Bart Vandekerckhove

Intracoronary Injection of CD133-Positive Enriched Bone Marrow Progenitor Cells Promotes Cardiac Recovery After Recent Myocardial Infarction Feasibility and Safety

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±2.2×106 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 18fluorodeoxyglucose 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.

Endothelial progenitor cells: identity defined?

•  Introduction •  The proof‐of‐concept in vivo: the cell, the read‐out and the animal model ‐  The CEPC: Still a putative cell ‐  Do CEPCs play an essential role in vascular (patho)physiology? ‐  The in vivo read‐out and animal model •  EPCs defined in vitro: the achilles heel in EPC biology ‐  EOCs and EC‐like cells ‐  What are potential caveats with in vitro defined cells? ‐  The search for the EOC precursor: lessons from embryonic development ‐  Do EOCs derive from an immature CEPC? ‐  Do EOCs derive from high proliferative vessel wall ECs? ‐  CEPCs and CECs: Different cells having the same identity? •  Summary

Endothelial Outgrowth Cells Are Not Derived From CD133 + Cells or CD45 + Hematopoietic Precursors

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.

Generation of T cells from human embryonic stem cell-derived hematopoietic zones

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 CD34highCD43low population, further substantiating the notion that hESC-derived CD34highCD43low 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.

Active Form of Notch Imposes T Cell Fate in Human Progenitor Cells

The crucial role of Notch signaling in cell fate decisions in hematopoietic lineage and T lymphocyte development has been well established in mice. Overexpression of the intracellular domain of Notch mediates signal transduction of the protein. By retroviral transduction of this constitutively active truncated intracellular domain in human CD34+ umbilical cord blood progenitor cells, we were able to show that, in coculture with the stromal MS-5 cell line, depending on the cytokines added, the differentiation toward CD19+ B lymphocytes was blocked, the differentiation toward CD14+ monocytes was inhibited, and the differentiation toward CD56+ NK cells was favored. The number of CD7+cyCD3+ cells, a phenotype similar to T/NK progenitor cells, was also markedly increased. In fetal thymus organ culture, transduced CD34+ progenitor cells from umbilical cord blood cells or from thymus consistently generated more TCR-γδ T cells, whereas the other T cell subpopulations were largely unaffected. Interestingly, when injected in vivo in SCID-nonobese diabetic mice, the transduced cells generated ectopically human CD4+CD8+ TCR-αβ cells in the bone marrow, cells that are normally only present in the thymus, and lacked B cell differentiation potential. Our results show unequivocally that, in human, Notch signaling inhibits the monocyte and B cell fate, promotes the T cell fate, and alters the normal T cell differentiation pathway compatible with a pretumoral state.

Both CD34+38+ and CD34+38- cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion.

Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38− umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38− cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38− cells.

An early decrease in Notch activation is required for human TCR-αβ lineage differentiation at the expense of TCR-γδ T cells

Although well characterized in the mouse, the role of Notch signaling in the human T-cell receptor alphabeta (TCR-alphabeta) versus TCR-gammadelta lineage decision is still unclear. Although it is clear in the mouse that TCR-gammadelta development is less Notch dependent compared with TCR-alphabeta differentiation, retroviral overexpression studies in human have suggested an opposing role for Notch during human T-cell development. Using the OP9-coculture system, we demonstrate that changes in Notch activation are differentially required during human T-cell development. High Notch activation promotes the generation of T-lineage precursors and gammadelta T cells but inhibits differentiation toward the alphabeta lineage. Reducing the amount of Notch activation rescues alphabeta-lineage differentiation, also at the single-cell level. Gene expression analysis suggests that this is mediated by differential sensitivities of Notch target genes in response to changes in Notch activation. High Notch activity increases DTX1, NRARP, and RUNX3 expression, genes that are down-regulated during alphabeta-lineage differentiation. Furthermore, increased interleukin-7 levels cannot compensate for the Notch dependent TCR-gammadelta development. Our results reveal stage-dependent molecular changes in Notch signaling that are critical for normal human T-cell development and reveal fundamental molecular differences between mouse and human.

Notch signaling is required for proliferation but not for differentiation at a well-defined β-selection checkpoint during human T-cell development

Notch signaling is absolutely required for beta-selection during mouse T-cell development, both for differentiation and proliferation. In this report, we investigated whether Notch has an equally important role during human T-cell development. We show that human CD34(+) thymocytes can differentiate into CD4(+)CD8beta(+) double positive (DP) thymocytes in the absence of Notch signaling. While these DP cells phenotypically resemble human beta-selected cells, they lack a T-cell receptor (TCR)-beta chain. Therefore, we characterized the beta-selection checkpoint in human T-cell development, using CD28 as a differential marker at the immature single positive CD4(+)CD3(-)CD8alpha(-) stage. Through intracellular TCR-beta staining and gene expression analysis, we show that CD4(+)CD3(-)CD8alpha(-)CD28(+) thymocytes have passed the beta-selection checkpoint, in contrast to CD4(+)CD3(-)CD8alpha(-)CD28(-) cells. These CD4(+)CD3(-)CD8alpha(-)CD28(+) thymocytes can efficiently differentiate into CD3(+)TCRalphabeta(+) human T cells in the absence of Notch signaling. Importantly, preselection CD4(+)CD3(-)CD8alpha(-)CD28(-) thymocytes can also differentiate into CD3(+)TCRalphabeta(+) human T cells without Notch activation when provided with a rearranged TCR-beta chain. Proliferation of human thymocytes, however, is clearly Notch-dependent. Thus, we have characterized the beta-selection checkpoint during human T-cell development and show that human thymocytes require Notch signaling for proliferation but not for differentiation at this stage of development.

Analysis of cytotoxic T cell precursor frequencies directed against individual HLA-A and -B alloantigens

We describe here a limiting dilution analysis to determine cytotoxic T lymphocyte precursor (CTLp) frequencies against individual HLA-A or -B antigens. This assay is reproducible and showed that the CTLp frequency of an individual remains stable with time. Significant variations in CTLp frequency against the same alloantigen were found in different individuals and even in monozygotic twins, showing that these differences were not (completely) genetically determined. Within an individual, a wide range of CTLp frequencies can be found against different allo-antigens. Serologically cross-reactivity seems not to interfere in this assay. This LDA is a practicable tool for a systematic analysis of CTLp response against selected individual HLA-A or -B antigens and can be used for the selection of HLA mismatched donors for transplantation patients.

Specific Notch receptor-ligand interactions control human TCR-αβ/γδ development by inducing differential Notch signal strength.

Jagged2 preferentially signals through Notch3 to promote γδ T cell development.