Dual Function of iPSC-Derived Pericyte-Like Cells in Vascularization and Fibrosis-Related Cardiac Tissue Remodeling In Vitro.

Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types-i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.

Generation of human induced pluripotent stem cell line encoding for a genetically encoded voltage indicator Arclight A242.

Genetically encoded voltage indicators (GEVIs) allow for monitoring membrane potential changes in neurons and cardiomyocytes (CMs) as an alternative to patch-clamp techniques. GEVIs facilitate non-invasive, high throughput screening of electrophysiological properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A dual transgenic hiPSC line with Arclight A242 (GEVI) and an antibiotic resistance cardiac selection cassette was successfully generated from an earlier established hiPSC line MHHi001-A. After cardiac differentiation and selection, purified populations of CMs with constitutive GEVI expression can be utilized for studying cardiac development, disease modeling, and drug testing.

A micro-LED array based platform for spatio-temporal optogenetic control of various cardiac models.

Optogenetics relies on dynamic spatial and temporal control of light to address emerging fundamental and therapeutic questions in cardiac research. In this work, a compact micro-LED array, consisting of 16 × 16 pixels, is incorporated in a widefield fluorescence microscope for controlled light stimulation. We describe the optical design of the system that allows the micro-LED array to fully cover the field of view regardless of the imaging objective used. Various multicellular cardiac models are used in the experiments such as channelrhodopsin-2 expressing aggregates of cardiomyocytes, termed cardiac bodies, and bioartificial cardiac tissues derived from human induced pluripotent stem cells. The pacing efficiencies of the cardiac bodies and bioartificial cardiac tissues were characterized as a function of illumination time, number of switched-on pixels and frequency of stimulation. To demonstrate dynamic stimulation, steering of calcium waves in HL-1 cell monolayer expressing channelrhodopsin-2 was performed by applying different configurations of patterned light. This work shows that micro-LED arrays are powerful light sources for optogenetic control of contraction and calcium waves in cardiac monolayers, multicellular bodies as well as three-dimensional artificial cardiac tissues.

Generation of human induced pluripotent stem cell lines encoding for genetically encoded calcium indicators RCaMP1h and GCaMP6f.

Calcium plays a key role in cardiomyocytes (CMs) for the translation of the electrical impulse of an action potential into contraction forces. A rapid, not-invasive fluorescence imaging technology allows for the monitoring of calcium transients in human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) to investigate the cardiac electrophysiology in vitro and after cell transplantation in vivo. The genetically encoded calcium indicators (GECIs) GCaMP6f or RCaMP1h were successfully transfected in the previously established hiPSC line MHHi001-A, together with a cardiac specific antibiotic selection cassette facilitating the monitoring of the calcium handling in highly pure populations of hiPSC-CMs.

Dextran-based scaffolds for in-situ hydrogelation: Use for next generation of bioartificial cardiac tissues.

In pursuit of a chemically-defined matrix for in vitro cardiac tissue generation, we present dextran (Dex)-derived hydrogels as matrices suitable for bioartificial cardiac tissues (BCT). The dextran hydrogels were generated in situ by using hydrazone formation as the crosslinking reaction. Material properties were flexibly adjusted, by varying the degrees of derivatization and the molecular weight of dextran used. Furthermore, to modulate dextran's bioactivity, cyclic pentapeptide RGD was coupled to its backbone. BCTs were generated by using a blend of modified dextran and human collagen (hColI) in combination with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and fibroblasts. These hColI + Dex blends with or without RGD supported tissue formation and functional maturation of CMs. Contraction forces (hColI + Dex-RGD: 0.27 ± 0.02 mN; hColI + Dex: 0.26 ± 0.01 mN) and frequencies were comparable to published constructs. Thus, we could demonstrate that, independent of the presence of RGD, our covalently linked dextran hydrogels are a promising matrix for building cardiac grafts.

iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome.

Therapeutic application of induced pluripotent stem cell (iPSC) derivatives requires comprehensive assessment of the integrity of their nuclear and mitochondrial DNA (mtDNA) to exclude oncogenic potential and functional deficits. It is unknown, to which extent mtDNA variants originate from their parental cells or from de novo mutagenesis, and whether dynamics in heteroplasmy levels are caused by inter- and intracellular selection or genetic drift. Sequencing of mtDNA of 26 iPSC clones did not reveal evidence for de novo mutagenesis, or for any selection processes during reprogramming or differentiation. Culture expansion, however, selected against putatively actionable mtDNA mutations. Altogether, our findings point toward a scenario in which intracellular selection of mtDNA variants during culture expansion shapes the mutational landscape of the mitochondrial genome. Our results suggest that intercellular selection and genetic drift exert minor impact and that the bottleneck effect in context of the mtDNA genetic pool might have been overestimated.

Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.

Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 108 CM in 150 mL and ∼4.0 × 108 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.

Pretreatment of therapeutic cells with poly(ADP-ribose) polymerase inhibitor enhances their efficacy in an in vitro model of cell-based therapy in myocardial infarct.

The potential of cell-based therapies in diseases involving ischemia-reperfusion is greatly hampered by the excessive loss of administered cells in the harsh and oxidative environment where these cells are supposed to act. Therefore, we investigated if inhibition of poly(ADP-ribose) polymerase (PARP) in the therapeutically added cells would lead to their increased viability and, subsequently, to an enhanced effect in an in vitro simulated ischemia-reperfusion (I-R) setting. Ischemic conditions were simulated by oxygen and glucose deprivation for 160 min using H9c2 rat cardiomyoblast cells. After 30 min of reperfusion, these cells received 4 types of treatments: no added cells (I-R model), fluorescently labeled (Vybrant DiD) therapeutic H9c2 cells with vehicle (H9c2) or PARP inhibitor (10 µM or 100 µM PJ34) pretreatment. We assessed viability (live, apoptotic and necrotic) of both 'postischemic' and therapeutic cells with flow cytometric analysis using calcein-AM/ethidium homodimer-2 fluorescent staining after 24 h of co-culture. Further measurements on necrosis and metabolic activity were performed using lactate dehydrogenase (LDH) release and resazurin based assays. The percentage of surviving therapeutic cells increased significantly with PARP inhibition (untreated, 52.02±5.01%; 10 µM PJ34, 63.38±4.50%; 100 µM PJ34, 64.99±3.47%). The percentage of necrotic cells decreased in a similar manner (untreated, 37.23±4.40%; 10 µM PJ34, 26.83±3.49%; 100 µM PJ34, 24.96±2.43%). Notably, the survival of the cells that suffered I-R injury was also significantly higher when treated with PARP-inhibited therapeutic cells (I-R model, 36.44±5.05%; H9c2, 42.81±5.11%; 10 µM PJ34, 52.07±5.80%; 100 µM PJ34, 54.95±5.55%), while necrosis was inhibited (I-R model, 43.64±4.00%; H9c2, 37.29±4.55%; 10 µM PJ34, 30.18±4.60%; 100 µM PJ34, 25.52±3.47%). In subsequent experiments, PARP inhibition decreased LDH-release of the observed combined cell population and enhanced the metabolic activity. Thus, our results suggest that pretreating the therapeutically added cells with a PARP inhibitor could be beneficial in the setting of cell-based therapies.

Comparison of the direct effects of human adipose- and bone-marrow-derived stem cells on postischemic cardiomyoblasts in an in vitro simulated ischemia-reperfusion model.

Regenerative therapies hold a promising and exciting future for the cure of yet untreatable diseases, and mesenchymal stem cells are in the forefront of this approach. However, the relative efficacy and the mechanism of action of different types of mesenchymal stem cells are still incompletely understood. We aimed to evaluate the effects of human adipose- (hASC) and bone-marrow-derived stem cells (hBMSCs) and adipose-derived stem cell conditioned media (ACM) on the viability of cardiomyoblasts in an in vitro ischemia-reperfusion (I-R) model. Flow cytometric viability analysis revealed that both cell treatments led to similarly increased percentages of living cells, while treatment with ACM did not (I-R model: 12.13 ± 0.75%; hASC: 24.66 ± 2.49%; hBMSC: 25.41 ± 1.99%; ACM: 13.94 ± 1.44%). Metabolic activity measurement (I-R model: 0.065 ± 0.033; hASC: 0.652 ± 0.089; hBMSC: 0.607 ± 0.059; ACM: 0.225 ± 0.013; arbitrary units) and lactate dehydrogenase assay (I-R model: 0.225 ± 0.006; hASC: 0.148 ± 0.005; hBMSC: 0.146 ± 0.004; ACM: 0.208 ± 0.009; arbitrary units) confirmed the flow cytometric results while also indicated a slight beneficial effect of ACM. Our results highlight that mesenchymal stem cells have the same efficacy when used directly on postischemic cells, and differences found between them in preclinical and clinical investigations are rather related to other possible causes such as their immunomodulatory or angiogenic properties.

Stem cell transplantation in an in vitro simulated ischemia/reperfusion model.

Stem cell transplantation protocols are finding their way into clinical practice. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.