Klaus Neef

Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells

AIMS Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as promising cell type for cardiac cell replacement therapy. We investigated long-term electrophysiological integration and maturation of transplanted iPSCM, which are essential for therapeutic benefit. METHODS AND RESULTS Murine iPSCM expressing enhanced green fluorescent protein and a puromycin resistance under control of the α-myosin heavy chain promoter were purified by antibiotic selection and injected into adult mouse hearts. After 6-12 days, 3-6 weeks, or 6-8 months, viable slices of recipient hearts were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue, and intracellular action potentials (APs) were recorded with glass microelectrodes in transplanted cells and neighbouring host tissue within the slices. Persistence and electrical integration of transplanted iPSCM into recipient hearts could be demonstrated at all time points. Quality of coupling improved, as indicated by a maximal stimulation frequency without conduction blocks of 5.77 ± 0.54 Hz at 6-12 days, 8.98 ± 0.38 Hz at 3-6 weeks and 10.82 ± 1.07 Hz at 6-8 months after transplantation. AP properties of iPSCM became more mature from 6-12 days to 6-8 months after transplantation, but still differed significantly from those of host APs. CONCLUSION Transplanted iPSCM can persist in the long term and integrate electrically into host tissue, supporting their potential for cell replacement therapy. Quality of electrical integration improves between 6-12 days and 6-8 months after transplantation, and there are signs of an electrophysiological maturation. However, even after 6-8 months, AP properties of transplanted iPSCM differ from those of recipient cardiomyocytes.

The influence of cardiovascular risk factors on bone marrow mesenchymal stromal cell fitness

BACKGROUND AIMS In the past, cell transplantation strategies for the treatment of heart failure have shown promising results in experimental and clinical studies. Bone marrow (BM)-derived stem cells represent the most frequently used cell population. Within this heterogeneous cell population, mesenchymal stromal cells (MSC) have been identified to induce therapeutic effects, mainly through paracrine mechanisms. Because of their low frequency in native tissues, in vitro cell culture expansion is mandatory prior to transplantation. We sought to identify patient-specific cardiovascular risk factors influencing the proliferative potential of MSC. METHODS BM aspirates from 51 patients undergoing elective cardiac surgery were analyzed for MSC frequency and cell culture expansion potential. Fibroblastic colony-forming units (CFU-F) were quantified for culture conditions applying autologous (AS) or fetal bovine serum (FBS) and different basic media. Univariate and multivariate analyzes were performed in order to determine the impact of patient-specific factors on CFU-F numbers. RESULTS Expanded MSC showed a specific immune phenotype and displayed adipogenic, chondrogeneic and osteogeneic differentiation potential. CFU-F numbers did not differ under AS or FBS supplementation. Elevated numbers of mononuclear cells, diabetes mellitus, steroid treatment, chronic obstructive pulmonary disease, renal failure, high euroSCORE and impaired left ventricular function were significant determinants for higher CFU-F numbers. CONCLUSIONS The impact of specific cardiovascular risk factors on MSC fitness could be determined. These results may help to establish patient profiling in order to identify patients suitable for autologous MSC transplantation, and might lead to the identification of disease-related mechanisms of stem cell activation.

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.

Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients

Cell loss after transplantation is a major limitation for cell replacement approaches in regenerative medicine. To assess the survival kinetics of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) we generated transgenic murine iPSC lines which, in addition to CM-specific expression of puromycin N-acetyl-transferase and enhanced green fluorescent protein (EGFP), also constitutively express firefly luciferase (FLuc) for bioluminescence (BL) in vivo imaging. While undifferentiated iPSC lines generated by random integration of the transgene into the genome retained stable FLuc activity over many passages, the BL signal intensity was strongly decreased in purified iPS-CM compared to undifferentiated iPSC. Targeted integration of FLuc-expression cassette into the ROSA26 genomic locus using zinc finger nuclease (ZFN) technology strongly reduced transgene silencing in iPS-CM, leading to a several-fold higher BL compared to iPS-CM expressing FLuc from random genomic loci. To investigate the survival kinetics of iPS-CM in vivo, purified CM obtained from iPSC lines expressing FLuc from a random or the ROSA26 locus were transplanted into cryoinfarcted hearts of syngeneic mice. Engraftment of viable cells was monitored by BL imaging over 4 weeks. Transplanted iPS-CM were poorly retained in the myocardium independently of the cell line used. However, up to 8% of cells survived for 28 days at the site of injection, which was confirmed by immunohistological detection of EGFP-positive iPS-CM in the host tissue. Transplantation of iPS-CM did not affect the scar formation or capillary density in the periinfarct region of host myocardium. This report is the first to determine the survival kinetics of drug-selected iPS-CM in the infarcted heart using BL imaging and demonstrates that transgene silencing in the course of iPSC differentiation can be greatly reduced by employing genome editing technology. FLuc-expressing iPS-CM generated in this study will enable further studies to reduce their loss, increase long-term survival and functional integration upon transplantation.

Noninvasive in vivo tracking of mesenchymal stem cells and evaluation of cell therapeutic effects in a murine model using a clinical 3.0 T MRI

Cardiac cell therapy with mesenchymal stem cells (MSCs) represents a promising treatment approach for endstage heart failure. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work is to determine the feasibility of magnetic resonance imaging (MRI) and in vivo monitoring after transplantation into infarcted mouse hearts using a clinical 3.0 T MRI device. The labeling procedure of bone marrow-derived MSCs with micron-sized paramagnetic iron oxide particles (MPIOs) did not affect the viability of the cells and their cell type-defining properties when compared to unlabeled cells. Using a clinical 3.0 T MRI scanner equipped with a dedicated small animal solenoid coil, 105 labeled MSCs could be detected and localized in the mouse hearts for up to 4 weeks after intramyocardial transplantation. Weekly ECG-gated scans using T1-weighted sequences were performed, and left ventricular function was assessed. Histological analysis of hearts confirmed the survival of labeled MSCs in the target area up to 4 weeks after transplantation. In conclusion, in vivo tracking of labeled MSCs using a clinical 3.0 T MRI scanner is feasible. In combination with assessment of heart function, this technology allows the monitoring of the therapeutic efficacy of regenerative therapies in a small animal model.

The influence of pre-operative risk on the number of circulating endothelial progenitor cells during cardiopulmonary bypass.

BACKGROUND AIMS The number of circulating endothelial progenitor cells (EPC) depends on cytokine release and is also associated with cardiovascular risk factors. During cardiopulmonary bypass (CPB) the endothelium is the first organ to be affected by mechanical and immunologic stimuli. We hypothesized that the magnitude of EPC mobilization by CPB correlates with the pre-operative cardiovascular morbidity profile. METHODS EPC were quantified in blood samples from 30 patients who underwent cardiac surgery by magnetic bead isolation and fluorescence-activated cell sorting (FACS) analysis, based on concomitant expression of CD34, CD133 and CD309. Patients were divided into two groups based on the European System for Cardiac Operative Risk Evaluation (EuroSCORE): low risk (LR) and high risk (HR). Ten healthy volunteers served as controls. Samples were obtained before the start of CPB and at 1 and 24 h post-operatively. Plasma samples were collected for determination of release levels of cytokines and growth factors. RESULTS All CPB patients showed a significantly reduced basal number of EPC compared with healthy individuals (LR 5.60 +/- 0.39/mL, HR 3.89 +/- 0.34/ mL, versus control 0.807 +/- 0.82/mL, P = 0.012 versus LR, P< 0.001 versus HR). CPB induced EPC release that peaked 1 h after surgery (pre-operative 4.79 +/- 0.32/mL, 1 h 57.49 +/- 5.31/mL, 24 h 6.67 +/- 1.05/mL, P< 0.001 pre-operative versus 1 h, P< 0.001 pre-operative versus 24 h) and was associated with the duration of CPB. However, EPC release was significantly attenuated in HR patients (33.09 +/- 3.58/mL versus 81.89 +/- 4.36/mL at 1 h after CPB, P < 0.0001) and inversely correlated with the pre-operative EuroSCORE. Serum granulocyte-colony-stimulating factor (G-CSF), stem cell factor (SCF) and vascular endothelial growth factor (VEGF) levels increased throughout the observation period and were also correlated with the EPC count. CONCLUSIONS Cardiovascular risk factors influence the mobilization of EPC from the bone marrow after stimulation by CPB. This could be secondary to impaired mobilization or the result of increased EPC turnover, and may have implications for future cell therapy strategies in cardiac surgical patients.

Rosuvastatin reloading before cardiac surgery with cardiopulmonary bypass.

Background/Purpose: Recent evidence suggests that statin-mediated cardioprotection after chronic statin therapy decreases over time and can be reactivated by preprocedural high-dose statin reloading therapy. We tested in a porcine cardiopulmonary bypass (CPB) model whether statin-related cardioprotection is further enhanced by a preoperative rosuvastatin reloading therapy. Methods: Control (n = 6), rosuvastatin-pretreated (n = 6; 20 mg/day for 7 days p.o.) and rosuvastatin-reloaded (n = 6; p.o. treatment plus 0.10 mg/kg/h i.v. during surgery) pigs (Deutsche Landrasse) were subjected to CPB for 2 h with 1 h of cardioplegic cardiac arrest. Systemic hemodynamics, cardiac index (CI), coronary blood flow (CBF) and left ventricular (LV) function [pressure-volume area (PVA), preload recruitable stroke work (PRSW)] were determined before and 4 h after CPB. Myocardial expression (PCR) and protein content (Western blot) of endothelial NO synthase (eNOS) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) were measured, and right coronary relaxation was assessed postmortem. All data are given as mean ± SD. Results: Preoperative plasma LDL, HDL and cholesterol did not differ between treatment groups. Compared to control, oral treatment improved post-CPB CI, CBF, first derivative of maximal LV-pressure (LVdp/dt) and PVA (p < 0.05). Significant enhancement was achieved with perioperative reloading therapy (CI: 5.2 ± 1.0 vs. 3.9 ± 1.5 l/min/m2; CBF: 76 ± 32 vs. 43 ± 8 ml/min; LVdp/dt: 1,980 ± 333 vs. 1,249 ± 461 mm Hg/s; PVA: 6,954 ± 941 vs. 3,252 ± 1,822 mm Hg·ml; p < 0.05) with improved in vitro NO-dependent coronary relaxation (102 ± 10 vs. 79 ± 14%; p = 0.003). Irrespective of recapture therapy statin pretreatment augmented myocardial eNOS and PTEN (p < 0.05), but failed to increase cardiac eNOS or PTEN expression after CPB. Conclusions: Periprocedural statin reloading therapy enhances myocardial and coronary function after cardiac surgery with CPB and may therefore provide a valuable therapeutic approach for the reduction of myocardial ischemia-reperfusion injury.

Mechanical preconditioning enables electrophysiologic coupling of skeletal myoblast cells to myocardium.

OBJECTIVE The effect of mechanical preconditioning on skeletal myoblasts in engineered tissue constructs was investigated to resolve issues associated with conduction block between skeletal myoblast cells and cardiomyocytes. METHODS Murine skeletal myoblasts were used to generate engineered tissue constructs with or without application of mechanical strain. After in vitro myotube formation, engineered tissue constructs were co-cultured for 6 days with viable embryonic heart slices. With the use of sharp electrodes, electrical coupling between engineered tissue constructs and embryonic heart slices was assessed in the presence or absence of pharmacologic agents. RESULTS The isolation and expansion procedure for skeletal myoblasts resulted in high yields of homogeneously desmin-positive (97.1% ± 0.1%) cells. Mechanical strain was exerted on myotubes within engineered tissue constructs during gelation of the matrix, generating preconditioned engineered tissue constructs. Electrical coupling between preconditioned engineered tissue constructs and embryonic heart slices was observed; however, no coupling was apparent when engineered tissue constructs were not subjected to mechanical strain. Coupling of cells from engineered tissue constructs to cells in embryonic heart slices showed slower conduction velocities than myocardial cells with the embryonic heart slices (preconditioned engineered tissue constructs vs embryonic heart slices: 0.04 ± 0.02 ms vs 0.10 ± 0.05 ms, P = .011), lower maximum stimulation frequencies (preconditioned engineered tissue constructs vs embryonic heart slices: 4.82 ± 1.42 Hz vs 10.58 ± 1.56 Hz; P = .0009), and higher sensitivities to the gap junction inhibitor (preconditioned engineered tissue constructs vs embryonic heart slices: 0.22 ± 0.07 mmol/L vs 0.93 ± 0.15 mmol/L; P = .0004). CONCLUSIONS We have generated skeletal myoblast-based transplantable grafts that electrically couple to myocardium.

Evaluation of the use of lower body perfusion at 28°C in aortic arch surgery

OBJECTIVES Although hypothermic circulatory arrest (HCA) and selective cerebral perfusion (SCP) are widely used for cerebral protection during aortic arch surgery, these strategies offer no protection for mesenteric ischaemia during prolonged circulatory arrest. This study explored mesenteric haemodynamics, metabolism, oxidative stress and inflammatory response levels during isolated SCP and combined cerebral and lower body perfusion (CLBP) in pigs. METHODS Fourteen pigs (35-45 kg) were cooled on CPB to 28°C. After 10 min of HCA, they were randomized to 60 min of isolated SCP (n = 7) and CLBP (n = 7) at low-flow pump rates: 10 ml/kg/min (SCP) and 20 ml/kg/min (LBP). Microspheres were injected at baseline, 5 and 60 min of SCP/CLBP and 5 and 60 min off CPB, to calculate mesenteric regional blood flow (RBF). Lactate levels and Oxy-DNA expression [fluorescence activated cell sorting (FACS)] in the portal venous blood were determined at the same time points. Semi-quantitative assessment of inflammatory cytokines was performed using real-time polymerase chain reaction (PCR) and immunhistochemical analyses. RESULTS At baseline mesenteric, RBF was 61 ± 31 ml/min/100 g in the jejunum and 78 ± 43 ml/min/100 g in the colon. Whereas SCP provided a residual mesenteric RBF of 5%, CLBP offered 47% of the baseline jejunal (34 ± 10 ml/min/100 g) and 68% of the colonic RBF (52 ± 34 ml/min/100 g; P = 0.001). Lactate levels were significantly higher in then SCP group (15 ± 2 vs. 11 ± 3 mmol/l; P = 0.01). Oxy-DNA increased, reaching 137% of baseline (SCP) and 129% (CLBP) at 60 min SCP/CLBP, but recovered promptly during reperfusion. Real-time PCR revealed a massive increase in early cytokine expression vs. baseline, showing significant higher interleukin (IL) -6 (29 vs.2; P = 0.027) and COX-relative expression (7 vs. 3, P = 0.016) in the SCP group. Immunhistochemical analysis confirmed a higher immunological activity in the SCP group, showing more intensive signal for tumour necrosis factor-α, IL-6 and p38 when compared with the CLBP group. CONCLUSIONS Low-flow CLBP provides a diminished but considerable mesenteric RBF, leading to lower lactate and oxidative stress levels and a diminished local inflammatory response reaction than isolated SCP.

Enhanced gap junction expression in myoblast-containing engineered tissue

Transplantation of skeletal myoblasts (SMs) has been investigated as a potential cardiac cell therapy approach. SM are available autologously, predetermined for muscular differentiation and resistant to ischemia. Major hurdles for their clinical application are limitations in purity and yield during cell isolation as well as the absence of gap junction expression after differentiation into myotubes. Furthermore, transplanted SMs do not functionally or electrically integrate with the host myocardium. Here, we describe an efficient method for isolating homogeneous SM populations from neonatal mice and demonstrate persistent gap junction expression in an engineered tissue. This method resulted in a yield of 1.4 × 10(8) high-purity SMs (>99% desmin positive) after 10 days in culture from 162.12 ± 11.85 mg muscle tissue. Serum starvation conditions efficiently induced differentiation into spontaneously contracting myotubes that coincided with loss of gap junction expression. For mechanical conditioning, cells were integrated into engineered tissue constructs. SMs within tissue constructs exhibited long term survival, ordered alignment, and a preserved ability to differentiate into contractile myotubes. When the tissue constructs were subjected to passive longitudinal tensile stress, the expression of gap junction and cell adherence proteins was maintained or increased throughout differentiation. Our studies demonstrate that mechanical loading of SMs may provide for improved electromechanical integration within the myocardium, which could lead to more therapeutic opportunities.