Olga Tura

Late Outgrowth Endothelial Cells Resemble Mature Endothelial Cells and Are Not Derived from Bone Marrow

A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte‐colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34+CD133−CD146+ cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real‐time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 ± 2.7 vs. 6.6 ± 3.7 vessels, respectively, n = 9). Bone marrow‐derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34+CD133−CD146+ mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrow‐derived circulating precursor for endothelial cells. STEM CELLS2013;31:338–348

Circulating endothelial progenitor cells are not affected by acute systemic inflammation

Vascular injury causes acute systemic inflammation and mobilizes endothelial progenitor cells (EPCs) and endothelial cell (EC) colony-forming units (EC-CFUs). Whether such mobilization occurs as part of a nonspecific acute phase response or is a phenomenon specific to vascular injury remains unclear. We aimed to determine the effect of acute systemic inflammation on EPCs and EC-CFU mobilization in the absence of vascular injury. Salmonella typhus vaccination was used as a model of acute systemic inflammation. In a double-blind randomized crossover study, 12 healthy volunteers received S. typhus vaccination or placebo. Phenotypic EPC populations enumerated by flow cytometry [CD34(+)VEGF receptor (VEGF)R-2(+)CD133(+), CD14(+)VEGFR-2(+)Tie2(+), CD45(-)CD34(+), as a surrogate for late outgrowth EPCs, and CD34(+)CXCR-4(+)], EC-CFUs, and serum cytokine concentrations (high sensitivity C-reactive protein, IL-6, and stromal-derived factor-1) were quantified during the first 7 days. Vaccination increased circulating leukocyte (9.8 + or - 0.6 vs. 5.1 + or - 0.2 x 10(9) cells/l, P < 0.0001), serum IL-6 [0.95 (0-1.7) vs. 0 (0-0) ng/l, P = 0.016], and VEGF-A [60 (45-94) vs. 43 (21-64) pg/l, P = 0.006] concentrations at 6 h and serum high sensitivity C-reactive protein at 24 h [2.7 (1.4-3.6) vs. 0.4 (0.2-0.8) mg/l, P = 0.037]. Vaccination caused a 56.7 + or - 7.6% increase in CD14(+) cells at 6 h (P < 0.001) and a 22.4 + or - 6.9% increase in CD34(+) cells at 7 days (P = 0.04). EC-CFUs, putative vascular progenitors, and the serum stromal-derived factor-1 concentration were unaffected throughout the study period (P > 0.05 for all). In conclusion, acute systemic inflammation causes nonspecific mobilization of hematopoietic progenitor cells, although it does not selectively mobilize putative vascular progenitors. We suggest that systemic inflammation is not the primary stimulus for EPC mobilization after acute vascular injury.

Granulocyte colony-stimulating factor (G-CSF) depresses angiogenesis in vivo and in vitro: implications for sourcing cells for vascular regeneration therapy

Summary.  Background: The most common source of hematopoietic progenitor cells (HPCs) for hematopoietic reconstitution comprises granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood stem cells (PBSCs). It has been proposed that endothelial progenitor cells (EPCs) share precursors with HPCs, and that EPC release may accompany HPC mobilization to the circulation following G‐CSF administration. Objective: To investigate EPC activity following HPC mobilization, and the direct effects of exogenous G‐CSF administration on human umbilical vein endothelial cells (HUVECs) and endothelial outgrowth cells (EOCs), using in vitro and in vivo correlates of angiogenesis. Patients/Methods: Heparinized venous blood samples were collected from healthy volunteers and from cord blood at parturition. G‐CSF‐mobilized samples were collected before administration, at apheresis harvest, and at follow‐up. PBSCs were phenotyped by flow cytometry, and cultured in standard colony‐forming unit (CFU)‐EPC and EOC assays. The effect of exogenous G‐CSF was investigated by addition of it to HUVECs and EOCs in standard tubule formation and aortic ring assays, and in an in vivo sponge implantation model. Results: Our data show that G‐CSF mobilization of PBSCs produces a profound, reversible depression of circulating CFU‐EPCs. Furthermore, G‐CSF administration did not mobilize CD34+CD133− cells, which include precursors of EOCs. No EOCs were cultured from any mobilized PBSCs studied. Exogenous G‐CSF inhibited CFU‐EPC generation, HUVEC and EOC tubule formation, microvessel outgrowth, and implanted sponge vascularization in mice. Conclusions: G‐CSF administration depresses both endothelial cell angiogenesis and monocyte proangiogenic activity, and we suggest that any angiogenic benefit observed following implantation of cells mobilized by G‐CSF may come only from a paracrine effect from HPCs.

Systematic assessment in an animal model of the angiogenic potential of different human cell sources for therapeutic revascularization

IntroductionEndothelial progenitor cells (EPC) capable of initiating or augmenting vascular growth were recently identified within the small population of CD34-expressing cells that circulate in human peripheral blood and which are considered hematopoietic progenitor cells (HPC). Soon thereafter human HPC began to be used in clinical trials as putative sources of EPC for therapeutic vascular regeneration, especially in myocardial and critical limb ischemias. However, unlike HPC where hematopoietic efficacy is related quantitatively to CD34+ cell numbers implanted, there has been no consensus on how to measure EPC or how to assess cellular graft potency for vascular regeneration. We employed an animal model of spontaneous neovascularization to simultaneously determine whether human cells incorporate into new vessels and to quantify the effect of different putative angiogenic cells on vascularization in terms of number of vessels generated. We systematically compared competence for therapeutic angiogenesis in different sources of human cells with putative angiogenic potential, to begin to provide some rationale for optimising cell procurement for this therapy.MethodsHuman cells employed were mononuclear cells from normal peripheral blood and HPC-rich cell sources (umbilical cord blood, mobilized peripheral blood, bone marrow), CD34+ enriched or depleted subsets of these, and outgrowth cell populations from these. An established sponge implant angiogenesis model was adapted to determine the effects of different human cells on vascularization of implants in immunodeficient mice. Angiogenesis was quantified by vessel density and species of origin by immunohistochemistry.ResultsCD34+ cells from mobilized peripheral blood or umbilical cord blood HPC were the only cells to promote new vessel growth, but did not incorporate into vessels. Only endothelial outgrowth cells (EOC) incorporated into vessels, but these did not promote vessel growth.ConclusionsThese studies indicate that, since EPC are very rare, any benefit seen in clinical trials of HPC in therapeutic vascular regeneration is predominantly mediated by indirect proangiogenic effects rather than through direct incorporation of any rare EPC contained within these sources. It should be possible to produce autologous EOC for therapeutic use, and evaluate the effect of EPC distinct from, or in synergy with, the proangiogenic effects of HPC therapies.

Absence of a relationship between immunophenotypic and colony enumeration analysis of endothelial progenitor cells in clinical haematopoietic cell sources

BackgroundThe discovery of adult endothelial progenitor cells (EPC) offers potential for vascular regenerative therapies. The expression of CD34 and VEGFR2 by EPC indicates a close relationship with haematopoietic progenitor cells (HPC), and HPC-rich sources have been used to treat cardiac and limb ischaemias with apparent clinical benefit. However, the laboratory characterisation of the vasculogenic capability of potential or actual therapeutic cell autograft sources is uncertain since the description of EPC remains elusive. Various definitions of EPC based on phenotype and more recently on colony formation (CFU-EPC) have been proposed.MethodsWe determined EPC as defined by proposed phenotype definitions (flow cytometry) and by CFU-EPC in HPC-rich sources: bone marrow (BM); cord blood (CB); and G-CSF-mobilised peripheral blood (mPB), and in HPC-poor normal peripheral blood (nPB).ResultsAs expected, the highest numbers of cells expressing the HPC markers CD34 or CD133 were found in mPB and least in nPB. The proportions of CD34+ cells co-expressing CD133 is of the order mPB>CB>BM≈nPB. CD34+ cells co-expressing VEGFR2 were also most frequent in mPB. In contrast, CFU-EPC were virtually absent in mPB and were most readily detected in nPB, the source lowest in HPC.ConclusionHPC sources differ in their content of putative EPC. Normal peripheral blood, poor in HPC and in HPC-related phenotypically defined EPC, is the richest source of CFU-EPC, suggesting no direct relationship between the proposed EPC immunophenotypes and CFU-EPC potential. It is not apparent whether either of these EPC measurements, or any, is an appropriate indicator of the therapeutic vasculogenic potential of autologous HSC sources.

Dissociation of phenotypic and functional endothelial progenitor cells in patients undergoing percutaneous coronary intervention

Objectives: Endothelial progenitor cells (EPCs) are circulating mononuclear cells with the capacity to mature into endothelial cells and contribute to vascular repair. We assessed the effect of local vascular injury during percutaneous coronary intervention (PCI) on circulating EPCs in patients with coronary artery disease. Design and setting: Prospective case-control study in a university teaching hospital. Patients: 54 patients undergoing elective coronary angiography. Interventions and main outcome measures: EPCs were quantified by flow cytometry (CD34+KDR+ phenotype) complemented by real-time polymerase chain reaction (PCR), and the colony forming unit (CFU-EC) functional assay, before and during the first 24 hours after diagnostic angiography (n = 27) or PCI (n = 27). Results: Coronary intervention, but not diagnostic angiography, resulted in an increase in blood neutrophil count (p<0.001) and C-reactive protein concentrations (p = 0.001) in the absence of significant myocardial necrosis. Twenty-four hours after PCI, CFU-ECs increased threefold (median [IQR], 4.4 [1.3–13.8] vs 16.0 [2.1–35.0], p = 0.01), although circulating CD34+KDR+ cells (0.019% (SEM 0.004%) vs 0.016% (0.003%) of leucocytes, p = 0.62) and leucocyte CD34 mRNA (relative quantity 2.3 (0.5) vs 2.1 (0.4), p = 0.21) did not. There was no correlation between CFU-ECs and CD34+KDR+ cells. Conclusions: Local vascular injury following PCI results in a systemic inflammatory response and increases functional CFU-ECs. This increase was not associated with an early mobilisation of CD34+KDR+ cells, suggesting these cells are not the primary source of EPCs involved in the immediate response to vascular injury.

Novel Biopolymers to Enhance Endothelialisation of Intra-vascular Devices

Rapid endothelisation is of critical importance in the prevention of adverse remodelling after device implantation. Currently, there is a need for alternative strategies to promote re-endothelialisation for intravascular stents and vascular grafts. Using polymer microarray technology 345 polymers are comprehensively assessed and a matrix is identified that specifically supports both progenitor and mature endothelial cell activity in vitro and in vivo while minimising platelet attachment.

Optimal ex vivo expansion of neutrophils from PBSC CD34+ cells by a combination of SCF, Flt3-L and G-CSF and its inhibition by further addition of TPO

BackgroundAutologous mobilised peripheral blood stem cell (PBSC) transplantation is now a standard approach in the treatment of haematological diseases to reconstitute haematopoiesis following myeloablative chemotherapy. However, there remains a period of severe neutropenia and thrombocytopenia before haematopoietic reconstitution is achieved. Ex vivo expanded PBSC have been employed as an adjunct to unmanipulated HSC transplantation, but have tended to be produced using complex cytokine mixtures aimed at multilineage (neutrophil and megakaryocyte) progenitor expansion. These have been reported to reduce or abrogate neutropenia but have little major effect on thrombocytopenia. Selective megakaryocyte expansion has been to date ineffective in reducing thrombocytopenia. This study was implemented to evaluate neutrophil specific rather than multilineage ex vivo expansion of PBSC for specifically focusing on reduction or abrogation of neutropenia.MethodsCD34+ cells (PBSC) were enriched from peripheral blood mononuclear cells following G-CSF-mobilisation and cultured with different permutations of cytokines to determine optimal cytokine combinations and doses for expansion and functional differentiation and maturation of neutrophils and their progenitors. Results were assessed by cell number, morphology, phenotype and function.ResultsA simple cytokine combination, SCF + Flt3-L + G-CSF, synergised to optimally expand and mature neutrophil progenitors assessed by cell number, phenotype, morphology and function (superoxide respiratory burst measured by chemiluminescence). G-CSF appears mandatory for functional maturation. Addition of other commonly employed cytokines, IL-3 and IL-6, had no demonstrable additive effect on numbers or function compared to this optimal combination. Addition of TPO, commonly included in multilineage progenitor expansion for development of megakaryocytes, reduced the maturation of neutrophil progenitors as assessed by number, morphology and function (respiratory burst activity).ConclusionGiven that platelet transfusion support is available for autologous PBSC transplantation but granulocyte transfusion is generally lacking, and that multilineage expanded PBSC do not reduce thrombocytopenia, we suggest that instead of multilineage expansion selective neutrophil expansion based on this relatively simple cytokine combination might be prioritized for development for clinical use as an adjunct to unmanipulated PBSC transplantation to reduce or abrogate post-transplant neutropenia.

Endothelial progenitor cells in patients with chronic obstructive pulmonary disease

The pathogenesis of chronic obstructive pulmonary disease is not fully understood. The objective of this study was to compare circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease to age, sex, and cigarette smoking matched healthy controls. Patients with chronic obstructive pulmonary disease (n = 37) and healthy controls (n = 19) were matched by age, sex, and smoking status. Circulating hematopoietic progenitor cells (CD34(+) or CD133(+) mononuclear cells) and endothelial progenitor cells (CD34(+)KDR(+) or CD34(+)CD133(+)KDR(+) mononuclear cells) were quantified by flow cytometry. Endothelial cell-colony forming units from peripheral blood mononuclear cells were quantified in vitro and phenotypic analysis carried out using immunocytochemistry. Patients with chronic obstructive pulmonary disease had more circulating mononuclear cells compared with controls (8.4 ± 0.6 vs. 5.9 ± 0.4 × 10(9) cells/l; P = 0.02). CD34(+) hematopoietic progenitor cells were reduced as a proportion of mononuclear cells in patients compared with controls (0.99 ± 0.12 vs. 1.9 ± 0.12%; P = 0.02); however, there were no differences in the absolute number of CD34(+), CD34(+)KDR(+), or CD34(+)CD133(+)KDR(+) cells (P > 0.05 for all). Endothelial cell-colony forming units were increased in patients with chronic obstructive pulmonary disease compared with controls (13.7 ± 5.2 vs. 2.7 ± 0.9 colonies; P = 0.048). In contrast to previous studies, the number of circulating progenitor cells was not reduced in patients with chronic obstructive pulmonary disease compared with carefully matched controls. It seems unlikely that circulating endothelial progenitor cells or failure of angiogenesis plays a central role in the development of emphysema.

264 CHARACTERISATION OF CARDIOMYOCYTES DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM (IPS) CELLS

Objective The ability to derive cardiomyocytes from human induced pluripotent stem (iPS) cells provides a novel approach to study inherited cardiac channelopathies. Fibroblasts from patients can be reprogrammed to iPS cells and differentiated into cardiomyocytes that carry the patients exact genetic background. Although this technique has enormous potential for modeling inherited channelopathies the derived cardiomyocytes have not been fully characterised and compared to adult and foetal cells. Knowledge about their maturity will be critical if assessments of cellular function are to provide insight into disease processes that manifest in the adult. Methods Healthy control human iPS cells were differentiated into cardiomyocytes using an unguided differentiation protocol. Embryoid bodies from undifferentiated iPS cells were cultured in suspension for 4 days before being plated onto 0.1% gelatin. Spontaneously contracting areas were disaggregated and single cardiomyocytes were characterised using immunocytochemistry, TEM, electrophysiology and Ca2+ imaging. Cells were stained for Troponin T, Troponin I and α-actinin. Ca2+ release into the cytoplasm was detected by fluo-4 and fluorescence intensity was traced before and after Tetrodotoxin (TTX) treatment. Sodium channel activity was measured in a low sodium buffer using perforated patch-clamping. Ultrastructure of cells was analysed with transmission electron microscopy while the presence of t-tubules was investigated using the fluorescent dye Di-8-ANEPPS. Cardiac cells derived from iPS cells were then compared to primary cardiomyocytes isolated from human foetal and adult cardiac tissue. Results iPS derived cardiomyocytes express a wide range of cardiac markers as well as Tetrodotoxin resistant voltage activated sodium channels that inactivate and recover from inactivation and exhibit action potential triggered Ca2+-induced-Ca2+-release. Spontaneous contraction and lack of t-tubules were observed in derived cardiomyocytes and immature human foetal cardiomyocytes. Conclusion iPS cells give rise to mostly immature cardiomyocytes, that express typical cardiac proteins and have functional cardiac sodium channels. This differentiation system may be utilised to investigate patients with and without known genetic mutations to provide a better understanding of the pathophysiology of inherited cardiac channelopathies.