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In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a BM cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13, and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs), as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age, with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.

Class 3 semaphorins were discovered as a family of axon guidance molecules, but are now known to be involved in diverse biologic processes. In this study, we investigated the anti-angiogenic potential of SEMA3E and SEMA3F (SEMA3E&F) in infantile hemangioma (IH). IH is a common vascular tumor that involves both vasculogenesis and angiogenesis. Our lab has identified and isolated hemangioma stem cells (HemSC), glucose transporter 1 positive (GLUT1(+)) endothelial cells (designated as GLUT1(sel) cells) based on anti-GLUT1 magnetic beads selection and GLUT1-negative endothelial cells (named HemEC). We have shown that these types of cells play important roles in hemangiogenesis. We report here that SEMA3E inhibited HemEC migration and proliferation while SEMA3F was able to suppress the migration and proliferation in all three types of cells. Confocal microscopy showed that stress fibers in HemEC were reduced by SEMA3E&F and that stress fibers in HemSC were decreased by SEMA3F, which led to cytoskeletal collapse and loss of cell motility in both cell types. Additionally, SEMA3E&F were able to inhibit vascular endothelial growth factor (VEGF)-induced sprouts in all three types of cells. Further, SEMA3E&F reduced the level of p-VEGFR2 and its downstream p-ERK in HemEC. These results demonstrate that SEMA3E&F inhibit IH cell proliferation and suppress the angiogenic activities of migration and sprout formation. SEMA3E&F may have therapeutic potential to treat or prevent growth of highly proliferative IH.

Background and Purpose Highly vascularized ovarian carcinoma secretes the putative endocannabinoid and GPR55 agonist, L-α-lysophosphatidylinositol (LPI), into the circulation. We aimed to assess the involvement of this agonist and its receptor in ovarian cancer angiogenesis. Experimental Approach Secretion of LPI by three ovarian cancer cell lines (OVCAR-3, OVCAR-5 and COV-362) was tested by mass spectrometry. Involvement of cancer cell-derived LPI on angiogenesis was tested in the in vivo chicken chorioallantoic membrane (CAM) assay along with the assessment of the effect of LPI on proliferation, network formation, and migration of neonatal and adult human endothelial colony-forming cells (ECFCs). Engagement of GPR55 was verified by using its pharmacological inhibitor CID16020046 and diminution of GPR55 expression by four different target-specific siRNAs. To study underlying signal transduction, Western blot analysis was performed. Key Results Ovarian carcinoma cell-derived LPI stimulated angiogenesis in the CAM assay. Applied LPI stimulated proliferation, network formation, and migration of neonatal ECFCs in vitro and angiogenesis in the in vivo CAM. The pharmacological GPR55 inhibitor CID16020046 inhibited LPI-stimulated ECFC proliferation, network formation and migration in vitro as well as ovarian carcinoma cell- and LPI-induced angiogenesis in vivo. Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis. These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase. Conclusions and Implications We conclude that inhibiting the pro-angiogenic LPI/GPR55 pathway appears a promising target against angiogenesis in ovarian carcinoma.

Background Hypoxia-induced genes are potential targets in cancer therapy. Responses to hypoxia have been extensively studied in vitro, however, they may differ in vivo due to the specific tumor microenvironment. In this study gene expression profiles were obtained from fresh human lung cancer tissue fragments cultured ex vivo under different oxygen concentrations in order to study responses to hypoxia in a model that mimics human lung cancer in vivo. Methods Non-small cell lung cancer (NSCLC) fragments from altogether 70 patients were maintained ex vivo in normoxia or hypoxia in short-term culture. Viability, apoptosis rates and tissue hypoxia were assessed. Gene expression profiles were studied using Affymetrix GeneChip 1.0 ST microarrays. Results Apoptosis rates were comparable in normoxia and hypoxia despite different oxygenation levels, suggesting adaptation of tumor cells to hypoxia. Gene expression profiles in hypoxic compared to normoxic fragments largely overlapped with published hypoxia-signatures. While most of these genes were up-regulated by hypoxia also in NSCLC cell lines, membrane metallo-endopeptidase (MME, neprilysin, CD10) expression was not increased in hypoxia in NSCLC cell lines, but in carcinoma-associated fibroblasts isolated from non-small cell lung cancers. High MME expression was significantly associated with poor overall survival in 342 NSCLC patients in a meta-analysis of published microarray datasets. Conclusions The novel ex vivo model allowed for the first time to analyze hypoxia-regulated gene expression in preserved human lung cancer tissue. Gene expression profiles in human hypoxic lung cancer tissue overlapped with hypoxia-signatures from cancer cell lines, however, the elastase MME was identified as a novel hypoxia-induced gene in lung cancer. Due to the lack of hypoxia effects on MME expression in NSCLC cell lines in contrast to carcinoma-associated fibroblasts, a direct up-regulation of stroma fibroblast MME expression under hypoxia might contribute to enhanced aggressiveness of hypoxic cancers.

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.

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.

Vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs) are key players in vascular homeostasis and regeneration. Due to their robust proliferative potential and profound vessel-forming capacity, ECFCs are considered to represent an attractive tool for vascular regenerative medicine and a promising target for antiangiogenic tumor therapy. Here, we describe an easily applicable method for isolating ECFCs directly from unmanipulated adult human blood and an animal protein-free large-scale expansion system to generate more than 100 million functional ECFCs.

The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.

Vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs) are key players in vascular homeostasis and regeneration. Due to their robust proliferative potential and profound vessel-forming capacity, ECFCs are considered to represent an attractive tool for vascular regenerative medicine and a promising target for antiangiogenic tumor therapy. Here, we describe an easily applicable method for isolating ECFCs directly from unmanipulated adult human blood and an animal protein-free large-scale expansion system to generate more than 100 million functional ECFCs.

Clinical trials for therapeutic angiogenesis use blood- or bone marrow-derived hematopoietic cells, endothelial progenitor cells (EPC) and mesenchymal stromal cells (MSC) for vascular regeneration. Recently concerns have emerged that all three cell types could also contribute to atherosclerosis by foam cell formation. Therefore, we asked whether human myelomonocytic cells, EPC or MSC can accumulate lipid droplets (LD) and develop into foam cells.LD accumulation was quantified by flow cytometry, confocal microscopy and cholesterol measurement in each of the cell types. The impact of an initial pro-angiogenic induction on subsequent foam cell formation was studied to mimic relevant settings already used in clinical trials. The phosphorylation state of intracellular signaling molecules in response to the pro-angiogenic stimulation was determined to delineate the operative mechanisms and establish a basis for interventional strategies.Foam cells were formed by monocytes but not by EPC or MSC after pro-angiogenic induction. Mitogen-activated protein kinase (MAPK) p38 phosphorylation was enhanced and kinase inhibition almost abrogated intracellular LD accumulation in monocytes.These data suggest that hematopoietic cell preparations containing monocytes bear the risk of foam cell formation after pro-angiogenic induction. Instead, EPC and MSC may drive vascular regeneration without atherogenesis aggravation. A thorough understanding of cell biology is necessary to develop new strategies combining pro-angiogenic and anti-atherogenic effects during cell therapy.

When violence has ceased it is often assumed that from this very moment peace has been restored. Definitions of peace and war have influenced earlier peace building interventions which in turn constituted the simple act of putting the weapons down as sufficient. Only recently have attempts to incorporate wider angles of facilitating peace and development been utilised. Critical approaches enhancing the perception of peace are put forward by Peace Theory scholars such as Johan Galtung and actual lessons learned serve to counter prevailing criticsm from Realist perspectives on peace. This paper demonstrates a rationale and case for supporting a more promising as well as more realistic view of peace and conflict management offered by peace theoretical considerations and approaches. Transformation of conflicts appears as preliminary requirement for lasting peace in the view of Peace Theory. Upholding this presumption, reconciliation is presented here as an essential process and likewise applicable method for transformation of conflict. The paper examines the advantages of reconciliation against Realist claims about power politics and international relations. After introducing the aim and outline of the paper, a theoretical framework will be established in the second chapter, acknowledging the Christian concept of reconciliation yet evolving towards modern interpretations within socio-political contexts. Scrutinising the applicability of reconciliation for conflict transformation, the third chapter presents reconciliation in practice drawing on the South African expample. Along this case study is highlighted how transitional justice is measured to default standards, what contextual constraints shape structure, mandate and process of applied reconciliation and why success is clearly visible in terms of socio-political reach. The model of the South African Truth and Reconciliation Commission (TRC) also serves to elaborate the feasibility of institutionalising reconciliation in conflict transformation on international level. This paper stresses that conditions and design of the TRC resemble the setting of current international conflict transformation processes. Consequently chapter four discusses the specific impediments for applied peace theory in international context before the last chapter concludes the significance of applied reconciliation and non-retributive justice models for international conflict transformation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion >1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4 This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony–forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum–free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

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 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% O2), venous oxygen (5% O2) and standard ambient air culture conditions (20% O2). ECFC and MSPC crosstalk in vivo was studied in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/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% O2, while MSPCs stabilized HIF-1α already at 5% O2. 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 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.

The microenvironment contributes and may regulate cancer development, progression and resistance to treatment. Our group reported first the contribution of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) for tumor development and metastasis. Accumulating evidence has shown that the BM microenvironment also plays a pivotal role in the pathophysiology and propagation of leukemia. We here describe a novel, genetically controlled bone and bone marrow model using human BM derived MSCs and endothelial colony-forming cells (ECFCs) subcutaneously injected into the flanks of NOD/SCID/IL-2rαnull mice, where they developed into bone-like tissues with high osteoblast activity after 10 weeks, with bone structures and typical bone marrow cavities constituting a robust hematopoietic environment. In vivo imaging with Osteosense confirmed the presence of hydroxylapatite, and luciferase imaging of firefly-luciferase labeled human leukemic cells demonstrated the engraftment of human MOLM13/Luc/GFP leukemic cells. The extramedullary leukemic BM was markedly hypoxic, as shown by Pimonidazole staining. Factors critical for MSC to support normal and leukemic hematopoiesis are largely unknown and cannot readily be studied since human MSC do not engraft reliably in xenograft models. We therefore investigated the possibility of genetically modifying MSC in this system and found a significant reduction (50 ± 6%, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2971. doi:1538-7445.AM2012-2971

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 1323 The importance of the tumor microenvironment for cancer development, progression and resistance to treatment has recently been recognized. Our group was first to report the contribution of bone marrow (BM) derived mesenchymal stromal cells (MSCs) for tumor development and metastasis. BM is also the dynamic microenvironment (niche) for normal and malignant hematopoietic stem cells (HSC) with high local concentrations of growth factors, chemokines and cytokines. The maintenance of HSCs quiescence and normal hematopoiesis require complex bidirectional interactions between the BM niches and HSCs. Accumulating evidence has shown that the BM microenvironment also plays a pivotal role in the pathophysiology and propagation of leukemia. Leukemia cells undergo spontaneous apoptosis once they are removed from the in vivo microenvironment and placed in suspension cultures without supportive stroma. The understanding of the interactions between leukemic cells and their BM niche is also critically important for leukemia therapy. We here describe a novel artificial bone and bone marrow model mimicking the human hematopoietic microenvironment by using human BM derived MSCs and endothelial colony-forming cells (ECFCs). MSCs and ECFCs were isolated from heparinized human bone marrow or peripheral blood through an initial adhesion step, grown in specific media and then subcutaneously injected into the flanks of the NOD/SCID/IL-2r-gammanull mice, where they developed into bone-like tissues with high osteoblast activity after 10 weeks (Figure 1). Histochemical stains confirmed the bone structures and also showed that these artificial bones contained typical bone marrow cavities constituting a robust hematopoietic environment. In vivo imaging with Osteosense confirmed the presence of hydroxylapatite, and luciferase imaging of firefly luciferase labeled human leukemic cells demonstrated the engraftment of MOLM13/Luc/GFP leukemic cells in the extramedullary BM sites. The extramedullary BM was markedly hypoxic, as shown by Pimonidazole staining, another critical feature of the BM microenvironment. Factors critical for MSC to support the normal and leukemic hematopoiesis are largely unknown and cannot be studied since human MSC do not engraft reliably in xenograft models. We therefore investigated the possibility of genetically modifying MSC in this system and found a significant reduction (50 ± 6%, p Disclosures: No relevant conflicts of interest to declare.

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 4442 Background: Clinical trials for therapeutic angiogenesis use blood- or marrow-derived transplants containing hematopoietic cells, endothelial progenitor cells (EPCs) and mesenchymal stem and progenitor cells (MSPCs) to support vascular regeneration. Recently concerns have emerged, as bone marrow-derived stem cell preparations also include these three cell types which probably may contribute to atherosclerosis. We therefore asked whether human myelomonocytic hematopoietic cells, EPCs or MSPCs after pro-angiogenic induction can accumulate lipid droplets (LDs) and develop into foam cells. Method: LD accumulation was quantified by flow cytometry, confocal microscopy and cholesterol measurement in each of the tested cell types. The impact of an initial three-day pro-angiogenic culture on subsequent foam cell formation was studied to mimic a relevant setting already being used in clinical trials. The phosphorylation state of intracellular signaling molecules in response to pro-angiogenic stimulation was determined to delineate the operative mechanisms and to establish a basis for interventional strategies. Result: Foam cells were formed by monocytes but neither by EPCs nor by MSPCs after pro-angiogenic induction. Mitogen-activated protein kinase (MAPK) p38 phosphorylation was enhanced in monocytes after pro-angiogenic stimulation. Kinase inhibition almost abrogated intracellular LD accumulation. Conclusion: These data suggest that hematopoietic cell preparations containing monocytes bear the risk of foam cell formation after pro-angiogenic induction. EPCs and MSPCs instead may drive vascular regeneration without atherogenesis aggravation. A thorough understanding of cell biology is necessary to develop new strategies combining pro-angiogenic and anti-atherogenic cellular effects during therapeutic angiogenesis. Disclosures: No relevant conflicts of interest to declare.

Circulating angiogenic cells (CACs) and several related hematopoietic cell types can mimic an endothelial progenitor cell (EPC) phenotype and facilitate vascular regeneration mainly by humoral and cell mediated support functions but do not form vessels. Despite a documented risk of tumor support and pre-metastatic niche formation, various types of hematopoietic CACs are currently tested in ongoing clinical trials. In sharp contrast, endothelial colony forming cells (ECFCs) have recently been described as the prototype of blood- and vessel-derived EPCs with a robust proliferative potential. Their profound vessel-forming capacity makes ECFCs a promising tool to study human vascular homeostasis, regeneration and tumor angiogenesis. This study was initiated to develop the first animal protein-free large-scale expansion system for adult human blood-derived ECFCs and to test their functionality in vitro and in vivo. We isolated ECFCs directly from whole blood with a novel recovery strategy. ECFC propagation was done under animal protein-free culture conditions with pooled human platelet lysate (pHPL) replacing fetal bovine serum (FBS) for clinical-scale expansion. ECFC long-term proliferation potential was monitored and phenotype was analyzed in detail by flow cytometry as well as immune cytochemistry. Functionality was studied during vascular network assembly in vitro and in two models for human vessel formation in immune-deficient mice in vivo. Genomic stability was assayed with chromosome G-banding and array-comparative genomic hybridization (array-CGH). A mean of four ECFC colonies/mL peripheral blood could be recovered repeatedly in seven donors. The progeny of these oligoclonal cultures could be expanded to mean 1.5 ± 0.5 x 108 ECFCs within 11–25 days in the humanized animal protein-free large scale culture system. Consecutive analysis confirmed ECFC purity, immune phenotype and sustained proliferation potential for >30 population doublings. Preserved progenitor hierarchy after oligoclonal large-scale expansion was confirmed by a mean 74% of high proliferation potential (HPP) and a mean 26% of low proliferation potential (LPP) colonies comprising >500 and 51–500 cells per day 14 colony, respectively. Monoclonal ECFC progeny could also be generated but was less suited for ECFC mass production than oligoclonal ECFCs due to the lower cumulative cell number recovered. Genomic stability was confirmed by karyotyping and array-CGH. Large-scale expanded ECFCs functioned even after cryopreservation to form complex vascular networks in vitro and assembled stable CD31+/Vimentin+/von Willebrand factor+ human vessels in vivo. These human vessels where firmly connected to the mouse circulation for the entire seven week study period as indicated by a rich content of Ter119+ murine red blood cells. This demonstrates for the first time that proliferating, functional, storable and genomically stable human ECFCs can be expanded to a relevant clinical quantity under GMP-compliant conditions in an animal protein-free system. This novel humanized procedure for largescale ECFC propagation represents a promising tool to develop innovative, experimental, diagnostic and therapeutic strategies. It should help to set a new standard to study therapeutic applicability and risk profile of vessel-forming EPC-based investigational new drugs.

The hematopoietic stem cell (HSC) niche is an anatomically confined space governing HSC proliferation, differentiation and self renewal. Recent research identified distinct compartments described as stromal and vascular niches. This study was initiated to directly compare mesenchymal stromal cell (MSC) and vascular endothelial progenitor cell (EPC) contribution to niche functions in a humanized co-culture system. MSCs and EPCs were propagated under animal protein-free conditions following recently established protocols. Autologous MSC-EPC pairs were established to avoid donor variation. Purified CD34+ hematopoietic progenitor cells (HPCs) and HSCs were used as responders in a cytokine-driven (IL-3, 6ng/mL; Flt-3-L, 50ng/mL; SCF, 20ng/mL) niche cell-regulated HPC co-culture system. Four different standard media were employed to compare fully serum-free conditions with humanized cultures that were supplemented with pooled human platelet lysate (pHPL) as a source of platelet-derived growth factors present within the niche. Primary expansion culture with and without niche cell support was followed by colony forming cell (CFC) assays to determine maintenance of clonogenicity. Humanized liquid cultures supplemented with 10% pHPL without niche cell support were more efficient than parallel serum-free cultures under three of four medium conditions resulting in a mean 16–84-fold increase in nucleated cell progeny within 11 days. Both MSCs and EPCs further amplified HPC proliferation, MSC actually up to 341-fold, confirming recently published results. Interestingly, the supportive effect of MSCs or EPCs was constantly more pronounced in humanized pHPL-supplemented compared to completely serum-free cultures, indicating a peculiar role of the natural platelet-derived growth factors in this process. Surprisingly, the combination of equal cumulative numbers of MSCs + EPCs constantly resulted in the highest HPC proliferation. This produced an up to 567-fold increase indicating at least nine population doublings within only 11 days. Under all tested conditions the HPC progeny mainly comprised differentiating myeloid cells. Consecutive CFC assays revealed that liquid culture supplemented with 10%HPL were more efficient than serum-free cultures resulting in a measurable 1.2–4.5 fold CFC expansion under three of four tested medium conditions. Both MSC and EPC initiated a more than 6-fold increase of CFCs. CFC expansion through combined action of MSCs + EPCs resulted in a maximum of 23.5-fold CFC increase in pHPL-supplemented cultures. We demonstrate for the first time that HPC expansion can be modulated in a fully humanized co-culture system based on the use of pHPL as a natural source of human platelet-derived growth factors. Our data indicate that the combination of MSCs + EPCs is at least as efficient in supporting HPC proliferation and CFC amplification as either niche cell compartment alone. We speculate that human platelet-derived factors may avoid premature HSC exhaustion in this system. The humanized co-culture thus provides a novel model system for subtractive analyses of HPC and HSC-niche cell interactions. It also builds the basis for further developments towards more effective animal serum-free HSC expansion strategies.

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.