Generation of three CRISPR/Cas9 edited human induced pluripotent stem cell lines (DHMi005-A-5, DHMi005-A-6 and DHMi005-A-7) carrying a Holt-Oram Syndrome patient-specific TBX5 mutation with known cardiac phenotype and a FLAG-tag after exon 9 of the TBX5 gene.

TBX5 is a transcription factor (TF) playing essential role during cardiogenesis. It is well known that TF mutations possibly result in non- or additional binding of the DNA due to conformational changes of the protein. We introduced a Holt-Oram Syndrome (HOS) patient-specific TBX5 mutation c.920_C > A heterozygously in a healthy induced pluripotent stell cell (iPSC) line. This TBX5 mutation results in conformational changes of the protein and displayed ventricular septal defects in the patient itself. Additionally we introduced a FLAG-tag on the TBX5 mutation-carrying allele. The resulting heterozygous TBX5-FLAG iPSC lines are a powerful tool to investigate altered TF activity bonding.

Generation of two CRISPR/Cas edited human induced pluripotent stem cell lines (DHMi005-A-3 and DHMi005-A-4) carrying a FLAG-tag after exon 9 of the TBX5 gene.

Although TBX5 plays a major role during human cardiogenesis and initiates and controls limb development, many of its interactions with genomic DNA and the resulting biological consequences are not well known. Existing anti-TBX5-antibodies work very inefficiently in certain applications such as ChIP-Seq analysis. To circumvent this drawback, we introduced a FLAG-tag sequence into the TBX5 locus at the end of exon 9 prior to the stop codon by CRISPR/Cas9. The expressed TBX5-FLAG fusion protein can effectively be precipitated by anti-FLAG antibodies. Therefore, these gene-edited iPSC lines represent powerful cellular in vitro tools to unravel TBX5:DNA interactions in detail.

Establishment of an induced pluripotent stem cell line DHMi005-A from a healthy male proband.

We generated an induced pluripotent stem cell (iPSC) line from a healthy male 29-year-old proband. Adipose fibroblasts (AFs) were reprogrammed using Sendai virus. Generated iPSCs showed typical stem cell morphology. From passage 9 on, iPSCs were free of virus. Pluripotency in the iPSCs was verified and spontaneous differentiation showed expression of all three germ layers. Karyotyping indicated no anomalies for the generated iPSCs. Many patient-specific iPSCs are generated from subcutaneous fat fibroblasts obtained during surgical procedure. The described control iPSC line was generated equally and therefore serves as an ideal control for adipose-fibroblast-based patient-specific iPSC lines in disease modeling.

Establishment of a patient-specific induced pluripotent stem cell line DHMi004-A from a male Holt-Oram syndrome patient with verified TBX5 mutation.

The Holt-Oram syndrome (HOS) is a rare autosomal dominant disorder, mostly based on mutations in the TBX5 gene. Patients show malformation of at least one upper limb along with congenital heart defects. The established induced pluripotent stem cell (iPSC) line was generated from a patient displaying pronounced and typical features of HOS and carrying a single-nucleotide change c.920_C>A leading to an amino acid change from proline to threonine at amino acid position 85, which appeared de novo. Adipose fibroblasts from the patient were reprogrammed using Sendai virus. Pluripotency of the iPSCs was fully demonstrated.

Human Engineered Heart Tissue Patches Remuscularize the Injured Heart in a Dose-Dependent Manner.

BACKGROUND: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose. METHODS: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility. RESULTS: We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9-15×106 cardiomyocytes) and pigs (5×7 cm, 450×106 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×106, or no cells were transplanted 7 days after cryo-injury (n=18-19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%-12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation. CONCLUSIONS: EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.

Development and validation of a measurement system for continuously monitoring postoperative reservoir levels.

Following cardiac surgical procedures, multiple drainage systems remain in place inside the patient's chest to prevent the development of pericardial effusion or pneumothorax. Therefore, postoperative bleeding must be diligently observed. Currently, observation of the exudate rate is performed through periodical visual inspection of the reservoir. To improve postoperative monitoring, a measurement system based on load cells was developed to automatically detect bleeding rates. A reservoir retaining bracket was instrumented with a load cell. The signal was digitized by a microcontroller and then processed and displayed on customized software written in LabView. In cases where bleeding rates reach critical levels, the device will automatically sound an alarm. Additionally, the bleeding rate is displayed on the screen with the status of the alarm, as well as the fluid level of the reservoir. These data are all logged to a file. The measurement system has been validated for gain stability and drift, as well as for sensor accuracy, with different in vitro examinations. Additionally, performance of the measurement device was tested in a clinical pilot study on patients recovering from cardiac surgical procedures. The in vitro investigation showed that the monitoring device had excellent gain and drift stability, as well as sensor accuracy, with a resolution of 2.6 mL/h for the bleeding rate. During the clinical examination, bleeding rates of all patients were correctly measured. Continuously recording drainage volume using the developed system was comparable to manual measurements performed every 30 min by a nurse. Implementation of continuous digital measurements could improve patient safety and reduce the workload of medical professionals working in intensive care units.

Corpuls cpr resuscitation device generates superior emulated flows and pressures than LUCAS II in a mechanical thorax model.

The provision of sufficient chest compression is among the most important factors influencing patient survival during cardiopulmonary resuscitation (CPR). One approach to optimize the quality of chest compressions is to use mechanical-resuscitation devices. The aim of this study was to compare a new device for chest compression (corpuls cpr) with an established device (LUCAS II). We used a mechanical thorax model consisting of a chest with variable stiffness and an integrated heart chamber which generated blood flow dependent on the compression depth and waveform. The method of blood-flow generation could be changed between direct cardiac-compression mode and thoracic-pump mode. Different chest-stiffness settings and compression modes were tested to generate various blood-flow profiles. Additionally, an endurance test at high stiffness was performed to measure overall performance and compression consistency. Both resuscitation machines were able to compress the model thorax with a frequency of 100/min and a depth of 5 cm, independent of the chosen chest stiffness. Both devices passed the endurance test without difficulty. The corpuls cpr device was able to generate about 10-40% more blood flow than the LUCAS II device, depending on the model settings. In most scenarios, the corpuls cpr device also generated a higher blood pressure than the LUCAS II. The peak compression forces during CPR were about 30% higher using the corpuls cpr device than with the LUCAS II. In this study, the corpuls cpr device had improved blood flow and pressure outcomes than the LUCAS II device. Further examination in an animal model is required to prove the findings of this preliminary study.

Corpuls CPR Generates Higher Mean Arterial Pressure Than LUCAS II in a Pig Model of Cardiac Arrest.

According to the European Resuscitation Council guidelines, the use of mechanical chest compression devices is a reasonable alternative in situations where manual chest compression is impractical or compromises provider safety. The aim of this study is to compare the performance of a recently developed chest compression device (Corpuls CPR) with an established system (LUCAS II) in a pig model. Methods. Pigs (n = 5/group) in provoked ventricular fibrillation were left untreated for 5 minutes, after which 15 min of cardiopulmonary resuscitation was performed with chest compressions. After 15 min, defibrillation was performed every 2 min if necessary, and up to 3 doses of adrenaline were given. If there was no return of spontaneous circulation after 25 min, the experiment was terminated. Coronary perfusion pressure, carotid blood flow, end-expiratory CO2, regional oxygen saturation by near infrared spectroscopy, blood gas, and local organ perfusion with fluorescent labelled microspheres were measured at baseline and during resuscitation. Results. Animals treated with Corpuls CPR had significantly higher mean arterial pressures during resuscitation, along with a detectable trend of greater carotid blood flow and organ perfusion. Conclusion. Chest compressions with the Corpuls CPR device generated significantly higher mean arterial pressures than compressions performed with the LUCAS II device.

Multicolor particle shadow accelerometry.

This paper describes the extension of multicolor particle shadow velocimetry (CPSV) to the measurement of local acceleration in an Eulerian frame of reference. A validation experiment was conducted on a pendulous disk undergoing unsteady rigid body rotation. Angular velocity and acceleration profiles by CPSA are presented along with a comparison to recordings by an accelerometer mounted on the pendulum. CPSA is also demonstrated in a fully-developed turbulent pipe flow. Profiles of standard deviation of the local acceleration in the near wall region (0< y + <75) are compared to similar measurements by Christensen and Adrian. A favorable comparison is found between CPSA and particle image accelerometry (PIA). The effect of acceleration time delay, or the time between two velocity estimates, on local acceleration estimates is discussed.

Correcting for color crosstalk and chromatic aberration in multicolor particle shadow velocimetry.

Color crosstalk and chromatic aberration can bias estimates of fluid velocity measured by color particle shadow velocimetry (CPSV), using multicolor illumination and a color camera. This article describes corrections to remove these bias errors, and their evaluation. Color crosstalk removal is demonstrated with linear unmixing. It is also shown that chromatic aberrations may be removed using either scale calibration, or by processing an image illuminated by all colors simultaneously. CPSV measurements of a fully developed turbulent pipe flow of glycerin were conducted. Corrected velocity statistics from these measurements were compared to both single-color PSV and LDV measurements and showed excellent agreement to fourth-order, to well into the viscous sublayer. Recommendations for practical assessment and correction of color aberration and color crosstalk are discussed.

Automation of a portable extracorporeal circulatory support system with adaptive fuzzy controllers.

The presented work relates to the procedure followed for the automation of a portable extracorporeal circulatory support system. Such a device may help increase the chances of survival after suffering from cardiogenic shock outside the hospital, additionally a controller can provide of optimal organ perfusion, while reducing the workload of the operator. Animal experiments were carried out for the acquisition of haemodynamic behaviour of the body under extracorporeal circulation. A mathematical model was constructed based on the experimental data, including a cardiovascular model, gas exchange and the administration of medication. As the base of the controller fuzzy logic was used allowing the easy integration of knowledge from trained perfusionists, an adaptive mechanism was included to adapt to the patient's individual response. Initial simulations show the effectiveness of the controller and the improvements of perfusion after adaptation.

Long-term results after transcatheter aortic valve implantation: what do we know today?

Transcatheter aortic valve implantation (TAVI) is evolving rapidly as a therapeutic option in patients deemed to be at high risk for surgical aortic valve replacement. Early outcome and survival of controlled feasibility trials and single- center experience with TAVI have been previously reported. Valve performance and hemodynamics seem to improve significantly after TAVI. Long-term outcome up to 3 years have been demonstrated in recent studies. Admittedly, the results are encouraging with a survival rate at 2 and 3 years ranging from 62 to 74% and from 56 to 61% respectively. The improvement in hemodynamical and clinical status sustained beyond the 3 years follows up. However, paravalvular leakage after TAVI remains an important issue in this rapidely evolving field.

Which way in? The necessity of multiple approaches to transcatheter valve therapy.

TAVI (transcatheter aortic valve implantation) is a less invasive treatment of the stenotic aortic valve while avoiding midline sternotomy and cardiopulmonary bypass. A crimped biological valve on a self-expanding or balloonexpandable stent is inserted antegradely or retrogradely under fluoroscopy, and deployed on the beating heart. Among the worldwide TAVI programs, many different concepts have been established for the choice of the access site. Whether retrograde or antegrade TAVI should be considered the superior approach is matter of an ongoing debate. The published literature demonstrates safety of all techniques if performed within a dedicated multidisciplinary team. Since there is no data providing evidence if one approach is superior to another, we conclude that an individualized patient-centered decision making process is most beneficial, taking advantage of the complementarity of the different access options. The aim of this article is to give an overview of the current practice of access techniques for transcatheter based valve treatment and to outline the respective special characteristics.

Development of an Exergame for individual rehabilitation of patients with cardiovascular diseases.

An Exergame prototype for improved and patient-adapted rehabilitation was developed. A target heart rate for individual users was defined and tracked using a chest belt. Physical activity was tracked by two 3-axis accelerometers, fixed to both wrists. Dependent on the recorded heart rate and by means of a supporting factor and linear regression the movement of the user within the game was supported or hindered. The Exergame was evaluated on 15 healthy users regarding entertaining aspects, physical effort, and impressions concerning the handling of the whole setup. The support factor algorithm to reach the target heart rate was reliable in all subjects.

Pressure shift freezing as potential alternative for generation of decellularized scaffolds.

Background. Protocols using chemical reagents for scaffold decellularization can cause changes in the properties of the matrix, depending on the type of tissue and the chemical reagent. Technologies using physical techniques may be possible alternatives for the production grafts with potential superior matrix characteristics. Material and Methods. We tested four different technologies for scaffold decellularization. Group 1: high hydrostatic pressure (HHP), 1 GPa; Group 2: pressure shift freezing (PSF); Group 3: pulsed electric fields (PEF); Group 4: control group: detergent (SDS). The degree of decellularization was assessed by histological analysis and the measurement of residual DNA. Results. Tissue treated with PSF showed a decellularization with a penetration depth (PD) of 1.5 mm and residual DNA content of 24% ± 3%. HHD treatment caused a PD of 0.2 mm with a residual DNA content of 28% ± .4%. PD in PEF was 0.5 mm, and the residual DNA content was 49% ± 7%. In the SDS group, PD was found to be 5 mm, and the DNA content was determined at 5% ± 2%. Conclusion. PSF showed promising results as a possible technique for scaffold decellularization. The penetration depth of PSF has to be optimized, and the mechanical as well as the biological characteristics of decellularized grafts have to be evaluated.

Combined spontaneous contralateral pneumothorax and post-pneumonectomy mediastinal shift-associated dextrocardia.

We report here on an unusual late postoperative presentation of extreme post-pneumonectomy dextrocardia and spontaneous contralateral pneumothorax presenting as late complications occurring approximately 2 years after right-sided pneumonectomy. Computed tomography is the diagnostic modality of choice to obtain information on anatomical changes within the post-pneumonectomy space. Knowledge of the spectrum of cardiopulmonary, pleural, and other complications after lung resection is important to properly manage complications in post-pneumonectomy patients.

Automation of an extracorporeal support system with adaptive fuzzy controllers.

For patients suffering from cardiogenic shock cardiopulmonary resuscitation may not be sufficient to restore normal heart function. However, their chances of survival may be increased with the use of an extracorporeal support system. With this system the patient's organs are perfused while being transported to the nearest hospital for proper treatment. In the automation of an extracorporeal support system the patient's vital signals are constantly monitored and proper adjustments are performed to improve organ perfusion. In this paper, an adaptive fuzzy controller is proposed that uses the knowledge and expertise of a perfusionist as a starting point and reference for regulation. Furthermore it is able to adapt to the patient's specific reactions by manipulating the rule base of the fuzzy controller. The performance of the adaptive fuzzy controller is tested with a simulation model of the cardiovascular system.

Design of a fuzzy controller for the automation of an extracorporeal support system with the use of a simulation environment.

The automation of a portable extracorporeal support system may greatly help people who suffer from cardiogenic shock by providing them with an optimal oxygen perfusion and avoid mult-organ failure while being transported to a hospital. This however requires the creation of tools that help in the design of the ideal controller. In this paper a simulation environment is described were a cardiovascular model from the ISR Physiome database was used together with a model of a portable extracorporeal support system. Additionally a model of an oxygenator/blender model is introduced to allow the simulation of oxygen perfusion. Fuzzy controlling was used for automation since it allows a straightforward implementation of expert knowledge. Through the simulation environment different scenarios may be created where intensive testing is possible and constant repetition for control optimization. Initial simulation results are given of the fuzzy controller adjusting the extracorporeal flow rate and oxygen administration for a case of low cardiac output.

Right ventricular fibroma in a 61-year-old man.

We report on a 61-year-old man who presented with new negative T-waves in V3 to V5. Coronary heart disease with a nonsignificant stenosis of the anterior interventricular artery was known for three years without any symptoms of heart failure. Harvested endomyocardial biopsies of the left ventricle during catheterization showed a chronic parvovirus B19-associated myocarditis. A magnetic resonance imaging was carried out and showed a 6 x 3 x 3-cm mass in the right ventricle extending from the apex. The tumor could be completely resected using cardiopulmonary bypass. Histopathological diagnosis was consistent with a benign fibroma.

Measurement and modeling of pulsed microchannel plate operation (invited).

Microchannel plates (MCPs) are a standard detector for fast-framing x-ray imaging and spectroscopy of high-temperature plasmas. The MCP is coated with conductive striplines that carry short duration voltage pulses to control the timing and amplitude of the signal gain. This gain depends on the voltage to a large exponent so that small reflections or impedance losses along the striplines can have a significant impact on the position-dependent amplitude and pulse width of the gain. Understanding the pulsed gain response therefore requires careful measurements of the position- and time-dependent surface voltage coupled with detailed modeling of the resulting electron cascade. We present measurements and modeling of the time- and space-dependent gain response of MCP detectors designed for use at Sandia National Laboratories' Z facility. The pulsed gain response is understood through measurements using a high impedence probe to determine the voltage pulse propagating along the stripline surface. Coupling the surface voltage measurements with Monte Carlo calculations of the electron cascade in the MCP provides a prediction of the time- and position-dependent gain that agrees with measurements made on a subpicosecond UV laser source to within the 25% uncertainty in the simulations.