Frank Timmermans

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.

Functionally Mature CD4 and CD8 TCRαβ Cells Are Generated in OP9-DL1 Cultures from Human CD34+ Hematopoietic Cells

Human CD34+ hematopoietic precursor cells cultured on delta-like ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4+ and cytotoxic CD8+ TCRαβ cells is not expected. Phenotypically mature CD27+CD1− TCRγδ as well as TCRαβ cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCRαβ cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8αβ and one expressing CD8αα dimers. TCRαβ CD8αα and TCRγδ cells both expressed the IL2Rβ receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8αβ+ and CD4+ TCRαβ cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8αβ+ and CD4+ T cells. These T cells had the characteristics of conventional T cells: CD4+ cells expressed ThPOK, CD40L, and high levels of IL-2 and IL-4; CD8+ cells expressed Eomes, Runx3, and high levels of granzyme, perforin, and IFN-γ. Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8+ cells. Similarly, the presence of dendritic cells was not required for the generation of mature CD4+ or CD8+ T cells. These data suggest that positive selection of these cells is induced by interaction between T precursor cells.

Time-dependent effects on coronary remodeling and epicardial conductance after intracoronary injection of enriched hematopoietic bone marrow stem cells in patients with previous myocardial infarction.

Bone marrow (BM) cells may interact with coronary endothelium and modulate coronary atherosclerosis. We investigated the time course of coronary luminal loss and changes in conductance after intracoronary injection of enriched hematopoietic BM stem cells in patients with previous myocardial infarction (MI). Among 24 patients with acute MI, 13 were randomized to early (<7 days) and 11 to late (4 months) intracoronary injection of CD133+ cells after the infarction. Segmental quantitative coronary angiography and fractional flow reserve (FFR) measurements of the infarct-related (IR) artery (A) and contralateral artery (control) were performed. In the early group, at 4 months, cumulative luminal loss (LL) of the minimal luminal diameter (MLD) of the IRA distal to the stented segment was −0.39 (−0.51−0.10) mm (p < 0.05 vs. control). There was no further change in LL between 4 and 8 months [−0.09 (−0.26−0.15) mm]. In parallel, FFR decreased at 4 months [−0.16 (−0.26−0.001), p < 0.05 vs. control] but slightly increased from 4 to 8 months follow-up [+0.05 (−0.10−0.09)]. In the late group, LL of the MLD of the IRA distal to the stented segments was −0.12 (—0.47−0.07) mm (NS vs. control) at 4 months and further −0.07 (−0.25−0.05) mm (NS) between 4 and 8 months. At 8 months, the total LL of the MLD in the early and late group was only slightly higher compared to control [−0.34 (−0.48—0.16), −0.36 (−0.69—0.09), and −0.12 (−0.39−0.05) mm, respectively, NS]. Early intracoronary administration of hematopoietic BM stem cells in patients with previous MI is associated with accelerated luminal loss and reduced conductance of the infarct-related artery.

Bone-marrow-derived cells for cardiac stem cell therapy: safe or still under scrutiny?

Cardiac stem cell therapy with bone-marrow-derived stem cells is a promising approach to facilitate myocardial regeneration after acute myocardial infarction or in congestive heart failure. The clinical data currently available seem to indicate that this approach is safe and is not associated with an increase in the number of adverse clinical events; nevertheless, the level of safety confidence is limited because of the small number of patients who have been treated and the absence of long-term clinical follow-up data. In order to establish the clinical safety of cardiac stem cell therapy, it will be necessary to collect additional data from both previous and ongoing clinical trials in subsets of patients relative to their background risk. Several conceptual safety concerns should also be addressed. These concerns relate to a number of operational mechanisms and include biological effects on differentiation, remote homing of transplanted stem cells, progression of atherosclerosis, and arrhythmias. The proactive scrutiny of these phenomena could eventually facilitate the translation of the promise of cardiac regeneration into a safe and effective therapy.

Biventricular Paced QRS Area Predicts Acute Hemodynamic CRT Response Better Than QRS Duration or QRS Amplitudes.

Vectorcardiographic (VCG) QRS area of left bundle branch block (LBBB) predicts acute hemodynamic response in cardiac resynchronization therapy (CRT) patients. We hypothesized that changes in QRS area occurring with biventricular pacing (BV) might predict acute hemodynamic CRT response (AHR).

Accuracy of computer-calculated and manual QRS duration assessments: Clinical implications to select candidates for cardiac resynchronization therapy

BACKGROUND QRS duration (QRSD) plays a key role in the field of cardiac resynchronization therapy (CRT). Computer-calculated QRSD assessments are widely used, however inter-manufacturer differences have not been investigated in CRT candidates. METHODS QRSD was assessed in 377 digitally stored ECGs: 139 narrow QRS, 140 LBBB and 98 ventricular paced ECGs. Manual QRSD was measured as global QRSD, using digital calipers, by two independent observers. Computer-calculated QRSD was assessed by Marquette 12SL (GE Healthcare, Waukesha, WI, USA) and SEMA3 (Schiller, Baar, Switzerland). RESULTS Inter-manufacturer differences of computer-calculated QRSD assessments vary among different QRS morphologies: narrow QRSD: 4 [2-9] ms (median [IQR]), p=0.010; LBBB QRSD: 7 [2-10] ms, p=0.003 and paced QRSD: 13 [6-18] ms, p=0.007. Interobserver differences of manual QRSD assessments measured: narrow QRSD: 4 [2-6] ms, p=non-significant; LBBB QRSD: 6 [3-12] ms, p=0.006; paced QRSD: 8 [4-18] ms, p=0.001. In LBBB ECGs, intraclass correlation coefficients (ICCs) were comparable for inter-manufacturer and interobserver agreement (ICC 0.830 versus 0.837). When assessing paced QRSD, manual measurements showed higher ICC compared to inter-manufacturer agreement (ICC 0.902 versus 0.776). Using guideline cutoffs of 130ms, up to 15% of the LBBB ECGs would be misclassified as <130ms or ≥130ms by at least one method. Using a cutoff of 150ms, this number increases to 33% of ECGs being misclassified. However, by combining LBBB-morphology and QRSD, the number of misclassified ECGs can be decreased by half. CONCLUSION Inter-manufacturer differences in computer-calculated QRSD assessments are significant and may compromise adequate selection of individual CRT candidates when using QRSD as sole parameter. Paced QRSD should preferentially be assessed by manual QRSD measurements.

Grading of mitral regurgitation based on intensity analysis of the continuous wave Doppler signal.

Objectives Echocardiographic methods are used to quantify mitral regurgitation (MR) severity; however, their applicability, accuracy and reproducibility have been debated. We aimed to develop and validate a novel custom-made transthoracic echocardiographic method for grading MR severity based on average pixel intensity (API) analysis of the continuous wave (CW) Doppler envelope. Methods MR was assessed in 290 patients using API, colour Doppler imaging, vena contracta width (VCW) and proximal iso-velocity surface area (PISA) method. For the validation of the API method, a pulsatile in vitro cardiac phantom was used. Results Indices of MR severity, such as left ventricular and atrial dimension, pulmonary arterial pressure, significantly cosegregate with API severity (p≤0.002). The API method showed a linear correlation with colour Doppler (r=0.79), VCW (r=0.68), PISA-effective regurgitant orifice area (r=0.72) and PISA-regurgitant volume (r=0.67); p<0.001 for all. The API was significantly more applicable than VCW (95% vs 75% of all patients; p<0.001) and PISA-based methods (65%; p<0.001). Additionally, the API showed a stronger intraobserver and interobserver agreement compared with other methods. Finally, in the in vitro validation, API values showed a strong linear correlation with increasing regurgitant volumes (r=0.81; p<0.001). Conclusions We showed the clinical feasibility and in vitro validation of a novel digital quantitative echocardiographic method to grade MR severity. This method is more applicable and has less interobserver and intraobserver variability compared with current quantitative methods.

The electrocardiographic characteristics of septal flash in patients with left bundle branch block.

Aims In patients with systolic heart failure and left bundle branch block (LBBB), septal flash (SF) movement has been described by echocardiography. We evaluated the prevalence of SF in LBBB and non-LBBB patients and evaluated whether specific electrocardiographic (ECG) characteristics within LBBB are associated with the presence of SF on echocardiography. Methods and results One hundred and four patients with probable LBBB on standard 12-lead ECG were selected, 40 patients with non-LBBB served as controls. Left bundle branch block and non-LBBB were defined, according to the most recent guidelines. The presence of SF was assessed by echocardiography. Strict LBBB criteria were met in 93.3% of the patients. Septal flash was present in 45.2% of LBBB patients and was not present in non-LBBB patients. This was more prevalent in patients without anterior ischaemic cardiomyopathy (ICMP) compared with those with anterior ICMP (P = 0.008). The duration of QRS was longer in SF patients compared with that of non-SF patients (P < 0.05). The presence of a mid-QRS notching in more than two consecutive leads was a good predictor for the presence of SF (P = 0.01), and when combined with an absent R-wave in lead V1, the presence of SF is very likely (P = 0.001). Conclusion Our data show that SF is present in 45.2% of LBBB patients, whereas it was absent in patients with non-LBBB. Patients with SF fulfilled more LBBB criteria compared with LBBB patients without SF. Our findings raise the provocative question of whether the presence of SF identifies patients with ‘true LBBB’ and whether this echocardiographic finding might be considered as a selection parameter in cardiac resynchronization therapy.