized culture conditions. In in vitro studies progenitor cell phenotype, long-term proliferation, molecular cellular response, wound repair as well as migratory and vasculogenic functions were monitored under severe hypoxia (1% O2), venous oxygen conditions (5% O2) and standard culture conditions (20% O2). ECFC and MSPC interaction in vivo were studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) after subcutaneous transplantation in various extracellular matrices (matrigel, collagen/fibronectin, human platelete lysate). To investigate the respective roles of MSPCs and ECFCs during vasculogenesis under hypoxia in vivo chemical and genetic inhibitors against protein synthesis (cycloheximide) and HIF-1α (YC-1, shRNA) were employed. Immune histochemistry, immune fluorescence and TUNEL assays were performed on plugs in the time course after transplantation. Results: In vitro studies showed that compared to 20% O2, proliferation of ECFCs and MSPCs in primary and long-term cultures was significantly reduced at 5% O2, and even more at 1% O2. Standard culture conditions resulted in a shift in the progenitor hierarchy with an augmented number of high proliferative potential (HPP)-ECFC colonies (60±18% of total colonies) as compared to venous oxygen conditions (9±6%) and a complete loss of HPP-ECFC colonies under severe hypoxia (0%). The absolute colony number remained unchanged independent of oxygen levels. Both ECFC vascular wound repair function in scratch assays and the ability to form vascular-like networks in matrigel assays in vitro were diminished with declining oxygen supply. The re-oxygenation to 20% O2 of ECFCs which where precultured at 1% or 5% O2 led to enhanced proliferation, colony size and function. Single cell analysis revealed that ECFCs stabilized hypoxia-inducing factor-1α (HIF-1α) only at 1% O2 while MSPCs stabilize HIF-1α at 1% O2 as well as 5% O2 conditions. In a mouse model, subcutaneously injected ECFCs underwent apoptosis after 24h and attracted mouse leucocytes. In contrast, ECFCs co-implanted in vivo with MSPCs were rescued from apoptotic death and formed perfused human vessels 7 days after transplantation independent of matrix. Perivascular cells, but not ECFCs, were positive for HIF-1α in vivo. Inhibition of MSPCs but not ECFCs protein synthesis and HIF-1α prior to co-implantation blocked vessel formation. Conclusion: These data demonstrates that hypoxic ECFCs alone show reduced functuionality in vitro and form patent vessels in vivo. In contrast, MSPCs react to the low oxygen environment more sensitively than ECFCs and promote vessel formation at least in part by rescuing ECFCs from hypoxia-induced apoptosis. Surprisingly, this study shows that therapeutic vasculogenesis can occur independent of endothelial HIF stabilization and protein synthesis. This data indicate that in addition to their established role regulating hematopoiesis, MSPCs oxygen sensing is crucial during vascular regeneration. This suggests a shift of focus from endothelial cells to perivascular cells as a therapeutic target in regenerative medicine and anti-angiogenic therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1322 Introduction. Mesenchymal Stem/Progenitor Cells (MSPC) are regarded as the universal skeletal progenitor, theoretically capable of differentiating into cartilage, bone, tendon and muscle. Their functions as pericytes as well as key bone marrow stromal cells are also well documented. Tremendous benefits could therefore be achieved by cell therapy with MSPC. Unfortunately, the very successful research aimed at isolating, expanding and differentiating these cells in vitro has so far failed to translate into significant clinical advances. In particular, bone regeneration studies are disappointing, because application of MSPC in vivo requires their osteogenic pre-differentiation in vitro and/or their co-implantation with bone chips, and because results are highly donor-dependant. In this context, recent studies indicate that MSPC expanded from human Bone Marrow (BM) in media supplemented by Human Platelet Lysate (HPL) in lieu of Fetal Bovine Serum (FBS) have a higher rate of bone differentiation. Here, we use a new, simplified mouse model of ectopic bone formation requiring neither pre-differentiation nor bone matrix. We show that human BM-MSPC expanded in HPL-supplemented medium from all donors spontaneously form bone through an endochondral mechanism. Importantly, the ossicles generated from MSPC from almost half of the donors become the site of ectopic bone marrow development. Further experiments suggest that maintenance of MSPC stemness by platelet-derived factors during cell expansion is paramount to this effect. Methods and Results. MSPC are expanded from human bone marrow following standard protocols, with culture media supplemented either with 10% FBS or with 10% HPL. Cultured MSPC are resuspended in a non-mineral collagen/laminin matrix (Matrigel®) and injected subcutaneously into immune-deficient NSG mice. Bone formation is monitored non-invasively by osteosensitive near-infrared imaging and/or by histology on paraffin-embedded ossicles. HPL-derived MSPC from 13 out of 13 donors form bone in vivo compared to only 2 out of 10 FBS-derived MSPC. Extensive cartilage formation is observed as early as one week after implantation, while signs of ossification appear from the third week onwards. In addition, ossicles generated by HPL-MSPC from 6 out of 13 donors become colonised by mouse bone marrow, indicating that platelet-derived factors maintain the capacity of MSPC to reconstitute a functional bone marrow niche. Bone formation in vivo is considerably delayed, but not blocked, by pre-treatment of the cells with Cholera Toxin, indicating that a Gα protein-coupled receptor (GPCR-α) ligand is partly responsible for the biological effect of HPL. In vitro , HPL and FBS-derived cells display remarkably little phenotypic differences, with the notable exception of the stemness-associated surface marker SSEA-4 which is consistently more expressed on HPL- than on FBS-derived MSPC. Interestingly, serial passage in FBS medium of cells first expanded in HPL medium leads to the rapid loss of surface SSEA-4 expression, paralleled by a loss of osteogenic and bone marrow support potential in vivo . Loss of SSEA-4 expression is also observed in cells serially passaged in HPL medium containing the PDGF receptor-β inhibitor Imatinib, and ossicles derived from these cells are no longer able to attract mouse bone marrow in vivo . These data suggest that human platelet-derived factors allow MSPC to retain their stem cell potential in culture as well as in the ectopic bone microenvironment. This hypothesis is further supported by the observation that human MSPC can be re-isolated and re-expanded from bone marrow-infiltrated ossicles and, remarkably, build bone again when re-injected into NSG mice. Conclusion. Our data suggest that platelet-derived factors, including PDGF and an (as yet unidentified) GPCR-α ligand, contribute in vitro to maintain BM-MSPC stemness. In vivo , they efficiently drive BM-MSPC to differentiate along the chondrogenic and osteogenic lineages, while preserving MSPC bone marrow-support function. We conclude that therapeutic approaches using MSPC for skeletal regeneration should preferentially use early passage BM-MSPC expanded in HPL-supplemented medium. Furthermore, the observed correlation between surface SSEA-4 expression and multipotency in BM-MSPC can be exploited to monitor the quality of the cell preparations. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 939 Background: Multilineage differentiation potential of mesenchymal stem and progenitor cells (MSPCs) make them attractive candidates for tissue regeneration purposes. Guiding the differentiation of MSPCs towards single lineages would facilitate their application for targeted therapies in vivo. We have previously shown that MSPCs are essential for endothelial colony-forming progenitor cell (ECFC)-derived patent vessel formation in vivo*[Blood 2009; 113 (26):6716-25]. Preliminary data indicate that the ratio of co-applied cells can change mesenchymal lineage differentiation from vascular support towards either osteogenesis with subsequent bone marrow (BM) ingrowth or chondrogenesis. We hypothesized that environmental conditioning by ECFCs plays an instructive role during the developmental fate decision of MSPCs in vivo. Methods: MSPCs as well as ECFCs were isolated from adult BM, white adipose tissue (WAT), umbilical cord blood (UCB) and perivascular cord tissue**[J Vis Exp. 2009;(32) pii: 1525]. Proliferation potential and clonogenicity were monitored. Phenotype was analyzed by flow cytometry and immune cytochemistry. Cell function was studied in differentiation assays and during vascular network assembly in vitro. Models for in vivo human vessel as compared to bone, BM or cartilage formation were established in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). Non-invasive imaging was performed using computed tomography (CT), magnetic resonance (MRI) and near-infrared fluorescence imaging to elucidate the time course of heterotopic tissue development. Immune histochemistry was applied for morphologic studies of organogenesis. Results: Baseline analysis confirmed MSPC and ECFC purity, immune phenotype and sustained proliferation potential. We could show that human BM-derived MSPCs are capable of forming bone in vivo. Osteogenic differentiation and heterotopic ossicle formation was followed by attraction of mouse hematopoiesis and the establishment of entire murine BM including red and white blood cells and megakaryocytes within a human endosteal niche. Co-transplanted human ECFCs could instruct the MSPCs to differentiate also into pericytes or chondrocytes in vivo, depending on the applied MSPC/ECFC cell ratio. Non-invasive imaging and histological staining revealed that ectopic organogenesis had already started after 2–4 weeks and was stable during the observation period of 20 weeks. Non-BM-derived populations, although phenotypically identical, invariably lacked the capacity to build bone and marrow environment in this model in vivo. Conclusion: These data indicate that human ECFCs can instruct MSPCs and induce developmental fate decisions early in the time course of organ regeneration after transplantation. We suppose that effective regenerative stem cell therapy in vivo requires more than the injection of one single cell population. For vascular repair as compared to bone and marrow environment reconstitution our model is a promising tool to study the therapeutic applicability and risk profile of such ECFC/MSPC-based transplantation strategies. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4313 Background: 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. Methods: 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-Prkdcscid Il2rgtm1Wjl/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. Results: 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. Background: 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. Disclosures: No relevant conflicts of interest to declare.