Stiff matrix induces switch to pure ß-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes.

Human pluripotent stem cell (hPSC)-derived cardiomyocytes hold great potential for in vitro modeling of diseases like cardiomyopathies. Yet, knowledge about expression and functional impact of sarcomeric protein isoforms like the myosin heavy chain (MyHC) in hPSC-cardiomyocytes is scarce. We hypothesized that ventricular ß-MyHC expression alters contraction and calcium kinetics and drives morphological and electrophysiological differentiation towards ventricular-like cardiomyocytes. To address this, we (1) generated human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that switched towards exclusive ß-MyHC, and (2) functionally and morphologically characterized these hESC-CMs at the single-cell level. MyHC-isoforms and functional properties were investigated during prolonged in vitro culture of cardiomyocytes in floating cardiac bodies (soft conditions) vs. culture on a stiff matrix. Using a specific anti-ß-MyHC and a newly generated anti-α-MyHC-antibody, we found individual cardiomyocytes grown in cardiac bodies to mostly express both α- and ß-MyHC-protein isoforms. Yet, 35 and 75 days of cultivation on laminin-coated glass switched 66 and 87 % of all cardiomyocytes to exclusively express ß-MyHC, respectively. Twitch contraction and calcium transients were faster for CMs on laminin-glass. Surprisingly, both parameters were only little affected by the MyHC-isoform, although hESC-CMs with only ß-MyHC had much lower ATP-turnover and tension cost, just as in human ventricular cardiomyocytes. Spontaneous contractions and no strict coupling of ß-MyHC to ventricular-like action potentials suggest that MyHC-isoform expression does not fully determine the hESC-CM differentiation status. Stiff substrate-induced pure ß-MyHC-protein expression in hESC-CMs, with several contractile parameters close to ventricular cardiomyocytes, provides a well-defined in vitro system for modeling of cardiomyopathies and drug screening approaches.

Substantial early loss of induced pluripotent stem cells following transplantation in myocardial infarction.

The limited success of cardiac stem cell therapy has lately generated discussion regarding its effectiveness. We hypothesized that immediate cell loss after intramyocardial injection significantly obscures the regenerative potential of stem cell therapy. Therefore, our aim was to assess the distribution and quantity of induced pluripotent stem cells after intramyocardial delivery using in vivo bioluminescence analysis. In this context, we wanted to investigate if the injection of different cell concentrations would exert influence on cardiac cell retention. Murine-induced pluripotent stem cells were transfected for luciferase reporter gene expression and transplanted into infarcted myocardium in mice after left anterior descending coronary artery ligation. Cells were delivered constantly in aqueous media (15 µL) in different cell concentrations (group A, n = 10, 5.0 × 10(5) cells; group B, n = 10, 1.0 × 10(6) cells). Grafts were detected using bioluminescence imaging. Organ explants were imaged 10 min after injection to quantify early cardiac retention and cell biodistribution. Bioluminescence imaging showed a massive early displacement from the injection site to the pulmonary circulation, leading to lung accumulation. Mean cell counts of explanted organs in group A were 7.51 × 10(4) ± 4.09 × 10(3) (heart), 6.44 × 10(4) ± 2.48 × 10(3) (left lung), and 8.06 × 10(5) ± 3.61 × 10(3) (right lung). Respective cell counts in group B explants were 1.69 × 10(5) ± 7.69 × 10(4) (heart), 2.11 × 10(5) ± 4.58 × 10(3) (left lung), and 3.25 × 10(5) ± 9.35 × 10(3) (right lung). Applying bioluminescence imaging, we could unveil and quantify massive early cardiac stem cell loss and pulmonary cell accumulation following intramyocardial injection. Increased injection concentrations led to much higher intracardiac cell counts; however, pulmonary biodistribution of transplanted cells still persisted. Therefore, we recommend applying tissue engineering techniques for cardiac stem cell transplantations in order to improve cardiac retention and limit biodistribution.

Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro.

AIMS: We explored the use of highly purified murine and human pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) to generate functional bioartificial cardiac tissue (BCT) and investigated the role of fibroblasts, ascorbic acid (AA), and mechanical stimuli on tissue formation, maturation, and functionality. METHODS AND RESULTS: Murine and human embryonic/induced PSC-derived CMs were genetically enriched to generate three-dimensional CM aggregates, termed cardiac bodies (CBs). Addressing the critical limitation of major CM loss after single-cell dissociation, non-dissociated CBs were used for BCT generation, which resulted in a structurally and functionally homogenous syncytium. Continuous in situ characterization of BCTs, for 21 days, revealed that three critical factors cooperatively improve BCT formation and function: both (i) addition of fibroblasts and (ii) ascorbic acid supplementation support extracellular matrix remodelling and CB fusion, and (iii) increasing static stretch supports sarcomere alignment and CM coupling. All factors together considerably enhanced the contractility of murine and human BCTs, leading to a so far unparalleled active tension of 4.4 mN/mm(2) in human BCTs using optimized conditions. Finally, advanced protocols were implemented for the generation of human PSC-derived cardiac tissue using a defined animal-free matrix composition. CONCLUSION: BCT with contractile forces comparable with native myocardium can be generated from enriched, PSC-derived CMs, based on a novel concept of tissue formation from non-dissociated cardiac cell aggregates. In combination with the successful generation of tissue using a defined animal-free matrix, this represents a major step towards clinical applicability of stem cell-based heart tissue for myocardial repair.

Transplantation and tracking of human-induced pluripotent stem cells in a pig model of myocardial infarction: assessment of cell survival, engraftment, and distribution by hybrid single photon emission computed tomography/computed tomography of sodium iodide symporter transgene expression.

BACKGROUND: Evaluation of novel cellular therapies in large-animal models and patients is currently hampered by the lack of imaging approaches that allow for long-term monitoring of viable transplanted cells. In this study, sodium iodide symporter (NIS) transgene imaging was evaluated as an approach to follow in vivo survival, engraftment, and distribution of human-induced pluripotent stem cell (hiPSC) derivatives in a pig model of myocardial infarction. METHODS AND RESULTS: Transgenic hiPSC lines stably expressing a fluorescent reporter and NIS (NIS(pos)-hiPSCs) were established. Iodide uptake, efflux, and viability of NIS(pos)-hiPSCs were assessed in vitro. Ten (±2) days after induction of myocardial infarction by transient occlusion of the left anterior descending artery, catheter-based intramyocardial injection of NIS(pos)-hiPSCs guided by 3-dimensional NOGA mapping was performed. Dual-isotope single photon emission computed tomographic/computed tomographic imaging was applied with the use of (123)I to follow donor cell survival and distribution and with the use of (99m)TC-tetrofosmin for perfusion imaging. In vitro, iodide uptake in NIS(pos)-hiPSCs was increased 100-fold above that of nontransgenic controls. In vivo, viable NIS(pos)-hiPSCs could be visualized for up to 15 weeks. Immunohistochemistry demonstrated that hiPSC-derived endothelial cells contributed to vascularization. Up to 12 to 15 weeks after transplantation, no teratomas were detected. CONCLUSIONS: This study describes for the first time the feasibility of repeated long-term in vivo imaging of viability and tissue distribution of cellular grafts in large animals. Moreover, this is the first report demonstrating vascular differentiation and long-term engraftment of hiPSCs in a large-animal model of myocardial infarction. NIS(pos)-hiPSCs represent a valuable tool to monitor and improve current cellular treatment strategies in clinically relevant animal models.

Myosin expression and contractile function are altered by replating stem cell-derived cardiomyocytes.

Myosin heavy chain (MyHC) is the main determinant of contractile function. Human ventricular cardiomyocytes (CMs) predominantly express the ß-isoform. We previously demonstrated that ∼80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express exclusively ß-MyHC after long-term culture on laminin-coated glass coverslips. Here, we investigated the impact of enzymatically detaching hESC-CMs after long-term culture and subsequently replating them for characterization of cellular function. We observed that force-related kinetic parameters, as measured in a micromechanical setup, resembled α- rather than ß-MyHC-expressing myofibrils, as well as changes in calcium transients. Single-cell immunofluorescence analysis revealed that replating hESC-CMs led to rapid upregulation of α-MyHC, as indicated by increases in exclusively α-MyHC- and in mixed α/ß-MyHC-expressing hESC-CMs. A comparable increase in heterogeneity of MyHC isoform expression was also found among individual human induced pluripotent stem cell (hiPSC)-derived CMs after replating. Changes in MyHC isoform expression and cardiomyocyte function induced by replating were reversible in the course of the second week after replating. Gene enrichment analysis based on RNA-sequencing data revealed changes in the expression profile of mechanosensation/-transduction-related genes and pathways, especially integrin-associated signaling. Accordingly, the integrin downstream mediator focal adhesion kinase (FAK) promoted ß-MyHC expression on a stiff matrix, further validating gene enrichment analysis. To conclude, detachment and replating induced substantial changes in gene expression, MyHC isoform composition, and function of long-term cultivated human stem cell-derived CMs, thus inducing alterations in mechanosensation/-transduction, that need to be considered, particularly for downstream in vitro assays.

Myosin-18B Regulates Higher-Order Organization of the Cardiac Sarcomere through Thin Filament Cross-Linking and Thick Filament Dynamics.

MYO18B loss-of-function mutations and depletion significantly compromise the structural integrity of striated muscle sarcomeres. The molecular function of the encoded protein, myosin-18B (M18B), within the developing muscle is unknown. Here, we demonstrate that recombinant M18B lacks motor ATPase activity and harbors previously uncharacterized N-terminal actin-binding domains, properties that make M18B an efficient actin cross-linker and molecular brake capable of regulating muscle myosin-2 contractile forces. Spatiotemporal analysis of M18B throughout cardiomyogenesis and myofibrillogenesis reveals that this structural myosin undergoes nuclear-cytoplasmic redistribution during myogenic differentiation, where its incorporation within muscle stress fibers coincides with actin striation onset. Furthermore, this analysis shows that M18B is directly integrated within the muscle myosin thick filament during myofibril maturation. Altogether, our data suggest that M18B has evolved specific biochemical properties that allow it to define and maintain sarcomeric organization from within the thick filament via its dual actin cross-linking and motor modulating capabilities.

Advanced Single-Cell Mapping Reveals that in hESC Cardiomyocytes Contraction Kinetics and Action Potential Are Independent of Myosin Isoform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an attractive model to investigate CM function and disease mechanisms. One characteristic marker of ventricular specificity of human CMs is expression of the ventricular, slow ß-myosin heavy chain (MyHC), as opposed to the atrial, fast α-MyHC. The main aim of this study was to investigate at the single-cell level whether contraction kinetics and electrical activity of hESC-CMs are influenced by the relative expression of α-MyHC versus ß-MyHC. For effective assignment of functional parameters to the expression of both MyHC isoforms at protein and mRNA levels in the very same hESC-CMs, we developed a single-cell mapping technique. Surprisingly, α- versus ß-MyHC was not related to specific contractile or electrophysiological properties of the same cells. The multiparametric cell-by-cell analysis suggests that in hESC-CMs the expression of genes associated with electrical activity, contraction, calcium handling, and MyHCs is independently regulated.

Generation of functional murine cardiac myocytes from induced pluripotent stem cells.

BACKGROUND: The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease- and human patient-specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line. METHODS AND RESULTS: With the use of a standard embryoid body-based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell-derived cardiomyocytes show typical features of ES cell-derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), alpha-myosin heavy chain (alpha-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric alpha-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca(2+) fluctuations with amplitudes of Ca(2+) transients comparable to ES cell cardiomyocytes. Simultaneous Ca(2+) release within clusters of iPS cell-derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the beta-adrenergic and muscarinic signaling cascade in these cells. CONCLUSIONS: iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.

Evaluating the Effect of Drug Compounds on Cardiac Spheroids Using the Cardiac Cell Outgrowth Assay.

The ideal cell culture model should mimic the cell physiology and the mechanical and the chemical cues that are present in specific tissues and organs, within a convenient high-throughput format. A possible key feature for such models is to recapture the cell polarity, the interactions between cells, and the interactions between the cells and the elastic extracellular matrix (ECM) by orienting the cells in a three-dimensional (3D) matrix. A common method to create 3D cell environments is to let the cells aggregate into spheroids with a diameter of around 200 µm. A major challenge for 3D cell cultures is to perform quick and easy imaging of the dense cell population, especially noninvasively. This protocol explains how to take advantage of the number of cells growing out from cell spheroids over time as a readout of the effect of a drug. The assay is compatible with standard imaging techniques and can be performed noninvasively using light microscopy or as a complement to other fluorescent imaging assays.

Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of NaV1.5 sodium channels.

Loss-of-function mutations of the SCN5A gene encoding for the sodium channel α-subunit NaV1.5 result in the autosomal dominant hereditary disease Brugada Syndrome (BrS) with a high risk of sudden cardiac death in the adult. We here engineered human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRISPR/Cas9 introduced BrS-mutation p.A735V-NaV1.5 (g.2204C > T in exon 14 of SCN5A) as a novel model independent of patient´s genetic background. Recent studies raised concern regarding the use of hiPSC-CMs for studying adult-onset hereditary diseases due to cells' immature phenotype. To tackle this concern, long-term cultivation of hiPSC-CMs on a stiff matrix (27-42 days) was applied to promote maturation. Patch clamp recordings of A735V mutated hiPSC-CMs revealed a substantially reduced upstroke velocity and sodium current density, a prominent rightward shift of the steady state activation curve and decelerated recovery from inactivation as compared to isogenic hiPSC-CMs controls. These observations were substantiated by a comparative study on mutant A735V-NaV1.5 channels heterologously expressed in HEK293T cells. In contrast to mutated hiPSC-CMs, a leftward shift of sodium channel inactivation was not observed in HEK293T, emphasizing the importance of investigating mechanisms of BrS in independent systems. Overall, our approach supports hiPSC-CMs' relevance for investigating channelopathies in a dish.

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.

Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells.

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.

Differential Expression of Cholinergic System Components in Human Induced Pluripotent Stem Cells, Bone Marrow-Derived Multipotent Stromal Cells, and Induced Pluripotent Stem Cell-Derived Multipotent Stromal Cells.

The components of the cholinergic system are evolutionary very old and conserved molecules that are expressed in typical spatiotemporal patterns. They are involved in signaling in the nervous system, whereas their functions in nonneuronal tissues are hardly understood. Stem cells present an attractive cellular system to address functional issues. This study therefore compared human induced pluripotent stem cells (iPSCs; from cord blood endothelial cells), mesenchymal stromal cells derived from iPSCs (iPSC-MSCs), and bone marrow-derived MSCs (BM-MSCs) from up to 33 different human donors with respect to gene expressions of components of the cholinergic system. The status of cells was identified and characterized by the detection of cell surface antigens using flow cytometry. Acetylcholinesterase expression in iPSCs declined during their differentiation into MSCs and was comparably low in BM-MSCs. Butyrylcholinesterase was present in iPSCs, increased upon transition from the three-dimensional embryoid body phase into monolayer culture, and declined upon further differentiation into iPSC-MSCs. In BM-MSCs a notable butyrylcholinesterase expression could be detected in only four donors, but was elusive in other patient-derived samples. Different nicotinic acetylcholine receptor subunits were preferentially expressed in iPSCs and during early differentiation into iPSC-MSCs, low expression was detected in iPS-MSCs and in BM-MSCs. The m2 and m3 variants of muscarinic acetylcholine receptors were detected in all stem cell populations. In BM-MSCs, these gene expressions varied between donors. Together, these data reveal the differential expression of cholinergic signaling system components in stem cells from specific sources and suggest the utility of our approach to establish informative biomarkers.

A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device.

Three-dimensional (3D) models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC)-derived cardiac spheroids can be quantified to serve as an indicator of a drug’s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput “cardiac cell outgrowth assay” with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.

Laser bioprinting of human induced pluripotent stem cells-the effect of printing and biomaterials on cell survival, pluripotency, and differentiation.

Research on human induced pluripotent stem cells (hiPSCs) is one of the fastest growing fields in biomedicine. Generated from patient's own somatic cells, hiPSCs can be differentiated towards all functional cell types and returned to the patient without immunological concerns. 3D printing of hiPSCs could enable the generation of functional organs for replacement therapies or realization of organ-on-chip systems for individualized medicine. Printing of living cells was demonstrated with immortalized cell lines, primary cells, and adult stem cells with different printing technologies and biomaterials. However, hiPSCs are more sensitive to handling procedures, in particular, when dissociated into single cells. Both pluripotency and directed differentiation are influenced by numerous environmental factors including culture media, biomaterials, and cell density. Notably, existing literature on the effect of applied biomaterials on pluripotency is rather ambiguous. In this study, laser bioprinting of undifferentiated hiPSCs in combination with different biomaterials was performed and the impact on cells' behavior, pluripotency, and differentiation was investigated. Our findings suggest that hiPSCs are indeed more sensitive to the applied biomaterials, but not to laser printing itself. With appropriate biomaterials, such as the hyaluronic acid based solutions applied in this study, hiPSCs can be successfully laser printed without losing their pluripotency.

Differences in Contractile Function of Myofibrils within Human Embryonic Stem Cell-Derived Cardiomyocytes vs. Adult Ventricular Myofibrils Are Related to Distinct Sarcomeric Protein Isoforms.

Characterizing the contractile function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is key for advancing their utility for cellular disease models, promoting cell based heart repair, or developing novel pharmacological interventions targeting cardiac diseases. The aim of the present study was to understand whether steady-state and kinetic force parameters of ß-myosin heavy chain (ßMyHC) isoform-expressing myofibrils within human embryonic stem cell-derived cardiomyocytes (hESC-CMs) differentiated in vitro resemble those of human ventricular myofibrils (hvMFs) isolated from adult donor hearts. Contractile parameters were determined using the same micromechanical method and experimental conditions for both types of myofibrils. We identified isoforms and phosphorylation of main sarcomeric proteins involved in the modulation of force generation of both, chemically demembranated hESC-CMs (d-hESC-CMs) and hvMFs. Our results indicate that at saturating Ca2+ concentration, both human-derived contractile systems developed forces with similar rate constants (0.66 and 0.68 s-1), reaching maximum isometric force that was significantly smaller for d-hESC-CMs (42 kPa) than for hvMFs (94 kPa). At submaximal Ca2+-activation, where intact cardiomyocytes normally operate, contractile parameters of d-hESC-CMs and hvMFs exhibited differences. Ca2+ sensitivity of force was higher for d-hESC-CMs (pCa50 = 6.04) than for hvMFs (pCa50 = 5.80). At half-maximum activation, the rate constant for force redevelopment was significantly faster for d-hESC-CMs (0.51 s-1) than for hvMFs (0.28 s-1). During myofibril relaxation, kinetics of the slow force decay phase were significantly faster for d-hESC-CMs (0.26 s-1) than for hvMFs (0.21 s-1), while kinetics of the fast force decay were similar and ~20x faster. Protein analysis revealed that hESC-CMs had essentially no cardiac troponin-I, and partially non-ventricular isoforms of some other sarcomeric proteins, explaining the functional discrepancies. The sarcomeric protein isoform pattern of hESC-CMs had features of human cardiomyocytes at an early developmental stage. The study indicates that morphological and ultrastructural maturation of ßMyHC isoform-expressing hESC-CMs is not necessarily accompanied by ventricular-like expression of all sarcomeric proteins. Our data suggest that hPSC-CMs could provide useful tools for investigating inherited cardiac diseases affecting contractile function during early developmental stages.

A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies.

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here, we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging.

Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells.

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up.

Stem cell derived in vivo-like human cardiac bodies in a microfluidic device for toxicity testing by beating frequency imaging.

Beating in vivo-like human cardiac bodies (CBs) were used in a microfluidic device for testing cardiotoxicity. The CBs, cardiomyocyte cell clusters derived from induced pluripotent stem cells, exhibited typical structural and functional properties of the native human myocardium. The CBs were captured in niches along a perfusion channel in the device. Video imaging was utilized for automatic monitoring of the beating frequency of each individual CB. The device allowed assessment of cardiotoxic effects of drug substances doxorubicin, verapamil and quinidine on the 3D clustered cardiomyocytes. Beating frequency data recorded over a period of 6 hours are presented and compared to literature data. The results indicate that this microfluidic setup with imaging of CB characteristics provides a new opportunity for label-free, non-invasive investigation of toxic effects in a 3D microenvironment.

Transplantation Effectiveness of Induced Pluripotent Stem Cells Is Improved by a Fibrinogen Biomatrix in an Experimental Model of Ischemic Heart Failure.

OBJECTIVES: The aim of this study was to investigate whether a fibrinogen biomatrix improves the transplantation effectiveness of induced pluripotent stem cells (iPSCs) in a model of myocardial infarction. BACKGROUND: Early retention, engraftment, and cell proliferation are important factors for successful cardiac stem cell therapy. Common transplantation techniques involve the direction injection of cells in aqueous media. However, this approach yields low retention and variable cell biodistribution, leading to reduced grafts that are unable to sufficiently regenerate damaged myocardium. Biologically compatible scaffolds that improve the retention of injected cells can improve cardiac stem cell therapy. METHODS: Murine iPSCs were transfected for luciferase reporter gene expression. First, in vitro experiments were performed comparing cell viability in fibrinogen and medium. Second, iPSCs were transplanted intramyocardially by direct injection into ischemic myocardium of immunodeficient mice, following permanent left coronary artery ligation. Cells were delivered in medium or fibrinogen. Follow-up included graft assessment by bioluminescence imaging, the evaluation of cardiac function by magnetic resonance imaging, and histology to evaluate graft size and determine the extent of myocardial scarring. RESULTS: In vitro experiments showed proliferation of iPSCs in fibrinogen from 6.4×10(3)±8.0×10(2) after 24 h to 2.1×10(4)±3.2×10(3) after 72 h. Early cardiac cell amount in control group animals was low (23.7%±0.7%) with massive cell accumulation in the right (46.3%±1.0%) and the left lung (30.0%±0.6%). When iPSCs were injected applying the fibrinogen biomatrix, intramyocardial cell amount was increased (66.3%±0.9%) with demonstrable graft proliferation over the experimental time course. Left ventricle-function was higher in the fibrinogen group (42.9%±2.8%), also showing a higher fraction of refilled infarcted-area (66.9%±2.7%). CONCLUSIONS: The fibrinogen biomatrix improved cardiac iPSc retention, sustaining functional improvement and cellular refill of infarcted myocardium. Therefore, fibrinogen can be considered an ideal biological scaffold for intramyocardial stem cell transplantations.