Abstract
Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.
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Introduction
Establishment of embryonic stem cell (ESC)-like cells (also know as induced pluripotent stem cells or iPSCs) by the reprogramming of adult somatic cells with a few defined transcription factors provides a fascinating route to generate patient-specific pluripotent cells as disease models and drug-testing systems1,2,3. Improvement of cardiac function by the transplantation of iPSC-derived cardiomyocytes (iPS-CMs) after myocardial infarction in animal models4 suggests a potential of using iPSCs in patient-specific cardiac regeneration5,6. However, to realize these application potentials, establishment of a highly efficient and easily practicable differentiation system is one of the prerequisites.
Cardiogenesis is a well-organized process tightly regulated by key developmental signals and extracellular microenvironment7,8. Although cardiomyocytes are successfully generated from mouse (m)9,10 and human (h)11,12 iPSCs in vitro, the cardiac differentiation efficiency remains very low5. Several attractive approaches focusing on the manipulation of critical signaling pathways to improve the cardiac differentiation efficiency of iPSCs have been reported currently13,14,15, while little is known about the contribution of manipulating extracellular microenvironments to the process of cardiac differentiation from iPSCs.
Another important obstacle hampering the utilization of iPSCs is the high interline variability in cardiac differentiation efficiency11,12,16, with some of the lines even showing no cardiac differentiation properties in vitro17. Therefore, a highly efficient and universal system must be developed to overcome or minimize such variations before the extensive use of iPSCs.
In addition, iPS-CMs have been proved to be less mature than those from ESCs or fetal hearts, reflected by the delayed development of sarcoplasmic reticulum and lower responses to β-adrenergic stimulus44.
To determine the apoptosis status of the cells, TUNEL staining was performed with the in situ Cell Death Detection kit (Roche, Mannheim, Germany) according to the manufacturer's instruction. Annexin V-PI double-stainings performed with PI (0.5 μg/ml) and APC-labeled Annexin V antibody (1:20; BD Biosciences) were further used to evaluate the apoptosis and necrosis levels. Cells were analyzed and quantified by flow cytometry.
Whole cell patch clamp
Whole cell patch clamps using EPC-10 amplifier (Heka Electronics, Bellmore, NY, USA) in current clamp mode were used to record APs in spontaneously beating iPS-CMs following the method described previously43. For AP recording, the pipette electrode (2∼6 MΩ) were filled with a solution containing (mmol/l): 50 KCl, 80 K-Asparate, 5 MgCl2, 5 EGTA, 10 Hepes, 5 Na2ATP (pH 7.2 adjusted with KOH); the extracellular bathing solution containing (mmol/l): 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 10.0 glucose and 10.0 HEPES (pH 7.4, adjusted with NaOH). The glass coverslips containing the cells were placed onto a temperature-controlled (35 °C) recording chamber and perfused continuously with extracellular solution.
Measurement of Ca2+ transients
Isolated mouse iPS-CMs were loaded with 5 μmol/l fura-2 AM and 0.45% pluronic F-127 (Molecular Probes, Eugene, OR, USA) for 10 min and washed in extracellular solution for 15 min at 35 °C room temperature. The cells were perfused continuously with extracellular solution at 35 °C. Fluorescence signals of fura-2 were detected by a Fluorescence System (IonOptix, Milton, MA). After subtraction of background fluorescence, the 340- to 380-nm fluorescence ratio (R) was recorded and analyzed by IonWizard 6.0 software (IonOptix).
Immunoblot analysis
Immunoblot analyses were performed according to the protocol described previously45. Protein samples were size fractionated by SDS-polyacrylamide gel electrophoresis and the separated proteins were electrophoretically transferred to polyvinylindene difluoride membranes (Bio-Rad, Hercules, CA, USA). Then the membrane was incubated with primary antibodies against p-ERK1/2 (1:1 000; Santa Cruz Biotechnology), total ERK1/2 (1:1 000; Cell Signaling), RyR2 (1:1 000; Abcam), SERCA2 (1:1 000; Santa Cruz Biotechnology), Phospholamban (1:2 000; Millipore), Connexin43 (1:500; Invitrogen), and GAPDH (1:1 000; Santa Cruz Biotechnology). Horseradish peroxidase-linked anti-rabbit (1:4 000; Santa Cruz Biotechnology) or anti-mouse antibodies (1:4 000; Sigma) were used as secondary antibodies.
Statistical analysis
Data were presented as means ± SEM. Statistical significance of differences was estimated by one way ANOVA or Student's t test by SigmaStat 3.5 software (Sigma). P < 0.05 was considered significant.
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Acknowledgements
This study was supported by grants from the National Basic Research Program of China (2011CB965300, 2009CB941100, 2010CB945600), the National Natural Science Foundation of China (31030050), Strategic Priority Research Program of CAS (XDA01000000), Science and Technology Committee of Shanghai Municipality (08DJ1400501), National Science and Technology Project of China (2012ZX09501-001-001), and Sanofi-Aventis Recherche & Développement-SIBS funding. We thank Dr Duanqing Pei (Guangzhou Institutes of Biomedicine and Health, China) for kindly providing the miPSC lines iPS-C5 and iPS-C12.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Table S1
iPSC lines used in this study (PDF 88 kb)
Supplementary information, Table S2
Action potential properties of iPS-CMs with or without AA-treatment (PDF 21 kb)
Supplementary information, Table S3
Primers used for RT-PCRs (PDF 99 kb)
Supplementary information, Table S4
Primers used for Q-PCRs (PDF 80 kb)
Supplementary information, Table S5
Target sequences of siRNAs used for specific gene knockdown experiments (PDF 80 kb)
Supplementary information, Figure S1
Systematic screening of suitable cardiomyocyte inducers of iPSCs. (PDF 53 kb)
Supplementary information, Figure S2
Characteristics of undifferentiated miPSCs. (PDF 247 kb)
Supplementary information, Figure S3
AA enhances cardiac differentiation of iPSCs in auto-aggregated EBs and serum-free cultivated EBs. (PDF 113 kb)
Supplementary information, Figure S4
AA increases the proportion of smooth muscle, endothelial but not hematopoietic progenitor cells. (PDF 68 kb)
Supplementary information, Figure S5
Ca2+ handling properties of iPS-CMs induced with or without AA. (PDF 103 kb)
Supplementary information, Figure S6
AA-promoted cardiogenesis is independent of its antioxidative property. (PDF 55 kb)
Supplementary information, Figure S7
Cardiomyocyte-promoting effect of AA is partially abolished by siRNA-mediated stable silencing of type I and IV collagen (Col I and ColIV). (PDF 77 kb)
Supplementary information, Figure S8
AA does not influence the apoptotic statues of differentiating iPSCs. (PDF 54 kb)
Supplementary information, Figure S9
AA does not affect the differentiation potential of sorted cardiac progenitors (CPCs). (PDF 73 kb)
Supplementary information, Figure S10
AA activates ERK signaling in a collagen synthesis-dependent manner. (PDF 110 kb)
Supplementary information, Movie S1
A control mouse EB of iPS-3F showing the contracting area at differentiation day 10. (MPG 1386 kb)
Supplementary information, Movie S2
An ascorbic acid-treated mouse EB of iPS-3F showing the contracting area at differentiation day 10. (MPG 2000 kb)
Supplementary information, Movie S3
A control mouse EB of iPS-4F showing the contracting area at differentiation day 10. (MPG 1574 kb)
Supplementary information, Movie S4
An ascorbic acid-treated mouse EB of iPS-4F showing the contracting area at differentiation day 10. (MPG 1810 kb)
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Cao, N., Liu, Z., Chen, Z. et al. Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells. Cell Res 22, 219–236 (2012). https://doi.org/10.1038/cr.2011.195
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DOI: https://doi.org/10.1038/cr.2011.195
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