Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a PITX2 p.M200V mutation

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the specific PITX2 gene have been identified in patients with early-onset AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a PITX2 p.M200V mutation, using a commercially available non-integrating expression system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the PITX2 p.M200V mutation

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a KCNA5 p.D322H mutation

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the atrial specific KCNA5 gene have been identified in patients with early-onset lone AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a KCNA5 p.D322H mutation, using a commercially available non-integrating system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the p.D322H mutation

NOVEL GELATIN-BASED HYDROGELS FOR TISSUE ENGINEERING APPLICATION

Introduction: Biomaterials play pivotal roles in modern strategies of tissue engineering as designable biophysical and biochemical milieus that control cell fate and function. The key strategy relies on the optimum combination of cells with a suitable biodegradable matrix that could support the cell viability and remodelling of tissues. In tissue engineering, hydrogels, 3D network of hydrophilic polymers, have received much attention due to their biocompatibility, biodegradability, structural similarity to the extracellular matrix (ECM). Driven by enormous potential of hydrogels, we have developed a novel gelatin (G)-based hydrogel with tunable mechanical, degradation and biological properties. Chitosan (CH) and hydroxyethyl cellulose (HEC) were added to better match the native ECM composition and mechanical properties as well as to tailor the degradation resistance and available cell binding motifs. The effects of different material composition on physico-chemical properties, mechanical behaviour, cell adhesion and viability were evaluated. Materials and methods: G-PEG hydrogel was prepared in aqueous solutions following a synthetic procedure which involves the reaction between gelatin amino-groups and the functional end groups of poly(ethylene glycol) (PEG). In order to obtain adducts with a number of reactive end groups able to produce crosslinking, an excess of PEG over protein amino-groups was employed. Moreover, in order to obtain insoluble materials with good mechanical properties, improved water resistance and controlled degradation rate, a specific crosslinking agent, i.e. ethylene diamine, able to react with unreacted epoxy groups, was added. G-PEG-HEC hydrogel and G-PEG-CH hydrogel were prepared adding the proper amount of HEC or CH solution to a G-PEG aqueous solution obtained starting from a 9% (w/v) solution of G. Results: Hydrogels have been fully characterized by FTIR spectroscopy that confirmed the expected structure. Mechanical tensile tests were performed and swelling and weight loss were also monitored over a period of about 30 days. Interestingly, G-PEG hydrogel as well as G-PEG-HEC and G-PEG-CH, with a G/PEG ratio of about 3.6:1, display good stiffness, flexibility and extensibility. They show non-linear J-shaped stress-strain curves, similar to those found for ECM, with initial elastic modulus and strain at break over the range of 1.5-6.5 MPa and 30-70%, respectively. The swelling test revealed a wide range of equilibrium swelling rate (200-450%). Hydrogels showed no significant change in dimension and shape during degradation tests carried out at 37oC, and the resistance to hydrolytic degradation was longer than 30 days for all the formulations. All the hydrogels showed good cell viability during long term culture of a human fibroblast cell line. Discussion: Functionalized PEG was chosen to verify the possibility of grafting on gelatin and chitosan to obtain hydrogel that could eventually undergo crosslinking in the presence of a suitable curing agent. The final purpose to obtain adequately stiff and strong biomaterials, which possessed at the same time limited solubility and degradation rate in comparison with pure gelatin or chitosan, was attained. This is in fact a perfect condition to obtain a suitable biodegradable/resorbable matrix promoting cell viability and remodelling of tissue

Preparation and properties of high performance gelatin-based hydrogels with chitosan or hydroxyethyl cellulose for tissue engineering applications

High performance gelatin-based biocompatible hybrid hydrogels are developed using functionalized polyethylene glycol as a cross-linker in presence of chitosan or hydroxyethyl cellulose. Tensile test shows robust and tunable mechanical properties and reveals non-linear and J-shaped stress-strain curves similar to those found for native extracellular matrix. Degradation study demonstrates that the mass loss and change in mechanical properties are dependent on hydrogel composition and cross-linking density. Structural features of the hydrogels are confirmed by infrared spectroscopy. A preliminary biological evaluation is carried out using rat myoblasts and human fibroblasts cell lines. The results show that all hydrogels allow cell adhesion and proliferation during four days culture, hence, they might have a great potential for use in the biomedical applications

Arrangement of live human cells through acoustic waves generated by piezoelectric actuators for tissue engineering applications

In this paper, the possibility to steer and confine live human cells by means of acoustic waves, such as flexural plate waves (FPWs), generated by piezoelectric actuators applied to non-piezoelectric substrates, has been explored. A device with two lead zirconate titanate (PZT) actuators with an interdigital transducer (IDT) screen-printed on an alumina (Al2O3) substrate has been fabricated and tested. The experimental results show that, by exciting the actuators at their resonant frequencies, FPW modes are generated in the substrate. By exploiting the device, arrangements of cells on lines at frequency-dependent distances have been obtained. To maintain the alignment after switching off the actuator, cells were entrapped in a fibrin clot that was cultured for several days, enabling the formation of cellular patterns

MEMS force microactuator with displacement sensing for mechanobiology experiments

This paper presents a Micro Electro-Mechanical System (MEMS) that performs electrostatic force actuation and capacitive microdisplacement sensing in the same chip. By driving the actuator with a given voltage, a known force can be applied to a microsample under test by using a silicon probe tip, while the obtained displacement is measured. This allows to extract the mechanical properties of the microsample entirely on chip, and to derive its force-displacement curve without external equipment. The proposed device is intended for mechanobiology experiments, where the microsample is made of biological tissues or cells. The device generates a force in the order of few micronewtons and a maximum displacement of 1.8 μm can be measured

Generation of the induced pluripotent stem cell line UNIBSi017-A from an individual with cardiospondylocarpofacial syndrome and the MAP3K7 c.737-7A > G variant

TAK1 is a serine threonine kinase that mediates signal transduction induced by TGFβ and bone morphogenetic proteins, and controls a variety of cell functions by modulating the downstream activation of NF-kkB, JNK, and p38. Heterozygous variants in the coding MAP3K7 gene cause the cardiospondylocarpofacial syndrome, characterized by various abnormalities. Skin fibroblasts derived from a patient carrying the MAP3K7 c.737-7A>G heterozygous variant were reprogrammed using Sendai viral vector system carrying the Yamanaka factors. The generated induced pluripotent stem cells (iPSC) line retained the original genotype, expressed pluripotency markers, and differentiated into cells of the three germ layers