Jiaoya Xi

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 Ca2+, Ni2+, nifedipine, ryanodine, β‐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 Ca2+ and decreased with nifedipine. Force was reduced by Ni2+. Isoproterenol (1 µM) increased the force most pronounced in embryonic tissue (207±31%, n=7;ESC‐CMs: 123±5%, n=4; iPS‐CMs: 120 ±4%, n=8). EC50 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 β‐adrenergic response of iPS‐CMs and ESC‐CMs.—Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel, M., Šarić, T., Wernig, M., Jaenisch, R., Brockmeier, K., Hescheler, J., Pillekamp, F. Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells. FASEB J. 24, 2739–2751 (2010). www.fasebj.org

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 β-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 INa, If, INCX, and ICaL, respectively, and suggested their involvement in cardiac pacemaking, with ICaL being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to β-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.

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.

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.

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.

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 β-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.

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.

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.