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Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling.

Kadari A, Mekala S, Wagner N, Malan D, Köth J, Doll K, Stappert L, Eckert D, Peitz M, Matthes J, Sasse P, Herzig S, Brüstle O, Ergün S, Edenhofer F - Stem Cell Rev (2015)

Bottom Line: In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T.Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis.We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, University of Würzburg, 97070, Würzburg, Germany.

ABSTRACT
Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

No MeSH data available.


Characterization of human iPSC (del-AR1034ZIMA 001) derived cardiomyocytes. a Scheme of efficient cardiac differentiation of human iPSC with combination of strong cardiac induction in early phase and cardiac enrichment in late phase. b RT-PCR analysis for mesendoderm, mesoderm, and cardiac specific gene expression c Immunohistochemical characterization of hiPS-derived cardiomyocytes using antibody against alpha-actinin (top) and cardiac troponin T (bottom). Scale bar: 40 μm. d Action potential recorded from a ventricular like cardiomyocyte. e Typical activation of voltage dependent inward and outward currents following a ramp protocol in voltage clamp (-100 to +60 mV in 250 ms). f Representative whole cell calcium current recording (2 mM extracellular Ca2+). Cells were depolarized from a holding potential of -80 to -40 mV for 45 ms in order to inactivate sodium channels. This prepulse was followed by test voltages ranging from -40 to +50 mV in 10 mV steps (pulse duration 150 ms). (G) Whole cell calcium current density-voltage relationship (n = 4)
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Fig3: Characterization of human iPSC (del-AR1034ZIMA 001) derived cardiomyocytes. a Scheme of efficient cardiac differentiation of human iPSC with combination of strong cardiac induction in early phase and cardiac enrichment in late phase. b RT-PCR analysis for mesendoderm, mesoderm, and cardiac specific gene expression c Immunohistochemical characterization of hiPS-derived cardiomyocytes using antibody against alpha-actinin (top) and cardiac troponin T (bottom). Scale bar: 40 μm. d Action potential recorded from a ventricular like cardiomyocyte. e Typical activation of voltage dependent inward and outward currents following a ramp protocol in voltage clamp (-100 to +60 mV in 250 ms). f Representative whole cell calcium current recording (2 mM extracellular Ca2+). Cells were depolarized from a holding potential of -80 to -40 mV for 45 ms in order to inactivate sodium channels. This prepulse was followed by test voltages ranging from -40 to +50 mV in 10 mV steps (pulse duration 150 ms). (G) Whole cell calcium current density-voltage relationship (n = 4)

Mentions: In conclusion our optimized protocol of cardiomyocyte differentiation from multiple human iPS lines represents a three phase protocol consisting of cardiac induction, specification and enrichment as outlined in Fig. 3a. During the induction phase iPS cells are treated with our formulation (BMP4 and CHIR) in a basal medium with insulin, which resulted in strong upregulation of the mesendodermal marker T-brachyury (Fig. 3b). Induction phase is followed by treatment with WNT inhibitors in basal medium devoid of insulin in order to achieve proper specification to cardiac mesoderm, which is confirmed by expression of early and late cardiac precursor markers ISL1 and Nkx2.5, respectively (Fig. 3b). Cells then further mature into beating cardiomyocytes expressing the mature cardiomyocyte marker cTNT (Fig. 3b). Once beating is observed cells are switched to basal medium with insulin followed by enrichment phase with basal medium devoid of glucose but supplemented with 4 mM lactate for 4 days (Fig. 3a).Fig. 3


Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling.

Kadari A, Mekala S, Wagner N, Malan D, Köth J, Doll K, Stappert L, Eckert D, Peitz M, Matthes J, Sasse P, Herzig S, Brüstle O, Ergün S, Edenhofer F - Stem Cell Rev (2015)

Characterization of human iPSC (del-AR1034ZIMA 001) derived cardiomyocytes. a Scheme of efficient cardiac differentiation of human iPSC with combination of strong cardiac induction in early phase and cardiac enrichment in late phase. b RT-PCR analysis for mesendoderm, mesoderm, and cardiac specific gene expression c Immunohistochemical characterization of hiPS-derived cardiomyocytes using antibody against alpha-actinin (top) and cardiac troponin T (bottom). Scale bar: 40 μm. d Action potential recorded from a ventricular like cardiomyocyte. e Typical activation of voltage dependent inward and outward currents following a ramp protocol in voltage clamp (-100 to +60 mV in 250 ms). f Representative whole cell calcium current recording (2 mM extracellular Ca2+). Cells were depolarized from a holding potential of -80 to -40 mV for 45 ms in order to inactivate sodium channels. This prepulse was followed by test voltages ranging from -40 to +50 mV in 10 mV steps (pulse duration 150 ms). (G) Whole cell calcium current density-voltage relationship (n = 4)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493626&req=5

Fig3: Characterization of human iPSC (del-AR1034ZIMA 001) derived cardiomyocytes. a Scheme of efficient cardiac differentiation of human iPSC with combination of strong cardiac induction in early phase and cardiac enrichment in late phase. b RT-PCR analysis for mesendoderm, mesoderm, and cardiac specific gene expression c Immunohistochemical characterization of hiPS-derived cardiomyocytes using antibody against alpha-actinin (top) and cardiac troponin T (bottom). Scale bar: 40 μm. d Action potential recorded from a ventricular like cardiomyocyte. e Typical activation of voltage dependent inward and outward currents following a ramp protocol in voltage clamp (-100 to +60 mV in 250 ms). f Representative whole cell calcium current recording (2 mM extracellular Ca2+). Cells were depolarized from a holding potential of -80 to -40 mV for 45 ms in order to inactivate sodium channels. This prepulse was followed by test voltages ranging from -40 to +50 mV in 10 mV steps (pulse duration 150 ms). (G) Whole cell calcium current density-voltage relationship (n = 4)
Mentions: In conclusion our optimized protocol of cardiomyocyte differentiation from multiple human iPS lines represents a three phase protocol consisting of cardiac induction, specification and enrichment as outlined in Fig. 3a. During the induction phase iPS cells are treated with our formulation (BMP4 and CHIR) in a basal medium with insulin, which resulted in strong upregulation of the mesendodermal marker T-brachyury (Fig. 3b). Induction phase is followed by treatment with WNT inhibitors in basal medium devoid of insulin in order to achieve proper specification to cardiac mesoderm, which is confirmed by expression of early and late cardiac precursor markers ISL1 and Nkx2.5, respectively (Fig. 3b). Cells then further mature into beating cardiomyocytes expressing the mature cardiomyocyte marker cTNT (Fig. 3b). Once beating is observed cells are switched to basal medium with insulin followed by enrichment phase with basal medium devoid of glucose but supplemented with 4 mM lactate for 4 days (Fig. 3a).Fig. 3

Bottom Line: In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T.Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis.We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology, University of Würzburg, 97070, Würzburg, Germany.

ABSTRACT
Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

No MeSH data available.