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Ca2+ Activated K Channels-New Tools to Induce Cardiac Commitment from Pluripotent Stem Cells in Mice and Men

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Abbreviations

EB:

Embryoid body

EBIO:

1-Ethyl-2-benzimidazolinone

ESC:

Embryonic stem cell

Hcn4:

Hyperpolarization-activated cyclic nucleotide-gated channel 4

iPSC:

Induced pluripotent stem cell

MEF:

Mouse embryonic fibroblast

NGFP:

Nanog-EGFP-iPSCs

SKCa:

Calcium activated potassium channel

TIPS:

Tail-tip murine fibroblast-derived iPS-cells

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Acknowledgements

The authors would like to thank Ralf Köhntop, Clair Weidgang and Sabine Seltenheim for excellent technical assistance. Alexander Kleger is supported by a fellowship provided by the Medical Faculty of Ulm University (Bausteinprogramm, L.SBR.0011). Main part of this study was funded by the Deutsche Forschungsgemeinschaft (DFG) (S.L., T.B. SFB 497) and (DFG BO1718/4-1) to S.L. and T.B. Part of this work was funded by a grant of the DFG Priority Program 1356 to M.Z. and a grant from the DFG to C.B. (BR-2891/4-1). M.W. is supported by “Deutsche Stiftung für Herzforschung” Projekt Nr. F/08/07 (BIOCARD). B. F. was supported by a grant of the DFG (FL 276/3-3) and a grant of the EU FP7 consortium CardioCell No223372.

Conflict of Interest Disclosures

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Author information

Authors and Affiliations

  1. Department of Internal Medicine I, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany

    Martin Müller, Michael Tischendorf, Götz von Wichert & Alexander Kleger

  2. Institute for Anatomy and Cell Biology, Ulm University, Albert-Einstein Allee 11, 89081, Ulm, Germany

    Marianne Stockmann, Leonhard Linta, Tobias M. Boeckers & Stefan Liebau

  3. Institute of Physiology I, Life and Brain Center, University of Bonn, Bonn, Germany

    Daniela Malan & Bernd K. Fleischmann

  4. Department of Internal Medicine I, University of Jena, Jena, Germany

    Anne Wolheim, Stephan Latz & Maria Wartenberg

  5. Institute of Molecular Medicine and Max-Planck-Research Department on Stem Cell Aging, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany

    Sarah-Fee Katz & Alexander Kleger

  6. Department of Cell Biology, Institute for Biomedical Engineering, Aachen, Germany

    Qiong Lin & Martin Zenke

  7. Institute for Physiological Chemistry, Ulm University, Ulm, Germany

    Cornelia Brunner

  8. Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

    Anna M. Wobus

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  1. Martin Müller
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Corresponding authors

Correspondence to Stefan Liebau or Alexander Kleger.

Additional information

Martin Müller and Marianne Stockmann contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure 1

FACS controls. (A-B) Unstained negative controls for control (A) and EBIO treated (B) CRG8 cultures at day 4+10+2 for FACS analyses were performed with cells labeled with secondary antibodies. No primary antibodies were added. (AVI 8.46 mb)

Supplementary Figure 2

Morphology of different murine iPSCs and expression of pluripotency markers. (A) Nanog-GFP (NGFP) cells exhibit typical ES cell colony morphology. Nanog positivity is shown via the endogenous EGFP reporter signal (green) (B) NGFP cells stain positive for SSEA1 (red). Nanog-EGFP (green). (C) TiPS cells exhibit typical ES cell colony morphology and (D) stain positive for SSEA1 (red). Scale bars, 10 μm. (AVI 8.09 mb)

Supplementary Figure 3

TiPS cells express SKCas on protein level. Immunocytochemistry of SKCa proteins. 48 hours after plating, undifferentiated NGFP cells were co-stained for F-actin (green), the indicated SKCa subtype (red); Scale bars, 5μm. (AVI 8.38 mb)

Supplementary Figure 4

SKCa-activation inhibits proliferation of murine iPSCs. (A) Monolayer assay of iPSCs. Undifferentiated iPSCs were cultured in leukemia inhibitory factor-containing medium. After seeding equal numbers of cells (5000 cells/cm2), EBIO was added and replaced every other day. (B-C) Proliferation/viability assay of EBIO-treated (EBIO) and untreated (Con) iPSCs. (B, NGFP and C, TiPS). (D) SKCa-activation induces G1 arrest in NGFP iPSCs cells and diminishes cells in S phase. Representative cell-cycle distribution blots are shown. (E) Quantification of cell cycle analysis data. (PDF 35 kb)

Supplementary Figure 5

SKCa-activation induces cardiac pacemaker-like cells from mouse iPSCs. NGFP (left column) and TIPS cells (right column) (A) 4-day-old (4d) EBs were plated and assayed as indicated. EB outgrowths were analyzed for mRNA levels. qPCR for Mesp1 (B) and Nkx2.5 (C). (D-E) Expression of early and mature pacemaker-like cell genes in control and EBIO-treated variants shown for Isl1 (D) and Cx30.2 (E) (n = 3). (PDF 29 kb)

Supplementary Figure 6

SKCa-activation induces cardiac pacemaker-like cells from mouse iPSCs (TiPS cells). (A) 4-day-old (4d) EBs were plated and assayed as indicated. EBIO was withdrawn on day 10. Culture days after EBIO removal are refereed as day 4+10. EB outgrowths were analyzed for mRNA levels and immunocytochemistry. (B-C) Induction of the cardiac marker gene Myh6 (B) and pacemaker gene (C, Hcn4) in TIPS cells. (D) Induction of HCN4 protein expression upon SKCa-activation and enhanced cardiac differentiation upon SKCa-activation in TiPS cells. Immunocytochemistry for HCN4 (red) and α-actinin (green) at day 4+10 cultures. Scale bars, 20 μm. (PDF 327 kb)

Supplementary Figure 7

SKCa-activation induces cardiac differentiation and pacemaker specification under virtually serum-free conditions in murine iPSCs. (A) 4-day-old (4d) EBs derived from NGFP iPSCs were plated and assayed as indicated. EBIO was withdrawn on day 10. Culture days after EBIO removal are refereed as day 4+10. (B-C) Induction of cardiac and pacemaker genes after SKCa-activation. qPCR for Myh6 (B) and Hcn4 (C) expression (n = 4). (D) Induction of the cardiac protein α-actinin and the pacemaker protein Hcn4 after SKCa-activation by EBIO-treatment. Immunocytochemistry for α-actinin (green) and Hcn4 (red) at indicated time points (n = 3). Scale bars, 20 μm. (PDF 192 kb)

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Müller, M., Stockmann, M., Malan, D. et al. Ca2+ Activated K Channels-New Tools to Induce Cardiac Commitment from Pluripotent Stem Cells in Mice and Men. Stem Cell Rev and Rep 8, 720–740 (2012). https://doi.org/10.1007/s12015-011-9324-9

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