CytoSpectre: a tool for spectral analysis of oriented structures on cellular and subcellular levels.

BACKGROUND: Orientation and the degree of isotropy are important in many biological systems such as the sarcomeres of cardiomyocytes and other fibrillar structures of the cytoskeleton. Image based analysis of such structures is often limited to qualitative evaluation by human experts, hampering the throughput, repeatability and reliability of the analyses. Software tools are not readily available for this purpose and the existing methods typically rely at least partly on manual operation. RESULTS: We developed CytoSpectre, an automated tool based on spectral analysis, allowing the quantification of orientation and also size distributions of structures in microscopy images. CytoSpectre utilizes the Fourier transform to estimate the power spectrum of an image and based on the spectrum, computes parameter values describing, among others, the mean orientation, isotropy and size of target structures. The analysis can be further tuned to focus on targets of particular size at cellular or subcellular scales. The software can be operated via a graphical user interface without any programming expertise. We analyzed the performance of CytoSpectre by extensive simulations using artificial images, by benchmarking against FibrilTool and by comparisons with manual measurements performed for real images by a panel of human experts. The software was found to be tolerant against noise and blurring and superior to FibrilTool when analyzing realistic targets with degraded image quality. The analysis of real images indicated general good agreement between computational and manual results while also revealing notable expert-to-expert variation. Moreover, the experiment showed that CytoSpectre can handle images obtained of different cell types using different microscopy techniques. Finally, we studied the effect of mechanical stretching on cardiomyocytes to demonstrate the software in an actual experiment and observed changes in cellular orientation in response to stretching. CONCLUSIONS: CytoSpectre, a versatile, easy-to-use software tool for spectral analysis of microscopy images was developed. The tool is compatible with most 2D images and can be used to analyze targets at different scales. We expect the tool to be useful in diverse applications dealing with structures whose orientation and size distributions are of interest. While designed for the biological field, the software could also be useful in non-biological applications.

The Junctophilin-2 Mutation p.(Thr161Lys) Is Associated with Hypertrophic Cardiomyopathy Using Patient-Specific iPS Cardiomyocytes and Demonstrates Prolonged Action Potential and Increased Arrhythmogenicity.

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases; it is primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. This study aimed at assessing the issue of modelling the rare Finnish founder mutation in cardiomyocytes (CMs) differentiated from iPSCs; therefore, presenting the same cardiac abnormalities observed in the patients. To explore the abnormal functions in JPH2-HCM, skin fibroblasts from a Finnish patient with JPH2 p.(Thr161Lys) were reprogrammed into iPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. Finally, iPSC-CMs were evaluated for the morphological and functional characteristics associated with JPH2 mutation. JPH2-hiPSC-CMs displayed key HCM hallmarks (cellular hypertrophy, multi-nucleation, sarcomeric disarray). Moreover, JPH2-hiPSC-CMs exhibit a higher degree of arrhythmia and longer action potential duration associated with slower inactivation of calcium channels. Functional evaluation supported clinical observations, with differences in beating characteristics when compared with isogenic-hiPSC-CMs. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as a translationally relevant platform to study genetic cardiac diseases.

Electrophysiological evaluation of human induced pluripotent stem cell-derived cardiomyocytes obtained by different methods.

The human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (CMs) (hiPSC-CMs) retain the same genetic information as the donor, and they have been shown to faithfully recapitulate the disease phenotypes of various genetic cardiac diseases. The hiPSC-CMs can be utilized in multiple types of studies and in most cases, the functionality of hiPSC-CMs is of interest. For the functional analyses, the hiPSC-CMs need to be manipulated after differentiation, e.g. enriched or dissociated into single-cell stage. For the functional assessments to be reliable and reproducible, the cell culture environment should support the cells in an optimal manner. The aim of the present study was to evaluate the effect of various differentiation methods, as well as coating materials used for the dissociated cells on the functionality of the differentiated hiPSC-CMs. The different protocols not only had different differentiation efficiencies, but they also yielded functionally different hiPSC-CMs. Additionally, the coating material had a major effect on the functionality of the hiPSC-CMs. The results of the present study emphasize that the cardiac differentiation method and the coating material have a major effect on hiPS-CMs' characteristics. Thus, when different hiPSC lines and results obtained in different labs are compared, extra care should be taken to check the conditions when results are compared.

Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes.

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes.

Divergent effects of adrenaline in human induced pluripotent stem cell-derived cardiomyocytes obtained from hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disease that affects the heart muscle with diverse clinical outcomes. HCM can cause sudden cardiac death (SCD) during or immediately after mild to rigorous physical activity in young patients. However, the mechanism causing SCD as a result of exercise remains unknown, but exercise-induced ventricular arrhythmias are thought to be responsible for this fatal consequence. To understand the disease mechanism behind HCM in a better way, we generated patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from HCM patients carrying either the MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. We extensively investigated the effects of low to high concentrations of adrenaline on action potential characteristics, and the occurrence of arrhythmias in the presence of various concentrations of adrenaline and in wash-out condition. We classified and quantified different types of arrhythmias observed in hiPSC-CMs, and found that the occurrence of arrhythmias was dependent on concentrations of adrenaline and positions of mutations in genes causing HCM. In addition, we observed ventricular tachycardia types of arrhythmias in hiPSC-CMs carrying the TPM1-Asp175Asn mutation. We additionally examined the antiarrhythmic potency of bisoprolol in HCM-specific hiPSC-CMs. However, bisoprolol could not reduce the occurrence of arrhythmias during administration or during the wash-out condition of adrenaline in HCM-specific hiPSC-CMs. Our study demonstrates hiPSC-CMs as a promising tool for studying HCM. The experimental design used in this study could be suitable and beneficial for studying other components and drugs related to cardiac disease in general.

Effects of cardioactive drugs on human induced pluripotent stem cell derived long QT syndrome cardiomyocytes.

Human induced pluripotent stem cells (hiPSC) have enabled a major step forward in pathophysiologic studies of inherited diseases and may also prove to be valuable in in vitro drug testing. Long QT syndrome (LQTS), characterized by prolonged cardiac repolarization and risk of sudden death, may be inherited or result from adverse drug effects. Using a microelectrode array platform, we investigated the effects of six different drugs on the electrophysiological characteristics of human embryonic stem cell-derived cardiomyocytes as well as hiPSC-derived cardiomyocytes from control subjects and from patients with type 1 (LQT1) and type 2 (LQT2) of LQTS. At baseline the repolarization time was significantly longer in LQTS cells compared to controls. Isoprenaline increased the beating rate of all cell lines by 10-73 % but did not show any arrhythmic effects in any cell type. Different QT-interval prolonging drugs caused prolongation of cardiac repolarization by 3-13 % (cisapride), 10-20 % (erythromycin), 8-23 % (sotalol), 16-42 % (quinidine) and 12-27 % (E-4031), but we did not find any systematic differences in sensitivity between the control, LQT1 and LQT2 cell lines. Sotalol, quinidine and E-4031 also caused arrhythmic beats and beating arrests in some cases. In summary, the drug effects on these patient-specific cardiomyocytes appear to recapitulate clinical observations and provide further evidence that these cells can be applied for in vitro drug testing to probe their vulnerability to arrhythmia.

Mutation-Specific Phenotypes in hiPSC-Derived Cardiomyocytes Carrying Either Myosin-Binding Protein C Or α-Tropomyosin Mutation for Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease, which affects the structure of heart muscle tissue. The clinical symptoms include arrhythmias, progressive heart failure, and even sudden cardiac death but the mutation carrier can also be totally asymptomatic. To date, over 1400 mutations have been linked to HCM, mostly in genes encoding for sarcomeric proteins. However, the pathophysiological mechanisms of the disease are still largely unknown. Two founder mutations for HCM in Finland are located in myosin-binding protein C (MYBPC3-Gln1061X) and α-tropomyosin (TPM1-Asp175Asn) genes. We studied the properties of HCM cardiomyocytes (CMs) derived from patient-specific human induced pluripotent stem cells (hiPSCs) carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. Both types of HCM-CMs displayed pathological phenotype of HCM but, more importantly, we found differences between CMs carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation in their cellular size, Ca(2+) handling, and electrophysiological properties, as well as their gene expression profiles. These findings suggest that even though the clinical phenotypes of the patients carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation are similar, the genetic background as well as the functional properties on the cellular level might be different, indicating that the pathophysiological mechanisms behind the two mutations would be divergent as well.

Comparative analysis of targeted differentiation of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells reveals variability associated with incomplete transgene silencing in retrovirally derived hiPSC lines.

Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus-derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.

Culture conditions affect cardiac differentiation potential of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are capable of differentiating into any cell type in the human body and thus can be used in studies of early human development, as cell models for different diseases and eventually also in regenerative medicine applications. Since the first derivation of hESCs in 1998, a variety of culture conditions have been described for the undifferentiated growth of hPSCs. In this study, we cultured both hESCs and hiPSCs in three different culture conditions: on mouse embryonic fibroblast (MEF) and SNL feeder cell layers together with conventional stem cell culture medium containing knockout serum replacement and basic fibroblast growth factor (bFGF), as well as on a Matrigel matrix in mTeSR1 medium. hPSC lines were subjected to cardiac differentiation in mouse visceral endodermal-like (END-2) co-cultures and the cardiac differentiation efficiency was determined by counting both the beating areas and Troponin T positive cells, as well as studying the expression of OCT-3/4, mesodermal Brachyury T and NKX2.5 and endodermal SOX-17 at various time points during END-2 differentiation by q-RT-PCR analysis. The most efficient cardiac differentiation was observed with hPSCs cultured on MEF or SNL feeder cell layers in stem cell culture medium and the least efficient cardiac differentiation was observed on a Matrigel matrix in mTeSR1 medium. Further, hPSCs cultured on a Matrigel matrix in mTeSR1 medium were found to be more committed to neural lineage than hPSCs cultured on MEF or SNL feeder cell layers. In conclusion, culture conditions have a major impact on the propensity of the hPSCs to differentiate into a cardiac lineage.

The effect of human and mouse fibroblast feeder cells on cardiac differentiation of human pluripotent stem cells.

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) are commonly used as feeder cells to maintain the pluripotent state of stem cells. The aim of the present study was to evaluate the effect of MEF and hFF feeders on the cardiac differentiation. Two human embryonic and two induced pluripotent stem cell lines were cultured on MEF and hFF before cardiac differentiation. The expression of Brachyury T was higher in cell lines cultured on MEF, than if cultured on hFF, suggesting enhanced mesoderm formation. However, significant positive influence of MEF feeders on cardiac differentiation was only seen with one cell line. Further, the ability of hFF to maintain pluripotency of stem cells originally cultured on MEF was quite poor. In conclusion, the cells behaved differently whether cultured on hFF or MEF feeders. However, the influence of the feeder cells on differentiation was less than the difference observed between the cell lines.

Comparison of biomaterials and extracellular matrices as a culture platform for multiple, independently derived human embryonic stem cell lines.

Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires a fibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESC production for potential application in regenerative medicine. Replacing feeder cells with defined human extracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production of clinical-grade hESCs. We tested and compared different feeder cell-free hESC culture methods based on different human ECM proteins, human and animal sera matrices, and a Matrigel matrix. Also selected biomaterials were tested for feeder cell-free propagation of undifferentiated hESCs. The matrices were tested together with conventional and modified hESC culture media, human foreskin fibroblast-conditioned culture medium, chemically defined medium, TeSR1, and modified TeSR1 media. The results showed the undefined, xenogeneic Matrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins, serum matrices, and the biomaterials tested. A long-term, feeder cell-free culture system was successful on Matrigel in combination with mTeSR1 culture medium, but a xeno-free, fully defined, and reproducible feeder cell-free hESC culture method still remains to be developed.