Introduction The proof‐of‐concept in vivo: the cell, the read‐out and the animal model EPCs defined in vitro: the achilles heel in EPC biology Summary In the past decade, researchers have gained important insights on the role of bone marrow (BM)‐derived cells in adult neovascularization. A subset of BM‐derived cells, called endothelial progenitor cells (EPCs), has been of particular interest, as these cells were suggested to home to sites of neovascularization and neoendothelialization and differentiate into endothelial cells (ECs) in situ, a process referred to as postnatal vasculogenesis. Therefore, EPCs were proposed as a potential regenerative tool for treating human vascular disease and a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. The focus of this review is on the controversial issues in the field of EPCs which are related to the lack of a unique EPC marker, identification challenges related to the paucity of EPCs in the circulation, and the important phenotypical and functional overlap between EPCs, haematopoietic cells and mature ECs. We also discuss our recent findings on the origin of endothelial outgrowth cells (EOCs), showing that this in vitro defined EC population does not originate from circulating CD133⁺ cells or CD45⁺ haematopoietic cells.

Background— Bone marrow CD133-postive (CD133 ⁺ ) cells possess high hematopoietic and angiogenic capacity. We tested the feasibility, safety, and functional effects of the use of enriched CD133 ⁺ progenitor cells after intracoronary administration in patients with recent myocardial infarction. Methods and Results— Among 35 patients with acute myocardial infarction treated with stenting, 19 underwent intracoronary administration of CD133 ⁺ progenitor cells (12.6±2.2×10 ⁶ cells) 11.6±1.4 days later (group 1) and 16 did not (group 2). At 4 months, left ventricular ejection fraction increased significantly in group 1 (from 45.0±2.6% to 52.1±3.5%, P <0.05), but only tended to increase in case-matched group 2 patients (from 44.3±3.1% to 48.6±3.6%, P =NS). Likewise, left ventricular regional chordae shortening increased in group 1 (from 11.5±1.0% to 16.1±1.3%, P <0.05) but remained unchanged in group 2 patients (from 11.1±1.1% to 12.7±1.3%, P =NS). This was paralleled by reduction in the perfusion defect in group 1 (from 28.0±4.1% to 22.5±4.1%, P <0.05) and no change in group 2 (from 25.0±3.0% to 22.6±4.1%, P =NS). In group 1, two patients developed in-stent reocclusion, 7 developed in-stent restenosis, and 2 developed significant de novo lesion of the infarct-related artery. In group 2, four patients showed in-stent restenosis. In group 1 patients without reocclusion, glucose uptake shown by positron emission tomography with ¹⁸ fluorodeoxyglucose in the infarct-related territory increased from 51.2±2.6% to 57.5±3.5% ( P <0.05). No stem cell-related arrhythmias were noted, either clinically or during programmed stimulation studies at 4 months. Conclusion— In patients with recent myocardial infarction, intracoronary administration of enriched CD133 ⁺ cells is feasible but was associated with increased incidence of coronary events. Nevertheless, it seems to be associated with improved left ventricular performance paralleled with increased myocardial perfusion and viability.

Objective— Two types of endothelial progenitor cells (EPCs), early EPCs and late EPCs (also called endothelial outgrowth cells [EOCs]), were described in vitro previously. In this report, we dissect the phenotype of the precursor(s) that generate these cell types with focus on the markers CD34, CD133, and vascular endothelial growth factor receptor-2 (VEGFR2) that have been used to identify putative circulating endothelial precursors. We also included CD45 in the analysis to assess the relation between CD34 ⁺ hematopoietic progenitors (HPC), CD34 ⁺ endothelial precursors, and both in vitro generated EPC types. Addressing this issue might lead to a better understanding of the lineage and phenotype of the precursor(s) that give rise to both cell types in vitro and may contribute to a consensus on their flowcytometric enumeration. Methods and Results— Using cell sorting of human cord blood (UCB) and bone marrow (BM) cells, we demonstrate that EOC generating precursors are confined to a small CD34 ⁺ CD45 ⁻ cell fraction, but not to the CD34 ⁺ CD45 ⁺ HPC fraction, nor any other CD45 ⁺ subpopulation. CD34 ⁺ CD45 ⁺ HPC generated monocytic cells that displayed characteristics typical for early EPCs. Phenotypic analysis showed that EOC generating CD34 ⁺ CD45 ⁻ cells express VEGFR2 but not CD133, whereas CD34 ⁺ CD45 ⁺ HPC express CD133 as expected, but not VEGFR2. Conclusion— EOCs are not derived from CD133 ⁺ cells or CD45 ⁺ hematopoietic precursors.

Human embryonic stem cells (hESC) are pluripotent stem cells. A major challenge in the field of hESC is the establishment of specific differentiation protocols that drives hESC down a particular lineage fate. So far, attempts to generate T cells from hESC in vitro were unsuccessful. In this study, we show that T cells can be generated in vitro from hESC-derived hematopoietic precursor cells present in hematopoietic zones (HZs). These zones are morphologically similar to blood islands during embryonic development, and are formed when hESC are cultured on OP9 stromal cells. Upon subsequent transfer of these HZs on OP9 cells expressing high levels of Delta-like 1 and in the presence of growth factors, cells expand and differentiate to T cells. Furthermore, we show that T cells derive exclusively from a CD34^highCD43^low population, further substantiating the notion that hESC-derived CD34^highCD43^low cells are formed in HZs and are the only population containing multipotent hematopoietic precursor cells. Differentiation to T cells sequentially passes through the physiological intermediates: CD34⁺CD7⁺ T/NK committed, CD7⁺CD4⁺CD8⁻ immature single positive, CD4⁺CD8⁺ double positive, and finally CD3⁺CD1⁻CD27⁺ mature T cell stages. TCRαβ⁺ and TCRγδ⁺ T cells are generated. Mature T cells are polyclonal, proliferate, and secrete cytokines in response to mitogens. This protocol for the de novo generation of T cells from hESC could be clinically and scientifically relevant.