Novel gene fusion discovery in Spitz tumours and its relevance in diagnostics.

In addition to morphologic analysis, molecular diagnostic work up of Spitz tumours is often of great value for their accurate diagnosis/classification. Nowadays, next-generation sequencing (NGS) is the predominant screening method in molecular diagnostics. Up to 80% of these melanocytic neoplasms comprise gene fusions as genetic anomalies for which the driver codes for a protein harbouring a kinase domain. However, because of the variety of fusion partners the use of PCR-based targeted enrichment NGS methods is not recommended. We describe a series of four Spitz tumour samples in which distinct gene fusions were detected by hybridisation-based capture NGS (TPM3::ALK, LIMA1::ROS1, LRRFIP2::ROS1 and MYO5A::RET). Two of these fusions are not previously described. All 4 fusions were confirmed by reverse transcription-PCR. These findings demonstrate the need for molecular analysis that can detect unknown fusions in Spitz neoplasms for optimal diagnosis.

Differentiation of Human Cardiac Atrial Appendage Stem Cells into Adult Cardiomyocytes: A Role for the Wnt Pathway?

Human cardiac stem cells isolated from atrial appendages based on aldehyde dehydrogenase activity (CASCs) can be expanded in vitro and differentiate into mature cardiomyocytes. In this study, we assess whether Wnt activation stimulates human CASC proliferation, whereas Wnt inhibition induces cardiac maturation. CASCs were cultured as described before. Conventional PCR confirmed the presence of the Frizzled receptors. Small-molecule inhibitors (IWP2, C59, XAV939, and IWR1-endo) and activator (CHIR99021) of the Wnt/ß -catenin signaling pathway were applied, and the effect on ß-catenin and target genes for proliferation and differentiation was assessed by Western blot and RT-qPCR. CASCs express multiple early cardiac differentiation markers and are committed toward myocardial differentiation. They express several Frizzled receptors, suggesting a role for Wnt signaling in clonogenicity, proliferation, and differentiation. Wnt activation increases total and active ß-catenin levels. However, this does not affect CASC proliferation or clonogenicity. Wnt inhibition upregulated early cardiac markers but could not induce mature myocardial differentiation. When CASCs are committed toward myocardial differentiation, the Wnt pathway is active and can be modulated. However, despite its role in cardiogenesis and myocardial differentiation of pluripotent stem-cell populations, our data indicate that Wnt signaling has limited effects on CASC clonogenicity, proliferation, and differentiation.

Archival May-Grünwald-Giemsa-Stained Bone Marrow Smears Are an Eligible Source for Molecular DNA Research.

Biobanking is increasingly important in studying complex heterogeneous diseases. Therefore, it is essential to ensure the sample quality after long-term storage for reliable downstream analyses. The Clinical Biobank of the Jessa Hospital and the University Biobank Limburg (UBiLim) hold a continuously growing collection of hematological samples, including May-Grünwald-Giemsa (MGG)- and Perls' Prussian Blue (PPB)-stained bone marrow (BM) smears, stored at room temperature (RT) for up to 20 years. In this study, we investigated the effect of short- and long-term storage on the quality of DNA and RNA extracted from these BM smears to assess their fitness-for-purpose in downstream molecular applications, including agarose gel electrophoresis, bio-analyzer analysis, quantitative polymerase chain reaction (qPCR), and targeted next-generation sequencing (NGS). The RNA quality was very low for all samples, independent of storage time or staining method. The DNA from PPB-stained BM smears was already degraded after 1 year of storage and correspondingly could not be used for reliable downstream molecular analysis. In contrast, DNA extracted from MGG-stained BM smears stored for up to 10 years was able to generate high-quality data in qPCR and targeted NGS analyses. Longer storage periods (>15 years) of these samples revealed a high degree of degradation and a significant amount of DNA transitions and transversions. In conclusion, the DNA extracted from archival MGG-stained BM smears with a storage time up to at least 10 years was qualitatively good and fit for downstream analysis, including targeted NGS. This indicates that these samples are an eligible source for molecular DNA research and for studying complex diseases.

Prognostic Biomarkers in the Progression From MGUS to Multiple Myeloma: A Systematic Review.

Multiple myeloma (MM), characterized by malignant plasma cells in the bone marrow, is consistently preceded by asymptomatic premalignant stage monoclonal gammopathy of undetermined significance (MGUS). These MGUS patients have an annual risk of 1% to progress to MM. Clinical, imaging, and genomic (genetic and epigenetic) factors were identified, whose presence increased the risk of progression from MGUS to MM. In this systematic review we summarize the currently identified clinical, imaging, and genomic biomarkers suggested to increase the progression risk or shown to be differentially expressed/present between both cohorts of patients. Despite the wide range of proposed markers, there are still no reliable biomarkers to individually predict which MGUS patient will progress to MM and which will not. Research on biomarkers in the progression from MGUS to MM will give more insight in the unknown pathogenesis of this hematological malignancy. This would improve research by elucidating new pathways and potential therapeutic targets as well as clinical management by closer follow-up and earlier treatment of high-risk MGUS patients.

Cardiac atrial appendage stem cells promote angiogenesis in vitro and in vivo.

Cardiac atrial appendage stem cells (CASCs) show extraordinary myocardial differentiation properties, making them ideal candidates for myocardial regeneration. However, since the myocardium is a highly vascularized tissue, revascularization of the ischemic infarct area is essential for functional repair. Therefore, this study assessed if CASCs contribute to cardiac angiogenesis via paracrine mechanisms. First, it was demonstrated that CASCs produce and secrete high levels of numerous angiogenic growth factors, including vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and insulin-like growth factor binding protein 3 (IGFBP-3). Functional in vitro assays with a human microvascular endothelial cell line (HMEC-1) and CASC CM showed that CASCs promote endothelial cell proliferation, migration and tube formation, the most important steps of the angiogenesis process. Addition of inhibitory antibodies against identified growth factors could significantly reduce these effects, indicating their importance in CASC-induced neovascularization. The angiogenic potential of CASCs and CASC CM was also confirmed in a chorioallantoic membrane assay, demonstrating that CASCs promote blood vessel formation in vivo. In conclusion, this study shows that CASCs not only induce myocardial repair by cardiomyogenic differentiation, but also stimulate blood vessel formation by paracrine mechanisms. The angiogenic properties of CASCs further strengthen their therapeutic potential and make them an optimal stem cell source for the treatment of ischemic heart disease.

From Bone Marrow to Cardiac Atrial Appendage Stem Cells for Cardiac Repair: A Review.

Traditionally the heart is considered a terminally differentiated organ. However, at the beginning of this century increased mitotic activity was reported in ischemic and idiopathic dilated cardiomyopathy hearts, compared to healthy controls, underscoring the potential of regeneration after injury. Due to the presence of adult stem cells in bone marrow and their purported ability to differentiate into other cell lineages, this cell population was soon estimated to be the most suited candidate for cardiac regeneration. Clinical trials with autologous bone marrow-derived mononuclear cells, using either an intracoronary or direct intramyocardial injection approach consistently showed only minor improvement in global left ventricular ejection fraction. This was explained by their limited cardiomyogenic differentiation potential. To obtain more convincing improvement in cardiac function, based on true myocardial regeneration, the focus of research has shifted towards resident cardiac progenitor cells. Several isolation procedures have been described: the c-kit surface marker was the first to be used, however experimental research has clearly shown that c-kit+ cells only marginally contribute to regeneration post myocardial infarction. Sphere formation was used to isolate the so-called cardiosphere derived cells (CDC), and also in this cell population cardiomyogenic differentiation is a rare event. Recently a new type of stem cells derived from atrial tissue (cardiac atrial stem cells - CASCs) was identified, based on the presence of the enzyme aldehyde dehydrogenase (ALDH). Those cells significantly improve both regional and global LV ejection fraction, based on substantial engraftment and consistent differentiation into mature cardiomyocytes (98%).

Cardiac atrial appendage stem cells engraft and differentiate into cardiomyocytes in vivo: A new tool for cardiac repair after MI.

BACKGROUND: This study assessed whether autologous transplantation of cardiac atrial appendage stem cells (CASCs) preserves cardiac function after myocardial infarction (MI) in a minipig model. METHODS AND RESULTS: CASCs were isolated from right atrial appendages of Göttingen minipigs based on high aldehyde dehydrogenase activity and expanded. MI was induced by a 2h snare ligation of the left anterior descending coronary artery. Upon reperfusion, CASCs were intramyocardially injected under NOGA guidance (MI-CASC, n=10). Non-transplanted pigs (MI, n=8) received sham treatment. 3D electromechanical mapping (EMM) and cardiac MRI were performed to assess left ventricular (LV) function. MI pigs developed LV dilatation at 2 months (2M), while in the MI-CASC group volumes remained stable. Global LV ejection fraction decreased by 16 ± 8% in MI animals vs 3 ± 10% in MI-CASC animals and regional wall thickening in border areas was better preserved in the MI-CASC group. EMM showed decreased viability and wall motion in the LV for both groups POST-MI, whereas at 2M these parameters only improved in the MI-CASC. Substantial cell retention was accompanied by cardiomyogenic differentiation in 98±1% of the transplanted CASCs, which functionally integrated. Second harmonic generation microscopy confirmed the formation of mature sarcomeres in transplanted CASCs. Absence of cardiac arrhythmias indicated the safety of CASC transplantation. CONCLUSION: CASCs preserve cardiac function by extensive engraftment and cardiomyogenic differentiation. Our data indicate the enormous potential of CASCs in myocardial repair.

Mesenchymal stem cell secreted platelet derived growth factor exerts a pro-migratory effect on resident Cardiac Atrial appendage Stem Cells.

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors, but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay, we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts, perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells, specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs), including a clonogenic potential of ~21.5% and expression of pluripotency associated genes like Oct-4, Nanog, c-Myc and Klf-4. Similar to CASCs, migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate, a PDGF receptor inhibitor, suggested a role for the PDGF-AA/PDGF receptor α axis in enhancing the migration process of CASCs. In conclusion, our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors, like PDGF-AA, can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury.

Differential secretome analysis of cancer-associated fibroblasts and bone marrow-derived precursors to identify microenvironmental regulators of colon cancer progression.

The identification of cancer-associated fibroblast (CAF)-derived proteins that mediate interactions between the tumor stroma and cancer cells is a crucial step toward the discovery of new molecular targets for therapy or molecular signatures that improve tumor classification and predict clinical outcome. CAF are α-smooth muscle actin positive, representing a myofibroblast phenotype that may differentiate from multiple precursor cells, including bone marrow-derived mesenchymal stem cells (MSC). Transforming growth factor-ß1 (TGF-ß1) is a crucial inducer of α-smooth muscle actin positive CAFs. In this study, we aimed to identify CAF-derived regulators of colon cancer progression by performing a high-throughput differential secretome profiling between CAF compared to noncancer-activated bone marrow-derived MSC. In addition, we explored the effect of TGF-ß1 on the secretion of proteins by bone marrow-derived MSC in comparison with the protein secretion profile of CAF. TGF-ß1 induced de novo secretion of 84 proteins in MSC, of which 16 proteins, including stromal-derived factor-1α and Rantes, were also present in CAF secretome. Immunohistochemistry further validated the expression of selected candidates such as tenascin C, fibronectin ED-A domain and stromal-derived factor-1 in clinical colon cancer specimens. In conclusion, this differential secretome approach enabled us to identify a series of candidate biomarkers for colon cancer that are associated with a CAF-specific phenotype.

The cardiac atrial appendage stem cell: a new and promising candidate for myocardial repair.

AIMS: Considerable shortcomings in the treatment of myocardial infarction (MI) still exist and therefore mortality remains high. Cardiac stem cell (CSC) therapy is a promising approach for myocardial repair. However, identification and isolation of candidate CSCs is mainly based on the presence or absence of certain cell surface markers, which suffers from some drawbacks. In order to find a more specific and reliable identification and isolation method, we investigated whether CSCs can be isolated based on the high expression of aldehyde dehydrogenase (ALDH). METHODS AND RESULTS: An ALDH(+) stem cell population, the cardiac atrial appendage stem cells (CASCs), was isolated from human atrial appendages. CASCs possess a unique phenotype that is clearly different from c-kit(+) CSCs but that seems more related to the recently described cardiac colony-forming-unit fibroblasts. Based on immunophenotype and in vitro differentiation studies, we suggest that CASCs are an intrinsic stem cell population and are not mobilized from bone marrow or peripheral blood. Indeed, they possess a clonogenicity of 16% and express pluripotency-associated genes. Furthermore, compared with cardiosphere-derived cells, CASCs possess an enhanced cardiac differentiation capacity. Indeed, differentiated cells express the most important cardiac-specific genes, produce troponin T proteins, and have an electrophysiological behaviour similar to that of adult cardiomyocytes (CMs). Transplanting CASCs in the minipig MI model resulted in extensive cardiomyogenic differentiation without teratoma formation. CONCLUSION: We have identified a new human CSC population able to differentiate into functional CMs. This opens interesting perspectives for cell therapy in patients with ischaemic heart disease.

Clinical benefits of the addition of lower extremity low-intensity resistance muscle training to early aerobic endurance training intervention in patients with coronary artery disease: a randomized controlled trial.

OBJECTIVE: Muscle resistance training is often combined with aerobic endurance training during rehabilitation of patients with coronary artery disease. However, the clinical effects of additional lower-extremity low-intensity muscle resistance training during early rehabilitation (within the first month after coronary revascularization) in patients with coronary artery disease remain unclear. DESIGN: Prospective randomized controlled trial. SUBJECTS: Sixty patients with coronary artery disease. METHODS: Subjects were randomly assigned to early aerobic endurance training (n = 30) or combined aerobic endurance and resistance muscle training (n = 30). Subjects performed 18 (standard deviation 2) exercise sessions (at 65% VO(2peak), for 40 mins/session). In resistance muscle training, additional low-intensity (12-20 repetition maximum) resistance muscle exercises were performed. The following parameters were evaluated: exercise capacity, body composition, blood lipid profile, glycaemic control, blood endothelial progenitor cell and cytokine content, and muscle performance. RESULTS: A total of 47 patients with coronary artery disease completed the intervention. Total body lean tissue mass tended to increase with greater magnitude (p = 0.07), and blood high-density lipid cholesterol content increased with significantly greater magnitude in resistance muscle training (p < 0.05), compared with aerobic endurance training. Maximal exercise capacity, ventilatory threshold, and muscle performance increased, and steady-state exercise respiratory exchange ratio, and adipose tissue mass reduced significantly (p < 0.05), without differences between groups (p < 0.05). CONCLUSION: In early aerobic endurance training intervention in patients with coronary artery disease, additional low-intensity resistance muscle training contributes to a greater increase in blood high-density lipid cholesterol content, and tends to affect lean tissue mass.

Mesenchymal stem cells or cardiac progenitors for cardiac repair? A comparative study.

In the past, clinical trials transplanting bone marrow-derived mononuclear cells reported a limited improvement in cardiac function. Therefore, the search for stem cells leading to more successful stem cell therapies continues. Good candidates are the so-called cardiac stem cells (CSCs). To date, there is no clear evidence to show if these cells are intrinsic stem cells from the heart or mobilized cells from bone marrow. In this study we performed a comparative study between human mesenchymal stem cells (hMSCs), purified c-kit(+) CSCs, and cardiosphere-derived cells (CDCs). Our results showed that hMSCs can be discriminated from CSCs by their differentiation capacity towards adipocytes and osteocytes and the expression of CD140b. On the other hand, cardiac progenitors display a greater cardiomyogenic differentiation capacity. Despite a different isolation protocol, no distinction could be made between c-kit(+) CSCs and CDCs, indicating that they probably derive from the same precursor or even are the same cells.

Human bone marrow stem cells co-cultured with neonatal rat cardiomyocytes display limited cardiomyogenic plasticity.

BACKGROUND AIMS: This study investigated whether neonatal rat cardiomyocytes (NRCM), when co-cultured, can induce transdifferentiation of either human mesenchymal stromal cells (MSC) or hematopoietic stem cells (HSC) into cardiomyocytes. Stem cells were obtained from patients with ischemic heart disease. METHODS: Ex vivo-expanded MSC or freshly isolated HSC were used to set-up a co-culture system between NRCM and MSC or HSC. 5-azacytidin (5-aza) or dimethylsulfoxide (DMSO) was used as differentiation-inducing factor. Co-cultured stem cells were separated from NRCM by flow sorting, and cardiac gene expression was analyzed by reverse transcriptase-polymerase chain reaction. Cellular morphology was analyzed by immunofluorescence and transmission electron microscopy (TEM). RESULTS: Co-culturing MSC induced expression of troponin T and GATA-4. However, no expression of alpha-actinin, myosin heavy chain or troponin I was detected. In the case of HSC, only expression of troponin T could be induced. Immunofluorescence and TEM confirmed the absence of sarcomeric organization in co-cultured MSC and HSC. Adding 5-aza or DMSO to the co-cultures did not influence differentiation. CONCLUSIONS: This in vitro co-culture study obtained no convincing evidence of transdifferentiation of either MSC or HSC into functional cardiomyocytes. Nevertheless, induction of troponin T was observed in MSC and HSC, and GATA-4 in MSC. However, no morphologic changes could be detected by immunofluorescence or by TEM. These data could explain why only limited functional improvement was reported in clinical stem cell trials.

In pursuit of B-cell synovial autoantigens in rheumatoid arthritis: Confirmation of citrullinated fibrinogen, detection of vimentin, and introducing carbonic anhydrase as a possible new synovial autoantigen.

We aimed to investigate potential synovial autoantigens in rheumatoid arthritis (RA) that could trigger the induction of B-cell autoantibodies. Total protein extract of synovial tissue obtained from seven RA patients was pooled and separated by 1-DE and 2-DE. The corresponding blots were probed with sera from RA (n = 30) and disease control samples (n = 30). Protein spots showing a sensitivity of >15% were identified by MS. 1-D immunoblots revealed one protein band with a specificity in RA of 100%, a sensitivity of 43%, which was identified as fibrinogen ß chain. 2-D analysis revealed the subunits of fibrinogen, especially the ß and γ chain, as the most prominent synovial autoantigens. We also identified vimentin, the Sa-antigen and carbonic anhydrase I as a potentially new synovial autoantigen. The protein patterns of these immunoreactive spots were observed as trains. The spots showing the highest autoimmune reactivity occurred at the acidic side of these trains and were recognized by anticitrullinated protein/peptide antibodies positive RA sera. Antimodified citrulline staining of these patterns confirmed protein citrullination. Therefore, PTMs such as citrullination due to alterations of peptidylarginine deiminase activity or generation of RA-specific epitopes, should be considered as a trigger in tolerance break.