Willy A. Noort

Cardiomyocyte progenitor cell-derived exosomes stimulate migration of endothelial cells

•  Cell therapy •  The paracrine hypothesis •  Exosomes •  Collection CMPC secreted exosomes •  Conditioned medium and exosomes in in vitro scratch wound assay •  Exosomal signalling via MMP and EMMPRIN •  Discussion

Human versus porcine mesenchymal stromal cells: phenotype, differentiation potential, immunomodulation and cardiac improvement after transplantation

Although mesenchymal stromal cells (MSCs) have been applied clinically to treat cardiac diseases, it is unclear how and to which extent transplanted MSCs exert their beneficial effects. To address these questions, pre‐clinical MSC administrations are needed for which pigs appear to be the species of choice. This requires the use of porcine cells to prevent immune rejection. However, it is currently unknown to what extent porcine MSCs (pMSCs) resemble human MSCs (hMSCs). Aim of this study was to compare MSC from porcine bone marrow (BM) with human cells for phenotype, multi‐lineage differentiation potential, immune‐modulatory capacity and the effect on cardiac function after transplantation in a mouse model of myocardial infarction. Flow cytometric analysis revealed that pMSC expressed surface antigens also found on hMSC, including CD90, MSCA‐1 (TNAP/W8B2 antigen), CD44, CD29 and SLA class I. Clonogenic outgrowth was significantly enriched following selection of CD271+ cells from BM of human and pig (129 ± 29 and 1961 ± 485 fold, respectively). hMSC and pMSC differentiated comparably into the adipogenic, osteogenic or chondrogenic lineages, although pMSC formed fat much faster than hMSC. Immuno‐modulation, an important feature of hMSC, was clearly demonstrated for pMSC when co‐cultured with porcine peripheral blood cells stimulated with PMA and pIL‐2. Finally, pMSC transplantation after myocardial infarction attenuated adverse remodelling to a similar extent as hMSC when compared to control saline injection. These findings demonstrate that pMSCs have comparable characteristics and functionality with hMSCs, making reliable extrapolation of pre‐clinical pMSC studies into a clinical setting very well possible.

Preclinical evidence for the therapeutic potential of CD38-targeted immuno-chemotherapy in multiple myeloma patients refractory to lenalidomide and bortezomib

Purpose: Novel therapeutic agents have significantly improved the survival of patients with multiple myeloma. Nonetheless, the prognosis of patients with multiple myeloma who become refractory to the novel agents lenalidomide and bortezomib is very poor, indicating the urgent need for new therapeutic options for these patients. The human CD38 monoclonal antibody daratumumab is being evaluated as a novel therapy for multiple myeloma. Prompted with the encouraging results of ongoing clinical phase I/II trials, we now addressed the potential value of daratumumab alone or in combination with lenalidomide or bortezomib for the treatment of lenalidomide- and bortezomib-refractory patients. Experimental Design: In ex vivo assays, mainly evaluating antibody-dependent cell-mediated cytotoxicity, and in an in vivo xenograft mouse model, we evaluated daratumumab alone or in combination with lenalidomide or bortezomib as a potential therapy for lenalidomide- and bortezomib-refractory multiple myeloma patients. Results: Daratumumab induced significant lysis of lenalidomide/bortezomib-resistant multiple myeloma cell lines and of primary multiple myeloma cells in the bone marrow mononuclear cells derived from lenalidomide- and/or bortezomib-refractory patients. In these assays, lenalidomide but not bortezomib, synergistically enhanced daratumumab-mediated multiple myeloma lysis through activation of natural killer cells. Finally, in an in vivo xenograft model, only the combination of daratumumab with lenalidomide effectively reduced the tumorigenic growth of primary multiple myeloma cells from a lenalidomide- and bortezomib-refractory patient. Conclusions: Our results provide the first preclinical evidence for the benefit of daratumumab plus lenalidomide combination for lenalidomide- and bortezomib-refractory patients. Clin Cancer Res; 21(12); 2802–10. ©2014 AACR. See related commentary by Laubach and Richardson, p. 2660

The Presence of Activated CD4+ T Cells Is Essential for the Formation of Colony-Forming Unit-Endothelial Cells by CD14+ Cells

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study, the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also, however, CD4+ T cells were found to be indispensable for the induction of CFU-EC colonies, mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4+ T cells or blocking MHC class II molecules on monocytes, it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes, the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14+ cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization.

Recovery and functional activity of mononuclear bone marrow and peripheral blood cells after different cell isolation protocols used in clinical trials for cell therapy after acute myocardial infarction.

AIMS Clinical trials showed contradictory results in functional recovery after intracoronary infusion of autologous mononuclear (bone marrow) cells in patients with acute myocardial infarction. A recent study suggests that this might be related to the isolation protocol used. In The Netherlands, a comparable randomised multicentre trial (HEBE) was designed. To validate the isolation method of bone marrow and peripheral blood-derived mononuclear cells, we compared our processing protocol with methods comparable to the ASTAMI (no beneficial effect) and the REPAIR-AMI study (beneficial effect). METHODS AND RESULTS The effect of several factors (density gradient, washing buffer and centrifugation speed) has been studied on recovery and function (migration and clonogenic capacity) of mononuclear cells. Significantly lower cell recoveries were found at a centrifugation speed of 250 g, compared to 600 or 800 g, respectively. Furthermore, washing buffer without supplemented human serum albumin and heparin resulted in significantly lower cell recovery and functional impairment as measured by clonogenic capacity. CONCLUSIONS The results of our study justify the cell-processing protocol as applied in the HEBE trial (600 g, human serum albumin supplemented washing buffer). This protocol results in viable and functional cells of which the quantity and quality is at least comparable to a successful study like the REPAIR-AMI.

Cell cycle and tissue of origin contribute to the migratory behaviour of human fetal and adult mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are potential cells for cellular therapies, in which the recruitment and migration of MSC towards injured tissue is crucial. Our data show that culture‐expanded MSC from fetal lung and bone marrow, adult bone marrow and adipose tissue contained a small percentage of migrating cells in vitro, but the optimal stimulus was different. Overall, fetal lung‐MSC had the highest migratory capacity. As fetal bone marrow‐MSC had lower migratory potential than fetal lung‐MSC, the tissue of origin may determine the migratory capacity of MSC. No additive effect in migration towards combined stimuli was observed, which suggests only one migratory MSC fraction. Interestingly, actin rearrangement and increased paxillin phosphorylation were observed in most MSC upon stromal cell‐derived factor‐1α or platelet‐derived growth factor‐BB stimulation, indicating that this mechanism involved in responding to migratory cues is not restricted to migratory MSC. Migratory MSC maintained differentiation and migration potential, and contained significantly less cells in S‐ and G2/M‐phase than their non‐migrating counterpart. In conclusion, our results suggest that MSC from various sources have different migratory capacities, depending on the tissue of origin. Similar to haematopoietic stem cells, cell cycle contributes to MSC migration, which offers perspectives for modulation of MSC to enhance efficacy of future cellular therapies.

Stem cell therapy for end-stage heart failure: indispensable role for the cell?

Purpose of reviewFor heart failure patients, the urgent need for heart transplantation exceeds the availability of donor hearts. Therefore, cell transplantation has emerged as an interesting and potential solution. This review will focus on the capability of different types of stem cells to regenerate the heart. Moreover, the mechanism for success will be addressed, focusing on the specific (and indispensable?) role of the cells. Recent findingsIn recent years, many types of stem cells have been described as a possible source for cell transplantation in failing hearts, with mixed outcomes. Cell transplantation is hampered by suboptimal delivery techniques, limited survival of cells, and reduced proliferation and differentiation rates in vivo. Interestingly, the number of injected cells that engrafted the heart successfully cannot explain the observed beneficial effects and, therefore, paracrine effects are suggested for the success in cell therapy. SummaryThis review summarizes the current types of stem or progenitor cells used in cardiac cell therapy and beneficial effects on heart function and morphology in preclinical studies. Currently, the observed effects suggest that paracrine effects might be responsible, thereby triggering mobilization and activation of resident (stem) cells, which challenges the classical concept and true regenerative capacity of cell therapy at this point.

Increasing short-term cardiomyocyte progenitor cell (CMPC) survival by necrostatin-1 did not further preserve cardiac function

AIMS One of the main limitations for an effective cell therapy for the heart is the poor cell engraftment after implantation, which is partly due to a large percentage of cell death in the hostile myocardium. In the present study, we investigated the utilization of necrostatin-1 (Nec-1) as a possible attenuator of cell death in cardiomyocyte progenitor cells (CMPCs). METHODS AND RESULTS In a mouse model of myocardial infarction, survival of CMPCs 3 days after intra-myocardial injection was 39 ± 9% higher in cells pretreated with the Nec-1 compound. However, the increase in cell number was not sustained over 28 days, and did not translate into improved cardiac function (ejection fraction %, 20.6 ± 2.1 vs. 21.4 ± 2.5 for vehicle and Nec-1-treated CMPC, respectively). Nonetheless, Nec-1 rescued CMPCs remained functionally competent. CONCLUSION A pharmacological pretreatment approach to solely enhance cell survival on the short term does not seem to be effective strategy to improve cardiac cell therapy with CMPCs.

Stem Cells from In- or Outside of the Heart: Isolation, Characterization, and Potential for Myocardial Tissue Regeneration

Heart failure emerges with a net loss of viable cardiomyocytes, and there is no current therapy to reverse this process to improve long-term cardiac function. Due to a change in viewpoint, that the human heart cannot be considered a terminally differentiated postmitotic organ, incapable of myocardial regeneration, a belief in a new approach for therapy evolved: regenerating the heart. Finding stem cells in the heart capable of replenishing lost cardiomyocytes became a holy grail for research. Heart stem cells were isolated and characterized, originally derived from in- or outside of the heart. Since the endogenous repair potential of the heart following injury is not sufficient, cellular therapy has been performed after myocardial infarction in clinical settings. Clinical therapies performed with autologous skeletal myoblasts, cardiomyocytes, and bone marrow, as well as the animal studies, showed improvements in cardiac function, although the clinical effects are still limited. These findings have stimulated optimism that progression of heart failure might be prevented or even reversed with cell-based therapy. For future research, it will be a challenge to isolate the most potent therapeutic cell with an intrinsic capacity to stimulate regeneration in the heart, by direct participation or by producing paracrine factors.

Abstract PR12: Biological insights into tumor-bone marrow microenvironment interactions derived from a humanized murine model

Interactions with the hematopoietic niche in the bone marrow (BM) microenvironment are essential for hematopoietic stem cell (HSC) self-renewal. In addition, this niche is considered to serve as a sanctuary site for leukemic stem cells during chemotherapy, and contributes to disease relapse. Although many advances have been made in understanding how the niche regulates HSC self-renewal and confers therapy resistance, most of this knowledge is based on genetically engineered murine models. Given the need for models that more closely resemble the human niche, we developed a humanized model in which a scaffold seeded with human BM stromal cells generates a bone microenvironment. Inoculation of these mice with human CD34+-progenitor cells resulted in homing to the human bone environment and the generation of hematopoietic cells of distinct lineages as well as the engraftment of CD34+ cells themselves. The functionality of these humanized niches was further investigated with primary samples obtained from patients diagnosed with MDS, AML, T-ALL and MM, malignancies of which the tumor cells are highly dependent on the BM microenvironment for survival and growth. In addition, by gene-marking MM and T-ALL cells with luciferase and using bioluminescent imaging, we were able to follow tumor burden over time as well as response to therapy. Importantly, in this model, the response of primary MM cells to established anti-MM agents correlates with clinical responses of the respective patients. Moreover, this model allows us to study bidirectional interactions between MM cells and stromal cells and the resulting impact. By analyzing gene expression in human BM stromal cells (CD73+ CD90+ CD105+) that we cultured from scaffolds containing MM tumors, we identified potential novel markers for osteogenesis in MM (e.g. OGN, OMD and ASPN), as well as adhesion molecules (e.g. ITGA2) and extracellular matrix proteins (e.g. STC1 and TGM2). Hence, our model allows to investigate essential interactions within the human BM microenvironment for the development of normal and malignant hematopoiesis and thus for therapy development. This abstract is also presented as Poster B41. Citation Format: Jessica Sigmans, Willy Noort, Coleman Lindsley, Li Pan, Linda Aalders, Megan Bariteau, Benjamin Ebert, Jon Aster, Uli Steidl, Jan Schuringa, Huipin Yuan, Joost de Bruijn, Reinier Raymakers, Henk Lokhorst, Tuna Mutis, Anton Martens, Constantine Mitsiades, Richard Groen. Biological insights into tumor-bone marrow microenvironment interactions derived from a humanized murine model. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR12. doi:10.1158/1538-7445.CHTME14-PR12