Assessing the role of phosphorylated S6 ribosomal protein in the pathological diagnosis of pulmonary antibody-mediated rejection.

BACKGROUND: Pulmonary antibody-mediated rejection is still a challenging diagnosis as C4d immunostaining has poor sensitivity. Previous studies have indicated that the phosphorylated S6 ribosomal protein, a component of the mammalian target of rapamycin (mTOR) pathway, is correlated with de novo donor-specific antibodies in lung transplantation. The objective of this study was to evaluate the phosphorylation of S6 ribosomal protein as a surrogate for antibody-mediated rejection diagnosis in lung transplant patients. METHODS: This multicentre retrospective study analyzed transbronchial biopsies from 216 lung transplanted patients, 114 with antibody-mediated rejection and 102 without (19 with acute cellular rejection, 17 with ischemia/reperfusion injury, 18 with infection, and 48 without post-transplant complications). Immunohistochemistry was used to quantify phosphorylated S6 ribosomal protein expression in macrophages, endothelium, epithelium, and inter-pathologist agreement was assessed. RESULTS: Median phosphorylated S6 ribosomal protein expression values were higher in antibody-mediated rejection cases than in controls for all cell components, with the highest sensitivity in macrophages (0.9) and the highest specificity in endothelial expression (0.8). The difference was mainly significant in macrophages compared to other post-lung transplantation complications. Inter-pathologist agreement was moderate for macrophages and endothelium, with higher agreement when phosphorylated S6 ribosomal protein expression was dichotomized into positive/negative. The inclusion of phosphorylated S6 ribosomal protein in the diagnostic algorithm could have increased antibody-mediated rejection certainty levels by 25%. CONCLUSIONS: The study supports the role of the mTOR pathway in antibody-mediated rejection-related graft injury and suggests that tissue phosphorylation of S6 ribosomal protein could be a useful surrogate for a more accurate pathological diagnosis of lung antibody-mediated rejection.

Lung allograft standardized histological analysis (LASHA) template: A research consensus proposal.

BACKGROUND: Routine monitoring of lung-transplanted patients is crucial for the identification of immunological and non-immunological complications. Determining the etiology of acute allograft dysfunction, particularly in alloimmune-mediated disorders, relies heavily on the lung biopsy with histopathologic analysis. Standardization of the pathologic diagnosis of rejection (e.g., cellular and antibody-mediated) is based on consensus statements and guidelines, indicating the importance of a multidisciplinary approach to achieve a definitive etiological diagnosis. In addition to these statements and guidelines, refinements and standardizations are feasible through systematic analysis morphological, immunophenotypic and molecular alterations observed in transbronchial biopsies. This study is to identify key morphologic features to be assessed, select consistent and reproducible terminology for each histological feature, and provide standardized definitions for pathological assessment and grading. METHODS: A template was created by experts in lung transplantation including pathologists, pulmonologists, immunologists. An initial draft was circulated, followed by discussions and multiple revisions by email and conference calls. RESULTS: The "lung allograft standardized histological analysis - LASHA" template was created and structured as multiple-choice questions with number of fields to be filled in to allow for standardization of results and easy transfer into a future electronic spreadsheet. CONCLUSION: This template will help facilitate multicenter studies through a uniform protocol and correlations with new diagnostic modalities. After validation in large-scale studies, an optimized template could be included in routine clinical practice to enhance graft assessment and medical decision-making.

Code of practice for medical autopsies: a minimum standard position paper for pathology departments performing medical (hospital) autopsies in adults.

The medical autopsy (also called hospital or clinical autopsy) is a highly specialised medical procedure, which requires professional expertise and suitably equipped facilities. To ensure high standards of performance, the Working Group of Autopsy Pathology of the European Society of Pathology (ESP) suggests a code of practice as a minimum standard for centres performing medical autopsies. The proposed standards exclusively address autopsies in adults, and not forensic autopsies, perinatal/or paediatric examinations. Minimum standards for organisation, standard of premises, and staffing conditions, as well as minimum requirements for level of expertise of the postmortem performing specialists, documentation, and turnaround times of the medical procedure, are presented. Medical autopsies should be performed by specialists in pathology, or by trainees under the supervision of such specialists. To maintain the required level of expertise, autopsies should be performed regularly and in a number that ensures the maintenance of good practice of all participating physicians. A minimum number of autopsies per dedicated pathologist in a centre should be at least 50, or as an average, at least one autopsy per working week. Forensic autopsies, but not paediatric/perinatal autopsies may be included in this number. Turnaround time for final reports should not exceed 3 weeks (14 working days) for autopsies without fixation of brain/spinal cord or other time-consuming additional examinations, and 6 weeks (30 working days) for those with fixation of brain/spinal cord or additional examinations.

ISHLT consensus document on lung transplantation in patients with connective tissue disease: Part I: Epidemiology, assessment of extrapulmonary conditions, candidate evaluation, selection criteria, and pathology statements.

Patients with connective tissue disease (CTD) and advanced lung disease are often considered suboptimal candidates for lung transplantation (LTx) due to their underlying medical complexity and potential surgical risk. There is substantial variability across LTx centers regarding the evaluation and listing of these patients. The International Society for Heart and Lung Transplantation-supported consensus document on lung transplantation in patients with CTD standardization aims to clarify definitions of each disease state included under the term CTD, to describe the extrapulmonary manifestations of each disease requiring consideration before transplantation, and to outline the absolute contraindications to transplantation allowing risk stratification during the evaluation and selection of candidates for LTx.

Protein Signatures of Remodeled Airways in Transplanted Lungs with Bronchiolitis Obliterans Syndrome Obtained Using Laser-Capture Microdissection.

Bronchiolitis obliterans syndrome, a common form of chronic lung allograft dysfunction, is the major limitation to long-term survival after lung transplantation. The histologic correlate is progressive, fibrotic occlusion of small airways, obliterative bronchiolitis lesions, which ultimately lead to organ failure. The molecular composition of these lesions is unknown. In this sutdy, the protein composition of the lesions in explanted lungs from four end-stage bronchiolitis obliterans syndrome patients was analyzed using laser-capture microdissection and optimized sample preparation protocols for mass spectrometry. Immunohistochemistry and immunofluorescence were used to determine the spatial distribution of commonly identified proteins on the tissue level, and protein signatures for 14 obliterative bronchiolitis lesions were established. A set of 39 proteins, identified in >75% of lesions, included distinct structural proteins (collagen types IV and VI) and cellular components (actins, vimentin, and tryptase). Each respective lesion exhibited a unique composition of proteins (on average, n = 66 proteins), thereby mirroring the morphologic variation of the lesions. Antibody-based staining confirmed these mass spectrometry-based findings. The 14 analyzed obliterative bronchiolitis lesions showed variations in their protein content, but also common features. This study provides molecular and morphologic insights into the development of chronic rejection after lung transplantation. The protein patterns in the lesions were correlated to pathways of extracellular matrix organization, tissue development, and wound healing processes.

[A clinical radiological score for femoral head grafts : Establishment of the Tabea FK score to ensure the quality of human femoral head grafts]. / Ein klinisch-radiologischer Score für Femurkopftransplantate : Etablierung des Tabea-FK-Scores zur Sicherung der Qualität humaner Femurkopftransplantate.

INTRODUCTION: Transplantation of cancellous tissue from human femoral heads (FK) is an established method in the reconstruction of bony defects in orthopedic and trauma surgery. Standardized rating systems with respect to the morphological quality of this tissue are not available. MATERIALS AND METHODS: In 91/105 patients who had been a regular, clinically-indicated surgery (arthroplasty of the hip joint) the respective femoral head (FK) was taken under standardized conditions. Using a checklist defined clinical and radiological criteria of FK are judged in terms of their quality (cysts, necrosis, calcification, deformities, osteoporosis) and divided by the Tabea FK score into three classes (best/middle/poor quality). This was followed by a blinded repeated scoring, now as macroscopic assessment of three sawed layers from the same femoral head. The femoral heads are examined by peripheral quantitative computed tomography (pQCT) and a standardized histological examination of the bony tissue. We evaluated the accordance of the Tabea FK score with complementary assessments by calculation of sensitivity and specificity. RESULTS: Femoral heads from 91/105 patients (ages: 68.4 ± 9.9 , n = 60 women, n = 31 men) were explanted and included in the study. The correlation between the primary radiologic clinical score (Tabea FK score) and the macroscopic second review of the sawn FK with respect to middle/best and poor/middle quality was classified as good (sensitivity 77% and 81%, respectively; specificity 76% and 84%, respectively). The correlation of histology and macroscopic second review was worse and in relation to discrimination of middle/best and poor/middle quality had a sensitivity of 85% and 54%, respectively, and a specificity of 66% and 97%, respectively. The pQCT showed a sensitivity of 82% only in discrimination of middle/best, while sensitivity in discrimination of poor/middle and poor/middle + best, respectively, was <10%. DISCUSSION: The corresponding correlation between the primary and the second clinical score was evaluated as good. This emphasizes the long-standing skills of operationally active orthopedic surgeons to classify the quality of cancellous bone correctly already on the basis of X­ray images and intraoperative findings. In this respect, the introduction of the Tabea FK score as a quality assurance tool in the routines of bone banks can be recommended.

Cardiac Myxomas Show Elevated Native T1, T2 Relaxation Time and ECV on Parametric CMR.

Introduction: While cardiac tumors are rare, their identification and differentiation has wide clinical implications. Recent cardiac magnetic resonance (CMR) parametric mapping techniques allow for quantitative tissue characterization. Our aim was to examine the range of values encountered in cardiac myxomas in correlation to histological measurements. Methods and Results: Nine patients with histologically proven cardiac myxomas were included. CMR (1.5 Tesla, Philips) including parametric mapping was performed in all patients pre-operatively. All data are reported as mean ± standard deviation. Compared to myocardium, cardiac myxomas demonstrated higher native T1 relaxation times (1,554 ± 192 ms vs. 1,017 ± 58 ms, p < 0.001), ECV (46.9 ± 13.0% vs. 27.1 ± 2.6%, p = 0.001), and T2 relaxation times (209 ± 120 ms vs. 52 ± 3 ms, p = 0.008). Areas with LGE showed higher ECV than areas without (54.3 ± 17.8% vs. 32.7 ± 18.6%, p = 0.042), with differences in native T1 relaxation times (1,644 ± 217 ms vs. 1,482 ± 351 ms, p = 0.291) and T2 relaxation times (356 ± 236 ms vs. 129 ± 68 ms, p = 0.155) not reaching statistical significance. Conclusions: Parametric CMR showed elevated native T1 and T2 relaxation times and ECV values in cardiac myxomas compared to normal myocardium, reflecting an increased interstitial space and fluid content. This might help in the differentiation of cardiac myxomas from other tumor entities.

Rare non-coding Desmoglein-2 variant contributes to Arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to variants in the coding sequence of desmosomal genes. The potential contribution of non-coding desmoglein-2 (DSG2) variants for development of ARVC is undescribed. We sequenced 1450 base pairs upstream of ATG in the DSG2 gene in 65 unrelated patients diagnosed with ARVC (10 borderline cases). Identified variants was evaluated by cosegregation and allele population frequency analysis, in silico tools, immunohistological investigations of myocardial biopsies, gene reporter assays, electrophoretic mobility shift assays (EMSA), and chromatin immunoprecipitation. The genetic analysis identified one novel, rare heterozygous DSG2 upstream variant (-317G > A) in a genetically unexplained ARVC patient. The variant segregated with signs of disease, was absent in publicly available databases, and affected a predicted binding site for activating protein-1 (AP-1). Immunohistochemical analysis of a myocardial biopsy from the -317G > A patient showed a marked reduction in DSG2 protein levels compared to healthy controls. Luciferase reporter gene assays showed promoter activity of the identified DSG2 upstream region and a general reduction in transcriptional activity in the presence of the minor DSG2_A allele (p < .01). Moreover, the DSG2_A allele reduced DSG2 activation by TGF-beta1 and a protein kinase C pathway activator (PMA; all p < .001 vs. DSG2_G). EMSAs showed altered transcription factor binding in presence of the DSG2_A allele. Chromatin immunoprecipitation assays in wild type epithelial cells identified AP-1 components c-FOS and c-JUN at the -317 locus. In conclusion, the non-coding DSG2 promoter variant -317G > A reduces DSG2 transcription in vitro and reduced myocardial DSG2 protein levels were observed in vivo. Our data support a contribution of non-coding DSG2 variants to the pathogenesis of ARVC.

Long-Term Lung Function and Exercise Capacity in Postinfectious chILD.

Background: Severe postinfectious diffuse pulmonary disease may clinically mimic other entities of children's interstitial lung disease and is clinically challenging comprising various disease severities despite treatment. Long-term lung function trend and physical capacity in children with postinfectious diffuse pulmonary disease are rarely reported. We investigated trends in pulmonary function by long-term follow-up and assessed physical capacity in such patients. Methods: We performed a descriptive, single-center follow-up study in children with biopsy-verified postinfectious diffuse pulmonary disease. Patients with completed primary treatment course were eligible for follow-up, including pulmonary function and exercise (VO2peak) testing. Results: Thirty patients with postinfectious diffuse pulmonary disease were identified and included. Median (range) age at diagnose was 27.5 (2-172) months after a mean lag time of 23 months. H. influenzae and rhinovirus were the most frequent pathogens. Fifteen patients were available for follow-up after mean (range) 7.6 (2-15) years of treatment completion. Lung clearance index (LCI2.5), forced expiratory volume in 1 second (FEV1), and bronchodilator responsiveness were abnormal in 80%, 53%, and 44%, respectively. Diffusion capacity for monoxide was abnormal in 7% and total lung capacity in 33%. Only 8% demonstrated low VO2peak, while 40% reported difficulties during physical exertion. Longitudinal data on spirometry (n = 14) remained unchanged from end of treatment throughout follow-up. A significant association was found between zLCI2.5 and zFEV1 (multiple linear regression; r 2 = 0.61; P = 0.0003). Conclusion: Postinfectious diffuse pulmonary disease in children carries a varying degree of chronic pulmonary impairment with onset of symptoms in the first months of life and a typical considerable lag time before diagnosis. Follow-up several years after the initial injury demonstrated moderate-to-severe peripheral airway impairment although no further lung function decline was found years after completion of treatment. Despite acceptable VO2peak, a considerable proportion struggled during heavy exercise.

Immune regulation by fibroblasts in tissue injury depends on uPARAP-mediated uptake of collectins.

Collectins such as mannose-binding lectin (MBL) and surfactant protein D (SP-D) become temporarily deposited in extravascular compartments after tissue injury and perform immune-stimulatory or inflammation-limiting functions. However, their turnover mechanisms, necessary to prevent excessive tissue damage, are virtually unknown. In this study, we show that fibroblasts in injured tissues undertake the clearance of collectins by using the endocytic collagen receptor uPARAP. In cellular assays, several types of collectins were endocytosed in a highly specific uPARAP-dependent process, not shared by the closely related receptor MR/CD206. When introduced into dermis or bleomycin-injured lungs of mice, collectins MBL and SP-D were endocytosed and routed for lysosomal degradation by uPARAP-positive fibroblasts. Fibroblast-specific expression of uPARAP governed endogenous SP-D levels and overall survival after lung injury. In lung tissue from idiopathic pulmonary fibrosis patients, a strong up-regulation of uPARAP was observed in fibroblasts adjacent to regions with SP-D secretion. This study demonstrates a novel immune-regulatory function of fibroblasts and identifies uPARAP as an endocytic receptor in immunity.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) in clinical practice.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited myocardial disease characterized by fibro-fatty replacement of the right ventricular myocardium, and associated with paroxysmal ventricular arrhythmias and sudden cardiac death (SCD). It is currently the second most common cause of SCD after hypertrophic cardiomyopathy in young people <35 years of age, causing up to 20% of deaths in this patient population. This condition has a male preponderance and is more commonly found in individuals of Italian and Greek descent. To date, there is no single diagnostic test for ARVC/D and the diagnosis is made based on clinical, electrocardiographic, and radiological findings according to the Revised 2010 Task Force Criteria. In this review, we will discuss the mainstay treatment which includes pharmacotherapy, implantable cardioverter-defibrillator insertion for abortion of sudden cardiac death, and in the advanced stages of the disease cardiac transplantation.

Identification of myocardial diffuse fibrosis by 11 heartbeat MOLLI T 1 mapping: averaging to improve precision and correlation with collagen volume fraction.

OBJECTIVES: Our objectives involved identifying whether repeated averaging in basal and mid left ventricular myocardial levels improves precision and correlation with collagen volume fraction for 11 heartbeat MOLLI T 1 mapping versus assessment at a single ventricular level. MATERIALS AND METHODS: For assessment of T 1 mapping precision, a cohort of 15 healthy volunteers underwent two CMR scans on separate days using an 11 heartbeat MOLLI with a 5(3)3 beat scheme to measure native T 1 and a 4(1)3(1)2 beat post-contrast scheme to measure post-contrast T 1, allowing calculation of partition coefficient and ECV. To assess correlation of T 1 mapping with collagen volume fraction, a separate cohort of ten aortic stenosis patients scheduled to undergo surgery underwent one CMR scan with this 11 heartbeat MOLLI scheme, followed by intraoperative tru-cut myocardial biopsy. Six models of myocardial diffuse fibrosis assessment were established with incremental inclusion of imaging by averaging of the basal and mid-myocardial left ventricular levels, and each model was assessed for precision and correlation with collagen volume fraction. RESULTS: A model using 11 heart beat MOLLI imaging of two basal and two mid ventricular level averaged T 1 maps provided improved precision (Intraclass correlation 0.93 vs 0.84) and correlation with histology (R 2 = 0.83 vs 0.36) for diffuse fibrosis compared to a single mid-ventricular level alone. ECV was more precise and correlated better than native T 1 mapping. CONCLUSION: T 1 mapping sequences with repeated averaging could be considered for applications of 11 heartbeat MOLLI, especially when small changes in native T 1/ECV might affect clinical management.

Development of nonfibrotic left ventricular hypertrophy in an ANG II-induced chronic ovine hypertension model.

Hypertension is a major risk factor for many cardiovascular diseases and leads to subsequent concomitant pathologies such as left ventricular hypertrophy (LVH). Translational approaches using large animals get more important as they allow the use of standard clinical procedures in an experimental setting. Therefore, the aim of this study was to establish a minimally invasive ovine hypertension model using chronic angiotensin II (ANG II) treatment and to characterize its effects on cardiac remodeling after 8 weeks. Sheep were implanted with osmotic minipumps filled with either vehicle control (n = 7) or ANG II (n = 9) for 8 weeks. Mean arterial blood pressure in the ANG II-treated group increased from 87.4 ± 5.3 to 111.8 ± 6.9 mmHg (P = 0.00013). Cardiovascular magnetic resonance imaging showed an increase in left ventricular mass from 112 ± 12.6 g to 131 ± 18.7 g after 7 weeks (P = 0.0017). This was confirmed by postmortem measurement of left ventricular wall thickness which was higher in ANG II-treated animals compared to the control group (18 ± 4 mm vs. 13 ± 2 mm, respectively, P = 0.002). However, ANG II-treated sheep did not reveal any signs of fibrosis or inflammatory infiltrates as defined by picrosirius red and H&E staining on myocardial full thickness paraffin sections of both atria and ventricles. Measurements of plasma high-sensitivity C-reactive protein and urinary 8-iso-prostaglandin F2α were inconspicuous in all animals. Furthermore, multielectrode surface mapping of the heart did not show any differences in epicardial conduction velocity and heterogeneity. These data demonstrate that chronic ANG II treatment using osmotic minipumps presents a reliable, minimally invasive approach to establish hypertension and nonfibrotic LVH in sheep.

Aortic Valve Pathology in Patients Supported by Continuous-Flow Left Ventricular Assist Device.

BACKGROUND: Continuous-flow left ventricular assist devices (CF-LVAD) may induce pathological changes to the aortic wall and aortic valve. We assessed histological changes in the relevant anatomic structures exposed to continuous flow over time and compared the histological results with clinical features in patients supported with CF-LVAD. METHODS AND RESULTS: A retrospective histological analysis was performed of 38 explanted hearts supported with CF-LVAD from patients who received heart transplantation between July 2003 and February 2014. Sections of formalin-fixed paraffin-embedded tissue showing the continuity of aortic wall and left-sided valves were examined histologically. Thickness of aorta, aortic root and aortic valve as well as 3 layers of the aortic cusps were measured individually on Elastica van Gieson-stained slides using specific software. Clinical parameters concerning aortic valve dysfunction were evaluated and validated against the histology. The aortic valve spongiosa and fibrosa layers showed no significant differences in thickness with regard to support duration or occurrence of aortic insufficiency. Longer CF-LVAD support duration correlated with a thinner aortic valve ventricularis layer (rS=-0.496). CONCLUSIONS: Long-term CF-LVAD support appears to cause involution of the ventricularis layer of the aortic valve cusp, consistent with more pronounced degenerative change with longer LVAD exposure, which may be explained by continuous coaptation of the cusps. (Circ J 2016; 80: 1371-1377).

Myocardial T1 maps reflect histological findings in acute and chronic stages of myocarditis in a rat model.

BACKGROUND: Cardiovascular magnetic resonance offers both diagnostic and prognostic information in myocarditis. Using an established animal model of myocarditis, the aim of this study was to measure myocardial T1 before the onset, in the acute and in the chronic phases of the disease and to compare its course with histological and immunohistochemistry findings. METHODS: Male young Lewis rats were immunized with 0.25 mg porcine myocardial myosin into the rear footpads on day 0. Native and contrast-enhanced ECG-triggered cardiac MRI examinations were performed before immunization on day 0 and on days 14, 21 and 35. Left ventricular function, pre- and post- contrast T1 parameters and LGE images were assessed using Small animal look-locker inversion recovery (SALLI). For each of the indicated time points a minimum of 4 rats were randomly sacrificed for pathological investigations including conventional histology (HE and Sirius-Red staining) and immunohistochemistry (CD 68) investigations. RESULTS: All immunized rats developed myocarditis (morbidity 100%). Histologically we observed increased wall thickness with biventricular macrophage-rich mixed inflammatory infiltrates. All rats with a histologically severe myocarditis showed increased native T1 and decreased post-contrast T1 of the myocardium. CONCLUSIONS: The assessment of native T1 and post-contrast T1 allows accurate differentiation between healthy myocardium and myocardium with inflammation and also between the acute and chronic phases of the disease.

Ultrastructural morphometric findings of cardiomyocytes in patients with impaired ventricular function--a comparative clinicopathological study.

AIM: The present study aims to analyze the differences in ultrastructural changes between right ventricular myocardium in clinically determined grades of heart failure (HF) [New York Heart Association (NYHA) classes I-IV] and their value in the routine diagnostic setting. METHODS: We investigated consecutive right ventricular endomyocardial biopsies of 12 patients presenting with HF (49±11.2years; male=10) by light microscopy and ultrastructural morphometric analysis. The patients were divided into four groups according to their NYHA classes (NYHA I: n=1, II: n=2, III: n=8, IV: n=1). We used a stereological point counting method on electron micrographs to determine the volume, surface, and numerical density of cardiomyocyte myofibrils; z-lines; mitochondria; and cristae as required. Further, secondary parameters were calculated. RESULTS: Myofibrillar parameters increased between NYHA class I and II (P<.01), which matched with more pronounced cardiomyocyte hypertrophy on the light microscopic level. In NYHA classes III and IV, the myofibrillar parameters dropped, while parameters concerning the mitochondria and their cristae rose (P<.01). This resulted in an elevated mitochondria to myofibril ratio (P<.05) and correlated with histologically evident atrophic cardiomyocytes, perinuclear loss of myofibrils and dot-like perinuclear staining positive on peroxide acid shift. CONCLUSION: In this present study, right ventricular myocardial ultrastructure differed between patients diagnosed with HF of different degrees in distinct subcellular changes. These findings suggest that ultrastructural analysis, while correlated with histopathological features, adds to the diagnosis in the routine diagnostic setting, specifically in lower NYHA grades, in which only minor changes are observed histologically.

Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells.

OBJECTIVES: Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure. METHODS: Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell attachment, apoptosis induction (Caspase 3/7 activity) and metabolic activity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium conversion) were followed. Transcriptional activation of genes involved in pluripotency; early and late myocardial development; and endothelial, ectodermal or endodermal commitment were monitored by quantitative real-time polymerase chain reaction (rtPCR). Protein expression of selected markers was confirmed by immunohistology. RESULTS: cECM supported the proliferation of ESCs and iPSCs, and Caspase 3/7 activity was significantly lower compared with standard culture. Cardiac lineage commitment was favoured when ESCs or iPSCs were grown on cECM, as evidenced by the significantly increased mRNA expression of cardiac alpha myosin heavy polypeptide 6 (Myh6), cardiac troponin T2 (Tnnt2) and NK2 homeobox 5 (Nkx2.5) as well as positive immunohistology for cardiac troponin T and heavy-chain cardiac myosin protein. In contrast, Matrigel or Geltrex did not induce cardiac-specific markers. MSCs showed no evidence of cardiomyocyte differentiation. CONCLUSIONS: Human cardiac ECM seems to direct differentiation of pluripotent stem cells towards a cardiomyocyte phenotype. This phenomenon supports the use of cardiac ECM preparations for guided stem cell differentiation and myocardial repair, and may ultimately increase the therapeutic efficacy of cell therapy in heart failure patients.