Reprogrammed mesenchymal stem cells derived from iPSCs promote bone repair in steroid-associated osteonecrosis of the femoral head.

BACKGROUND: Cellular therapy based on mesenchymal stem cells (MSCs) is a promising novel therapeutic strategy for the osteonecrosis of the femoral head (ONFH), which is gradually becoming popular, particularly for early-stage ONFH. Nonetheless, the MSC-based therapy is challenging due to certain limitations, such as limited self-renewal capability of cells, availability of donor MSCs, and the costs involved in donor screening. As an alternative approach, MSCs derived from induced pluripotent stem cells (iPSCs), which may lead to further standardized-cell preparations. METHODS: In the present study, the bone marrow samples of patients with ONFH (n = 16) and patients with the fracture of the femoral neck (n = 12) were obtained during operation. The bone marrow-derived MSCs (BMSCs) were isolated by density gradient centrifugation. BMSCs of ONFH patients (ONFH-BMSCs) were reprogrammed to iPSCs, following which the iPSCs were differentiated into MSCs (iPSC-MSCs). Forty adult male rats were randomly divided into following groups (n = 10 per group): (a) normal control group, (b) methylprednisolone (MPS) group, (c) MPS + BMSCs treated group, and (d) MPS + iPSC-MSC-treated group. Eight weeks after the establishment of the ONFH model, rats in BMSC-treated group and iPSC-MSC-treated group were implanted with BMSCs and iPSC-MSCs through intrabone marrow injection. Bone repair of the femoral head necrosis area was analyzed after MSC transplantation. RESULTS: The morphology, immunophenotype, in vitro differentiation potential, and DNA methylation patterns of iPSC-MSCs were similar to those of normal BMSCs, while the proliferation of iPSC-MSCs was higher and no tumorigenic ability was exhibited. Furthermore, comparing the effectiveness of iPSC-MSCs and the normal BMSCs in an ONFH rat model revealed that the iPSC-MSCs was equivalent to normal BMSCs in preventing bone loss and promoting bone repair in the necrosis region of the femoral head. CONCLUSION: Reprogramming can reverse the abnormal proliferation, differentiation, and DNA methylation patterns of ONFH-BMSCs. Transplantation of iPSC-MSCs could effectively promote bone repair and angiogenesis in the necrosis area of the femoral head.

Skeletal Extracellular Matrix Supports Cardiac Differentiation of Embryonic Stem Cells: a Potential Scaffold for Engineered Cardiac Tissue.

BACKGROUND/AIMS: Decellularized cardiac extracellular matrix (cECM) has been widely considered as an attractive scaffold for engineered cardiac tissue (ECT), however, its application is limited by immunogenicity and shortage of organ donation. Skeletal ECM (sECM) is readily available and shows similarities with cECM. Here we hypothesized that sECM might be an alternative scaffold for ECT strategies. METHODS: Murine ventricular tissue and anterior tibial muscles were sectioned into 300 mm-thick, and then cECM and sECM were acquired by pretreatment/SDS/TritonX-100 three-step-method. Acellularity and morphological properties of ECM was assessed. SECM was recellularized with murine embryonic stem cells (mESCs) or mESC-derived cardiomyocytes (mESC-CMs), and was further studied by biocompatibility assessment, immunofluorescent staining, quantitative real-time PCR and electrophysiological experiment. RESULTS: The relative residual contents of DNA, protein and RNA of sECM were comparable with cECM. The morphological properties and microstructure of sECM were similar to cECM. SECM supported mESCs to adhere, survive, proliferate and differentiate into functional cardiac microtissue with both electrical stimulated response and normal adrenergic response. Purified mESC-CMs also could adhere, survive, proliferate and form a sECM-based ECT with synchronized contraction within 6 days of recellularization. CONCLUSION: ECMs from murine skeletal muscle support survival and cardiac differentiation of mESCs, and are suitable to produce functional ECT patch. This study highlights the potential of patient specific of sECM to replace cECM for bioengineering ECT.

Puerarin Enhances Ca2+ Reuptake and Ca2+ Content of Sarcoplasmic Reticulum in Murine Embryonic Stem Cell-Derived Cardiomyocytes via Upregulation of SERCA2a.

BACKGROUND/AIMS: The embryonic stem cell-derived cardiomyocytes (ES-CMs) serve as potential sources for cardiac regenerative therapy. However, the immature sarcoplasmic reticulum (SR) function of ES-CMs prevents its application. In this report, we examined the effect of puerarin, an isoflavone compound, on SR function of murine ES-CMs. METHODS: Murine ES-CMs were harvested by embryoid body-based differentiation method. Confocal calcium imaging and whole-cell patch clamps were performed to assess the function of SR. The mRNA expression levels of SR-related genes were examined by quantitative PCR. The protein expression of sarcoplasmic reticulum calcium-ATPase 2a (SERCA2a) was evaluated by immunofluorescent and western blot. RESULTS: Long-term application of puerarin promotes basic properties of spontaneous calcium transient with increased amplitude, decay velocity, and decreased duration. Puerarin fails to alter ICa,L but increases the Ca2+ content of SR. Puerarin-treated ES-CMs have intact SR Ca2+ cycling with more SR Ca2+ reuptake. Long-term application of puerarin asynchronously upregulates the mRNA and protein expression of SERCA2a, as well as the transcripts of calsequestrin and triadin in developing ES-CMs. Application of puerarin during the stage of post-cardiac differentiation upregulates dose-dependently the transcripts of SERCA2a, phospholamban and tridin which can be reversed by the inhibitors of the PI3K/Akt and MAPK/ERK signaling pathways, but shows no effect on the protein expression of SERCA2a. CONCLUSION: This study demonstrates that long-term puerarin treatment enhances Ca2+ reuptake and Ca2+ content via upregulation of SERCA2a.

Puerarin Exerts a Delayed Inhibitory Effect on the Proliferation of Cardiomyocytes Derived from Murine ES Cells via Slowing Progression through G2/M Phase.

OBJECTIVE: Puerarin, which shows beneficial and protective effects on cardiovascular diseases, is the main isoflavone extracted from Pueraria lobata (kudzu) root. The aim of this study was to investigate the effects of puerarin on in vitro myocardial proliferation and its underlying mechanism. METHODS: Myocardial differentiation of transgenic embryonic stem (ES) cells was performed by embryoid body-based differentiation method. The proliferation assay of cardiomyocytes (CMs) derived from ES cells (ES-CMs) was performed by EdU (5-Ethynyl-2'-deoxyuridine) staining. Flow cytometry was employed to determine the cell cycle distribution and apoptosis of purified ES-CMs. Quantitative real-time PCR was utilized to study the transcription of genes related to cell cycle progression. Signaling pathways relating to proliferation were studied by western blot analysis and application of specific inhibitors. RESULTS: Puerarin exerted a delayed inhibitory effect on the proliferation of ES-CMs at the early-stage differentiation. Meanwhile, puerarin slowed progression through G2/M phase without inducing apoptosis of ES-CMs. Further assays showed that puerarin up-regulated the transcription of Cyclin A2, Cyclin B1 and Cdk1 in ES-CMs. The ERK1/2 specific inhibitor PD0325901 and the PI3K specific inhibitor Wortmannin successfully reversed puerarin-induced up-regulation of Cdk1 but not Cyclin A2 and B1. CONCLUSION: These findings suggest that puerarin inhibits CM proliferation via slowing progression through G2/M phase during early-stage differentiation.

Puerarin Suppresses the Self-Renewal of Murine Embryonic Stem Cells by Inhibition of REST-MiR-21 Regulatory Pathway.

BACKGROUND/AIMS: Puerarin shows a wide range of biological activities, including affecting the cardiac differentiation from murine embryonic stem (mES) cells. However, little is known about its effect and mechanism of action on the self-renewal of mES cells. This study aimed to determine the effect of puerarin on the self-renewal and pluripotency of mES cells and its underlying mechanisms. METHODS: RT-PCR and real-time PCR were used to detect the transcripts of core transcription factors, specific markers for multiple lineages, REST and microRNA-21 (miR-21). Colony-forming assay was performed to estimate the self-renewal capacity of mES cells. Western blotting and wortmannin were employed to explore the role of PI3K/Akt signaling pathway in the inhibitory action of puerarin on REST transcript. Transfected mES cells with antagomir21 were used to confirm the role of miR-21 in the action of puerarin on cell self-renewal. RESULTS: Puerarin significantly decreased the percentage of the self-renewal colonies, and suppressed the transcripts of Oct4, Nanog, Sox2, c-Myc and REST. Besides, PECAM, NCAM and miR-21 were up-regulated both under the self-renewal conditions and at day 4 of differentiation. The PI3K inhibitor wortmannin successfully reversed the mRNA expression changes of REST, Nanog and Sox2. Transfection of antagomir21 efficiently reversed the effects of puerarin on mES cells self-renewal. CONCLUSION: Inhibition of REST-miR-21 regulatory pathway may be the key mechanism of puerarin-induced suppression of mES cells self-renewal.

[Acquirement and evaluation of murine ventricular extracellular matrix].

Cardiac extracellular matrix (ECM), generated from the process of decellularization, has been widely considered as an ideal source of biological scaffolds. However, current ECM preparations are generally difficult to be applied to generate cardiac tissue. Our research was aimed to improve decellularization protocols to prepare cardiac ECM slices. Adult murine ventricular tissues were embedded in low melting agarose and cut into 300 µm slices, and then were divided randomly into three groups: normal cardiac tissue, SDS treated group (0.1% SDS) and SDS+Triton X-100 treated group (0.1% SDS+0.5% Triton X-100). Total RNA content and protein content quantification, HE staining and immunostaining were used to evaluate the removal of cell components and preservation of vital ECM components. Furthermore, murine embryonic stem cell-derived cardiomyocytes (mES-CMs) and mouse embryonic fibroblasts (MEFs) were co-cultured with ECM slices to evaluate biocompatibility. The relative residual RNA and protein contents of ECM slices significantly decreased after decellularization. HE staining showed that SDS+Triton X-100 treatment better destroyed cellular structure and removed nuclei of ECM slices, compared with SDS treatment. Immunostaining showed that collagen IV and laminin were better preserved and presented better similarity to original cardiac tissue in ECM slices acquired by SDS+Triton X-100 treatment. However, collagen IV and laminin were significantly decreased and arranged disorderly in SDS treated group. We observed effective survival (≥ 12 days) of MEFs and mES-CMs on ECM slices acquired by SDS+Triton X-100 treatment, and signs of integration, whereas those signs were not found in SDS treated group. We concluded that, compared with traditional SDS method, new combined protocol (SDS+Triton X-100) generated ECM slices with better component and structural preservation, as well as better biocompatibility.

Puerarin facilitates T-tubule development of murine embryonic stem cell-derived cardiomyocytes.

AIMS: The embryonic stem cell-derived cardiomyocytes (ES-CM) is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. METHODS: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules) was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. RESULTS: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. CONCLUSION: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

[Progress on PI3K/Akt signaling pathway regulating self-renewal and pluripotency of embryonic stem cells].

The phosphatidylinositol 3-kinase (PI3K) and its downstream target protein kinase B (Akt/PKB) can be activated by a variety of extracellular and intracellular signals. They are important signaling molecules and key survival factors involved in cell proliferation, differentiation, apoptosis and other cellular processes. Recently, many reports demonstrate that type I PI3K/Akt signaling pathway plays an important role in maintenance of self-renewal and pluripotency of embryonic stem (ES) cells. Further studies with regard to the self-renewal and pluripotency of ES cells and underlying molecular mechanisms are crucial to its application in cell replacement therapy, regenerative medicine and tissue engineering. The present review focuses on the recent progress on the mediation of PI3K/Akt signaling pathway on the maintenance of self-renewal and pluripotency of ES cells.

Effects of puerarin on cardiac differentiation and ventricular specialization of murine embryonic stem cells.

AIMS: It is important to screen and identify chemical compounds to improve the efficiency of cardiac differentiation and specialization of embryonic stem (ES) cells. The objective of this study was to investigate the effect of puerarin, a natural phytoestrogen, on the in vitro cardiac differentiation and ventricular specialization of murine ES cells. METHODS: Cardiac differentiation of murine ES cells was performed by embryoid body (EB)-based differentiation method. Quantitative RT-PCR, flow cytometry and immunofluorescence were employed to identify cardiomyocytes (CMs) derived from murine ES cells (mES-CMs). Patch clamp was used to study the electrophysiological properties of CMs. RESULTS: We found that continuous puerarin treatment significantly increased the population of ES-CMs which express typical cardiac markers and are electrophysiological intact. Puerarin treatment shifted the cardiac phenotype from pacemaker-like cells to ventricular-like cells, which were Mlc2v-positive and present typical ventricular-like AP. Puerarin up-regulated transcripts involved in cardiac differentiation and ventricular specialization of ES cells. CONCLUSION: Our results suggest that puerarin promotes cardiac differentiation, and significantly enhances the specialization of mES cells into ventricular-like CMs. Puerarin may be used to increase the yield of ventricular mES-CMs during in vitro differentiation.

Coculture of embryonic ventricular myocytes and mouse embryonic stem cell enhance intercellular signaling by upregulation of connexin43.

BACKGROUND: Stem cell therapy has been proposed as a potential treatment strategy for ischemic cardiomyopathy in recent years. A variety of stem cells or stem cell-derived cells can potentially be used for transplantation. Despite improved cardiac function after treatment, one of the major problems is the poor integration between host and donor cells which can lead to post-transplantation arrhythmia and poor long-term outcome. METHODS: In the present study, we cocultured murine embryonic stem cells (mES) with murine embryonic ventricular myocytes (mEVs) in hanging drops to assess the cellular interaction and function of mES-derived cardiomyocytes under these conditions. RESULTS: We found that when mEVs are added to a culture system of embryonic stem cells, the number of spontaneously beating areas in embryoid bodies (EBs) increases, intercellular gap junction communication is enhanced by upregulation of Cx43 expression at the mid-developmental stage and Cx43 is distributed more orderly between cardiomyocytes. CONCLUSIONS: Our findings suggest mES-derived cardiomyocytes are able to form effective signaling pathways through coculture with mEVs which is important for providing more functional grafts for cardiac cell therapy by improving the integration between transplanted and host cells.

Baicalin maintains late-stage functional cardiomyocytes in embryoid bodies derived from murine embryonic stem cells.

BACKGROUND/AIMS: Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. METHODS: The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. RESULTS: Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to ß-adrenergic and muscarinic stimulations. CONCLUSION: Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells.

Properties and functions of KATP during mouse perinatal development.

BACKGROUND: Prevailing data suggest that ATP-sensitive potassium channels (K(ATP)) contribute to a surprising resistance to hypoxia in mammalian embryos, thus we aimed to characterize the developmental changes of K(ATP) channels in murine fetal ventricular cardiomyocytes. METHODS: Patch clamp was applied to investigate the functions of K(ATP). RT-PCR, Western blot were used to further characterize the molecular properties of K(ATP) channels. RESULTS: Similar K(ATP) current density was detected in ventricular cardiomyocytes of late development stage (LDS) and early development stage (EDS). Molecular-biological study revealed the upregulation of Kir6.1/SUR2A in membrane and Kir6.2 remained constant during development. Kir6.1, Kir6.2, and SUR1 were detectable in the mitochondria without marked difference between EDS and LDS. Acute hypoxia-ischemia led to cessation of APs in 62.5% of tested EDS cells and no APs cessation was observed in LDS cells. SarcK(ATP) blocker glibenclamide rescued 47% of EDS cells but converted 42.8% of LDS cells to APs cessations under hypoxia-ischemic condition. MitoK(ATP) blocker 5-HD did not significantly influence the response to acute hypoxia-ischemia at either EDS or LDS. In summary, sarcK(ATP) played distinct functional roles under acute hypoxia-ischemic condition in EDS and LDS fetal ventricular cardiomyocytes, with developmental changes in sarcK(ATP) subunits. MitoK(ATP) were not significantly involved in the response of fetal cardiomyocytes to acute hypoxia-ischemia and no developmental changes of K(ATP) subunits were found in mitochondria.

Adenylyl cyclase/cAMP-PKA-mediated phosphorylation of basal L-type Ca(2+) channels in mouse embryonic ventricular myocytes.

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of ß-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (I(Ca,L)) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca(2+) channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5-E11.5) than late developmental stage (LDS, E16.5-E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, I(Ca,L) increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca(2+) influx via LTCCs on AC activity, leading to the inability to inhibit AC.

Molecular and functional changes in voltage-gated Na⁺ channels in cardiomyocytes during mouse embryogenesis.

BACKGROUND: Embryonic cardiomyocytes undergo profound changes in their electrophysiological properties during development. However, the molecular and functional changes in Na⁺ channel during cardiogenesis are not yet fully explained. METHODS AND RESULTS: To study the functional changes in the Na⁺ channel during cardiogenesis, Na⁺ currents were recorded in the early (EDS) and late (LDS) developmental stages of cardiomyocytes in embryonic mice. Compared with EDS myocytes, LDS myocytes exhibited a larger peak current density, a more negative shift in the voltage of half inactivation, a larger fast inactivation component and a smaller slow inactivation component, and smaller time constants for recovery from inactivation. Additionally, multiple Na⁺ channel α-subunits (Nav 1.1-1.6) and ß-subunits (Nav ß1-ß3) of mouse embryos were investigated. Transcripts of Nav 1.1-1.3 were absent or present at very low levels in embryonic hearts. Transcripts encoding Nav 1.4-1.6 and Nav ß1-ß3 increased during embryogenesis. Data on the sensitivity of total Na⁺ currents to tetrodotoxin (TTX) showed that TTX-resistant Nav 1.5 is the predominant isoform expressed in the heart of the mouse embryo. CONCLUSIONS: The results indicate that significant changes in the functional properties of Na⁺ channels develop in the cardiomyocytes of the mouse embryo, and that different Na⁺ channel subunit genes are strongly regulated during embryogenesis, which further support a physiological role for voltage-gated Na⁺ channels during heart development.

Fibroblasts support functional integration of purified embryonic stem cell-derived cardiomyocytes into avital myocardial tissue.

Transplantation of purified pluripotent stem cell-derived cardiomyocytes into damaged myocardium might become a therapy to improve contractile function after myocardial infarction. However, engraftment remains problematic. Aim of this study was to investigate whether murine embryonic fibroblasts (MEFs) support the functional integration of purified embryonic stem cell-derived cardiomyocytes (ES-CMs). Neonatal murine ventricular tissue slices were subjected to oxygen and glucose deprivation to simulate irreversible ischemia. Vital tissue slices served as control. Vital and avital tissue slices were cultured with or without MEFs before coculturing with clusters of puromycin-selected ES-CMs. Integration of ES-CM clusters was assessed morphologically, motility by long-term microscopy, and functional integration by isometric force measurements. We observed a good morphological integration into vital but a poor integration into avital slices. Adding MEFs improved morphological integration into irreversibly damaged slices and enabled purified ES-CMs to migrate and to confer force. We conclude that noncardiomyocytes like MEFs support morphological integration and force transmission of purified ES-CMs by enabling adhesion and migration.

Functional characterization of inward rectifier potassium ion channel in murine fetal ventricular cardiomyocytes.

AIMS: Previous studies have shown the dramatic changes in electrical properties of murine fetal cardiomyocytes, while details on inward rectifier potassium current (IK1) are still seldom discussed. Thus we aimed to characterize the functional expression and functional role of IK1 in murine fetal ventricular cardiomyocytes. METHODS: Whole cell patch clamp was applied to investigate the electrophysiological properties of IK1. Quantitative real-time PCR, western blotting and double-label immunofluorescence were further utilized to find out the molecular basis of IK1. RESULTS: Compared to early developmental stage (EDS), IK1 at late developmental stage (LDS) displayed higher current density, stronger rectifier property and faster activation kinetics. It was paralleled with the downregulation of Kir2.3 and the upregulation of Kir2.1/Kir2.2. IK1 contributed to maintain the maximum diastolic potential (MDP), late repolarization phase (LRP) as well as the action potential duration (APD). However, the contribution to MDP and velocity of LRP did not change significantly with maturation. CONCLUSIONS: During fetal development, the switch of IK1 subtypes from Kir2.1/Kir2.3 to Kir2.1 resulted in the dramatic changes in IK1 electrophysiological properties.

WITHDRAWN: Capsaicin inhibits beta-adrenergically-activated Na(+) current through endogenous calcineurin in cardiac myocytes.

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Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells.

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC-CMs) and iPS cells (iPS-CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5-7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force-frequency relationship (FFR), effects of Ca(2+), Ni(2+), nifedipine, ryanodine, beta-adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC-CMs and iPS-CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca(2+) and decreased with nifedipine. Force was reduced by Ni(2+). Isoproterenol (1 microM) increased the force most pronounced in embryonic tissue (207+/-31%, n=7; ESC-CMs: 123+/-5%, n=4; iPS-CMs: 120+/-4%, n=8). EC(50) values were similar. Contractile properties of iPS-CMs and ESC-CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the beta-adrenergic response of iPS-CMs and ESC-CMs.

Cardiac myocytes derived from murine reprogrammed fibroblasts: intact hormonal regulation, cardiac ion channel expression and development of contractility.

AIMS: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. METHODS AND RESULTS: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to beta-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of I(Na), I(f), I(NCX), and I(CaL), respectively, and suggested their involvement in cardiac pacemaking, with I(CaL) being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to beta-adrenergic stimulation. CONCLUSIONS: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty.

Neonatal murine heart slices. A robust model to study ventricular isometric contractions.

BACKGROUND/AIMS: Cardiac function is increasingly studied using murine models. However, current multicellular preparations to investigate contractile properties have substantial technical and biological limitations and are especially difficult to apply to the developing murine heart. METHODS: Newborn murine hearts were cut with a vibratome into viable tissue slices. The structural and functional integrity of the tissue was shown by histology, ATP content and sharp electrode recordings. RESULTS: Within the first 48 hours after slicing structure remained intact without induction of apoptosis. ATP concentrations and action potential parameters were comparable to those of physiological tissue. Isometric force measurements demonstrated a physiological force-frequency relationship with a ;primary-phase' negative force-frequency relationship up to 1-2 Hz and a ;secondary-phase' positive force-frequency relationship up to 8 Hz. (-)-Isoproterenol (10(-6) mol/l) increased active force to 251 +/- 35% (n=15) of baseline values and shortened relaxation times indicating a preserved beta-adrenergic regulation of contraction. Changes of the force-frequency relationship after application of ryanodine and nifedipine indicated functionality of calcium release from the sarcoplasmic reticulum and of L-type calcium channels. CONCLUSION: Generation of viable, physiological intact ventricular slices from neonatal hearts is feasible and provides a robust model to study loaded contractions.