Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker

Abstract

The sinoatrial node (SAN) is the primary pacemaker of the heart and controls heart rate throughout life. Failure of SAN function due to congenital disease or aging results in slowing of the heart rate and inefficient blood circulation, a condition treated by implantation of an electronic pacemaker. The ability to produce pacemaker cells in vitro could lead to an alternative, biological pacemaker therapy in which the failing SAN is replaced through cell transplantation. Here we describe a transgene-independent method for the generation of SAN-like pacemaker cells (SANLPCs) from human pluripotent stem cells by stage-specific manipulation of developmental signaling pathways. SANLPCs are identified as NKX2-5 cardiomyocytes that express markers of the SAN lineage and display typical pacemaker action potentials, ion current profiles and chronotropic responses. When transplanted into the apex of rat hearts, SANLPCs are able to pace the host tissue, demonstrating their capacity to function as a biological pacemaker.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Identification and characterization of NKX2-5SIRPA+ SANLPCs.
Figure 2: BMP and RA signaling promote SANLPC development.
Figure 3: SANLPCs display functional characteristics of pacemaker cells.
Figure 4: SANLPCs can pace human ventricular-like cardiomyocytes in vitro.
Figure 5: SANLPCs can engraft and function as a biological pacemaker in vivo.
Figure 6: Generation and isolation of SANLPCs from non-genetically modified hPSC lines.
Figure 7: Summary of strategy used for the specification and isolation of SANLPCs and VLCMs.

Similar content being viewed by others

References

  1. Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Laflamme, M.A. et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25, 1015–1024 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Kattman, S.J. et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011).

    Article  CAS  PubMed  Google Scholar 

  4. Burridge, P.W. et al. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. He, J.Q., Ma, Y., Lee, Y., Thomson, J.A. & Kamp, T.J. Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ. Res. 93, 32–39 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Ma, J. et al. High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents. Am. J. Physiol. Heart Circ. Physiol. 301, H2006–H2017 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rosen, M.R., Brink, P.R., Cohen, I.S. & Robinson, R.B. Genes, stem cells and biological pacemakers. Cardiovasc. Res. 64, 12–23 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Li, R.A. Gene- and cell-based bio-artificial pacemaker: what basic and translational lessons have we learned? Gene Ther. 19, 588–595 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wiese, C. et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ. Res. 104, 388–397 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Mommersteeg, M.T. et al. The sinus venosus progenitors separate and diversify from the first and second heart fields early in development. Cardiovasc. Res. 87, 92–101 (2010).

    Article  CAS  PubMed  Google Scholar 

  11. Blaschke, R.J. et al. Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2 in sinoatrial and pacemaking development. Circulation 115, 1830–1838 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Espinoza-Lewis, R.A. et al. Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx2-5. Dev. Biol. 327, 376–385 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mommersteeg, M.T. et al. Molecular pathway for the localized formation of the sinoatrial node. Circ. Res. 100, 354–362 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Hoogaars, W.M. et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev. 21, 1098–1112 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sizarov, A. et al. Molecular analysis of patterning of conduction tissues in the developing human heart. Circ Arrhythm Electrophysiol 4, 532–542 (2011).

    Article  PubMed  Google Scholar 

  16. Christoffels, V.M. et al. Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18. Circ. Res. 98, 1555–1563 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Schott, J.J. et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281, 108–111 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Horsthuis, T. et al. Gene expression profiling of the forming atrioventricular node using a novel tbx3-based node-specific transgenic reporter. Circ. Res. 105, 61–69 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Christoffels, V.M. & Moorman, A.F. Development of the cardiac conduction system: why are some regions of the heart more arrhythmogenic than others? Circ Arrhythm Electrophysiol 2, 195–207 (2009).

    Article  PubMed  Google Scholar 

  20. Birket, M.J. et al. Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells. Nat. Biotechnol. 33, 970–979 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Dubois, N.C. et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat. Biotechnol. 29, 1011–1018 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Christoffels, V.M., Smits, G.J., Kispert, A. & Moorman, A.F. Development of the pacemaker tissues of the heart. Circ. Res. 106, 240–254 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Elliott, D.A. et al. NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat. Methods 8, 1037–1040 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Witty, A.D. et al. Generation of the epicardial lineage from human pluripotent stem cells. Nat. Biotechnol. 32, 1026–1035 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Piedra, M.E. & Ros, M.A. BMP signaling positively regulates Nodal expression during left right specification in the chick embryo. Development 129, 3431–3440 (2002).

    CAS  PubMed  Google Scholar 

  26. Katsu, K., Tatsumi, N., Niki, D., Yamamura, K. & Yokouchi, Y. Multi-modal effects of BMP signaling on Nodal expression in the lateral plate mesoderm during left-right axis formation in the chick embryo. Dev. Biol. 374, 71–84 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Xavier-Neto, J. et al. A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart. Development 126, 2677–2687 (1999).

    CAS  PubMed  Google Scholar 

  28. Rosenthal, N. & Xavier-Neto, J. From the bottom of the heart: anteroposterior decisions in cardiac muscle differentiation. Curr. Opin. Cell Biol. 12, 742–746 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Pildner von Steinburg, S. et al. What is the “normal” fetal heart rate? PeerJ 1, e82 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Näbauer, M., Beuckelmann, D.J., Uberfuhr, P. & Steinbeck, G. Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation 93, 168–177 (1996).

    Article  PubMed  Google Scholar 

  31. Liu, J., Laksman, Z. & Backx, P.H. The electrophysiological development of cardiomyocytes. Adv. Drug Deliv. Rev. 96, 253–273 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Marionneau, C. et al. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J. Physiol. (Lond.) 562, 223–234 (2005).

    Article  CAS  Google Scholar 

  33. Mesirca, P., Torrente, A.G. & Mangoni, M.E. Functional role of voltage gated Ca(2+) channels in heart automaticity. Front. Physiol. 6, 19 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Keren-Politansky, A., Keren, A. & Bengal, E. Neural ectoderm-secreted FGF initiates the expression of NKX2-5 in cardiac progenitors via a p38 MAPK/CREB pathway. Dev. Biol. 335, 374–384 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Barron, M., Gao, M. & Lough, J. Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative. Dev. Dyn. 218, 383–393 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Robey, T.E., Saiget, M.K., Reinecke, H. & Murry, C.E. Systems approaches to preventing transplanted cell death in cardiac repair. J. Mol. Cell. Cardiol. 45, 567–581 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Jung, J.J. et al. Programming and isolation of highly pure physiologically and pharmacologically functional sinus-nodal bodies from pluripotent stem cells. Stem Cell Reports 2, 592–605 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Ionta, V. et al. SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells, improving biological pacing ability. Stem Cell Reports 4, 129–142 (2015).

    Article  CAS  PubMed  Google Scholar 

  39. Kapoor, N., Liang, W., Marbán, E. & Cho, H.C. Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18. Nat. Biotechnol. 31, 54–62 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Hu, Y.F., Dawkins, J.F., Cho, H.C., Marbán, E. & Cingolani, E. Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block. Sci. Transl. Med. 6, 245ra94 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kehat, I. et al. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat. Biotechnol. 22, 1282–1289 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Opthof, T. The normal range and determinants of the intrinsic heart rate in man. Cardiovasc. Res. 45, 177–184 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Park, I.H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Kennedy, M., D'Souza, S.L., Lynch-Kattman, M., Schwantz, S. & Keller, G. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109, 2679–2687 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Okada, Y. (ed.) Springer Protocols Handbooks (Springer Japan, Tokyo, 2012).

  47. Liu, J., Kim, K.H., London, B., Morales, M.J. & Backx, P. H. Dissection of the voltage-activated potassium outward currents in adult mouse ventricular myocytes: l(to,f), l(to,s), I(K,slow1), I(K,slow2), and l(ss). Basic Res. Cardio. 106, 189–204 (2011).

    Article  CAS  Google Scholar 

  48. Nicklas, J.A. & Buel, E. Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples. J. Forensic Sci. 48, 936–944 (2003).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank members of the Keller laboratory for experimental advice and critical comments on the manuscript, M. Gagliardi for assistance with large-scale tissue culture experiments for the in vivo rat heart transplantation studies, A. Elefanty and E. Stanley (Monash University, Victoria, AU) for providing the HES3-NKX2-5gfp/w reporter cell line, G. Daley (Harvard Medical School, Boston) for providing the MSC-iPS1 cell line, R. Hamilton (Sick Kids, Toronto, ON, Canada) for assistance in obtaining fetal tissue samples, R. Li (UHN, Toronto, ON, Canada) for use of the MEA apparatus and the Sick Kids/UHN Flow Cytometry Facility for assistance with cell sorting. This work was supported by grants from Canadian Institute of Health Research (CIHR, MOP-84524 to G.M.K. and MOP-83453 to P.H.B.) and the European Research Council Ideas-Program (ERC, ERC-2010-StG-260830-Cardio-iPS to L.G.) as well as by donors to Toronto General and Western Hospital Foundation including Joyce Mason and the supporters of the Technion-UHN International Centre for Cardiovascular Innovation collaboration. S.I.P. was supported by a Banting postdoctoral fellowship.

Author information

Authors and Affiliations

Authors

Contributions

S.I.P. and G.M.K. designed the study and wrote the paper. S.I.P., U.N., J.L. and L.O. designed and performed experiments and analyzed data. P.H.B. and L.G. provided conceptual advice, discussed results and edited the manuscript.

Corresponding author

Correspondence to Gordon M Keller.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of NKX2-5-SIRPA+ SANLPCs.

(a) Representative flow cytometric analyses showing the proportion of PDGFRα+KDR+ mesodermal cells at day 4 of differentiation and cTNT+ cells at day 20. (b) qRT-PCR analysis of mesoderm, pan-cardiomyocyte and pacemaker genes in whole embryoid body populations at the indicated times of differentiation. Values represent expression levels relative to the housekeeping gene TBP (n = 4). (c) Representative flow cytometric analyses of cTNT expression in day 20 NKX2-5+SIRPA+ and NKX2-5-SIRPA+ sorted populations. (d) Representative flow cytometric analyses of MLC2V expression in the day 20 NKX2-5+SIRPA+ and NKX2-5-SIRPA+ sorted populations, bar graph indicates average proportion of MLC2V+cTNT+ cells from multiple experiments (n = 3). (e,f) Photomicrographs showing immunostaining of (e) the atrial/posterior cardiomyocyte marker COUP-TFII and (f) the pacemaker transcription factor TBX3 in NKX2-5+SIRPA+ and NKX2-5-SIRPA+ cells isolated from day 20 cultures. Cells were counterstained with cTNT to visualize all cardiomyocytes and DAPI to visualize all cells. Scale bars represent 100 μm. Error bars represent s.e.m. t-test: **P < 0.01 vs Nkx2-5+SIRPA+ cells.

Supplementary Figure 2 Characterization of NKX2-5-SIRPA+ SANLPCs after 30 days of additional culture.

(a-d) qRT-PCR analysis of the expression levels of: pan-cardiomyocyte and ventricular cardiomyocyte (a), SAN pacemaker (b), AVN pacemaker (c) and cardiac ion channel (d) genes in day 20 NKX2-5+SIRPA+ and NKX2-5-SIRPA+ sorted cells cultured for an additional 30 days (n = 4). Values represent expression levels relative to housekeeping gene TBP. (e) Beating rate of day 20 NKX2-5+SIRPA+ and NKX2-5-SIRPA+ sorted cells following additional 30 days of culture (n = 5). (f) Photomicrographs showing immunostaining of cTNT in day 20 NKX2-5+SIRPA+ and NKX2-5-SIRPA+ sorted cells following additional 30 days of culture. NKX2-5:GFP expression was visualized to demonstrate that the cardiomyocytes that were isolated based on their lack of NKX2-5 expression (NKX2-5-SIRPA+) remain NKX2-5 negative following the additional 30 day culture period. Scale bars represent 100 μm. (g) Representative flow cytometric analyses showing the proportion of NKX2-5:GFP+cTNT+ cells in the day 20 NKX2-5+SIRPA+ and NKX2-5-SIRPA+ populations following additional 30 days of culture. Error bars represent s.e.m. t-test: *P < 0.05, **P < 0.01 vs Nkx2-5+SIRPA+ cells.

Supplementary Figure 3 BMP signaling promotes SANLPC development.

(a) The total number of cTNT+ cells and the proportion of NKX2-5+cTNT+ and NKX2-5-cTNT+ cells in day 20 populations generated from mesoderm induced with the indicated amounts of BMP and ACTA between days 1-3 of differentiation (n = 3). (b) Flow cytometric analyses showing the proportion of NKX2-5+cTNT+ and NKX2-5-cTNT+ cells in day 20 populations generated with and without (Control) the addition of the TGFβ/Activin/Nodal inhibitor SB-431542 (SB) (5.4 μM) between days 3-5 from mesoderm that was induced with 3 ng/ml BMP4 and 2 ng/ml ACTA (n = 4). (c-e) Flow cytometric analyses of the proportion of NKX2-5+cTNT+ and NKX2-5-cTNT+ cells in day 20 populations that were specified by addition of the indicated amounts of dorsomorphin or BMP4 together with SB (5.4 μM) between days 3-5 from mesoderm that was induced with either 3 ng/ml BMP4, 2 ng/ml ACTA (3B/2A) (n = 4) (c) or 5 ng/ml BMP4, 4 ng/ml ACTA (5B/4A) (n = 3) (d) or 10 ng/ml BMP4, 6 ng/ml ACTA (10B/6A) (n = 4) (e). t-test: *P < 0.05, **P < 0.01 vs Nkx2-5-cTNT+ cells at endogenous (E) BMP4 levels. (f) Total number of cells in day 20 populations specified from mesoderm by addition of the indicated amounts of dorsomorphin or BMP4 together with SB (5.4 μM) between days 3-5 (n = 4). (g) qRT-PCR analysis of TBX18 expression in populations at indicated time points specified from mesoderm with no additional treatment (control) or by the addition of 2.5 ng/ml BMP4 + 5.4 μM SB (BMP) (days 3-5). t-test: **P < 0.01 vs indicated sample (n = 4). (h) Quantification of TBX18+ cells in immunostained day 6 populations specified from mesoderm with no additional treatment (control) or by the addition of 2.5 ng/ml BMP4 + 5.4 μM SB (BMP) (days 3-5). t-test: **P < 0.01 vs control (n = 11 images from N = 3 cell culture replicates). (i) Photomicrograph showing immunostaining of TBX18 in day 6 populations specified from mesoderm with no additional treatment (control) or by the addition of 2.5 ng/ml BMP4 + 5.4 μM SB (BMP) (days 3-5). DAPI was used to visualize cell nuclei. Scale bars represent 100 μm. All error bars represent s.e.m. d, day; Dorso, dorsomorphin; E, endogenous

Supplementary Figure 4 RA signaling does not affect the efficiency of SANLPC development.

(a) Individual data point dot plots of the flow cytometric analyses presented as stacked bar graph in Fig. 2e showing the proportion of NKX2-5+cTNT+ and NKX2-5-cTNT+ cells at day 20 of differentiation generated from mesoderm (3 ng/ml BMP4, 2 ng/ml ACTA) treated with retinoic acid (RA) on days 2-12 (n = 4). (b) Individual data point dot plots of the flow cytometric analyses presented as stacked bar graph in Fig. 2g showing the proportion of NKX2-5+cTNT+ and NKX2-5-cTNT+ cells at day 20 of differentiation generated from mesoderm (3 ng/ml BMP4, 2 ng/ml ACTA) treated with BMP4 (2.5 ng/ml) together with SB (5.4 μM) and/or different concentrations of retinoic acid (RA) between days 3-5. t-test: **P < 0.01 vs Nkx2-5-cTNT+ cells in untreated control condition (n = 4). All error bars represent s.e.m. d, day

Supplementary Figure 5 Electrophysiological characterization of SANLPCs.

(a-c) Analysis of pacemaker funny current densities (If): (a) Current-voltage relationship for If current densities in SANLPCs and VLCMs. (b,c) Representative recordings of funny current (If) in a SANLPC (inset: voltage protocol) made at different membrane potentials in Tyrode’s solution (b) and in the presence of the If blocker Cesium (Cs+) (c) at a concentration of 2 mM. (d) Analysis of acetylcholine activated inward rectifier potassium current densities (IKACh): Current-voltage relationship for IKACh current densities in SANLPCs and VLCMs (inset: voltage protocol). (e) Analysis of sodium current densities (INa): Current-voltage relationship for INa current densities in SANLPCs and VLCMs (inset: voltage protocol). (f,g) Analysis of barium (Ba2+)-sensitive inward rectifier potassium current densities (IK1): (f) Representative recording of the barium sensitive inward rectifier potassium current component in a VLCM (inset: voltage protocol) made at different membrane potentials. (g) Right: Current-voltage relationship for barium-sensitive current densities and left: summary of maximum IK1 current densities recorded at -120 mV in VLCMs and SANLPCs. (h-l) Analysis of outward potassium current densities (IK) and dissection and quantification of the transient outward potassium current (Ito): (h) Representative recording of IK current in a SANLPC and a VLCM (inset: voltage protocol). (i) Peak IK current densities recorded in VLCMs and SANLPCs. (j) Maximum Ito current densities and (k) Ito inactivation time constant determined by curve fitting in VLCMs and SANLPCs. (l) Properties of Ito recovery from inactivation fitted with a bi-exponential function used to separate the fast and slow components of Ito as detailed in the methods. (inset: voltage protocol and parameters of recovery form inactivation determined by curve fitting). All error bars represent s.e.m. t-test: *P < 0.05 **P < 0.01 vs VLCMs.

Supplementary Figure 6 Electrophysiological characterization of SANLPCs.

(a-d) Analysis of total calcium current densities (ICa) and nickel-sensitive T-type calcium current densities (ICaT): (a,b) Representative recording of calcium currents in a SANLPC (a) and VLCM (b) made at different membrane potentials before (Control) and after the application of 100 μM Nickel (Ni2+) (inset: voltage protocol). (c,d) Current-voltage relationship for calcium current densities before (Control) and after the application of 100 μM Nickel (Ni2+) in SANLPCs (c) and VLCMs (d) (n = 6). (e,f) Dose response curve for changes in average beating rates after β-adrenergic stimulation with Isoproterenol (e) and muscarinic stimulation with Carbachol (f) (n = 10). SANLPC and VLCM aggregates were cultured on multi-electrode arrays (MEAs) and beating rates were recorded at 37°C. All error bars represent s.e.m.

Supplementary Figure 7 Confocal imaging of Connexin 43 channels shared between graft and host cardiomyocytes.

(a) Brightfield image of a rat heart 14 days after transplantation of SANLPCs. The scar tissue that developed at the injection site was used as indicator for location of the graft (white arrow). Scale bar represents 10 mm. (b-g) Photomicrographs showing immunostaining of CX43 on cryosections of rat hearts with a SANLPC transplant (b-d): (c) high magnification of boxed region in b. (d) confocal sectioning in x-y-z dimension of boxed region in c; a VLCM transplant (e-g): (f) high magnification of boxed region in e. (g) confocal sectioning in x-y-z dimension of boxed region in f. An antibody specifically recognizing human cTNT (hcTNT) was used to identify the human graft. Sections were counterstained with a pan-species cTNT antibody to mark rat and human cardiomyocytes. DAPI was used to visualize cell nuclei. White arrows indicate CX43 shared between rat cardiomyocytes. Yellow arrows indicate CX43 shared between the human graft and the rat host cardiomyocytes.

Supplementary Figure 8 Inhibition of FGF signaling represses the emergence of NKX2-5+ cardiomyocytes

(a) Individual data point dot plots of the flow cytometric analyses presented as stacked bar graph in Fig. 6b showing the proportion of of NKX2-5+cTNT+ cells and NKX2-5-cTNT+ SANLPCs in day 20 populations generated from mesoderm specified with either 20 ng/ml bFGF, no additional FGF (endogenous) or the small molecule FGF inhibitor PD173074 (480 nM) between days 4-6 in the background of BMP4 (2.5 ng/ml), SB (5.4 μM) and RA (0.25 μM) signaling (days 3-6). t-test: **P < 0.01 vs Nkx2-5-cTNT+ cells at endogenous (E) FGF levels, ##P < 0.01 vs Nkx2-5+cTNT+ cells at endogenous (E) FGF levels (n = 5). (b) Flow cytometric analyses showing the proportion of NKX2-5+cTNT+ cells and NKX2-5-cTNT+ SANLPCs in day 20 populations generated from mesoderm specified with different concentrations of the small molecule FGF inhibitor PD173074 added between days 4-6 in the background of BMP4+SB (2.5 ng/ml, 5.4 μM) and RA (0.25 μM) signaling (days 3-6). t-test: *P < 0.05, **P < 0.01 vs Nkx2-5-cTNT+ cells at endogenous (E) FGF levels, #P < 0.05, ##P < 0.01 vs Nkx2-5+cTNT+ cells at endogenous (E) FGF levels (n = 4). (c) Number of total and NKX2-5-cTNT+ cells in day 20 populations generated from mesoderm specified with 480 nM FGFi from days 4-6. t-test: **P < 0.01 vs indicated sample (n = 6). (d) Flow cytometric analyses showing the proportion of NKX2-5+cTNT+ cells and NKX2-5-cTNT+ SANLPCs in day 20 populations generate from mesoderm specified with FGFi (480 nM) at indicated time points in the background of BMP4+SB and RA signaling (days 3-5). t-test: *P < 0.05, **P < 0.01 vs Nkx2-5-cTNT+ cells in untreated control, #P < 0.05, ##P < 0.01 vs Nkx2-5+cTNT+ cells in untreated control (n = 4). (e) Flow cytometric analyses showing the proportion of NKX2-5+cTNT+ cells and NKX2-5-cTNT+ SANLPCs in day 20 populations generated from mesoderm specified with 960 nM of FGFi alone or in the background of BMP+SB and RA signaling (days 3-6). t-test: *P < 0.05 vs Nkx2-5-cTNT+ cells in FGFi only (n = 4). All error bars represent s.e.m. d, day; E, endogenous; FGFi, FGF inhibitor (PD173074)

Supplementary Figure 9 Generation of SANLPCs from different hPSC lines.

(a) A comparison of the proportion of NKX2-5+ cells in a day 20 population identified based on NKX2-5:GFP expression and with an anti-NKX2.5 antibody (APC). Cells stained with secondary antibody alone are shown as the negative control. (b-d) Generation of SANLPCs and VLCMs from the HES2 hPSC line. (b) Flow cytometric analyses of the proportion of NKX2-5+cTNT+ cells and NKX2-5-cTNT+ SANLPCs in day 20 HES2-hPSC-derived populations specified from mesoderm with different concentrations of FGFi between days 4-6 or 3-6 in the background of BMP4+SB and RA signaling (days 3-6). t-test: #P < 0.05, ##P < 0.01 vs Nkx2-5+cTNT+ cells in untreated control or indicated sample (n = 3). (c) Flow cytometric analyses showing the proportion of NKX2-5+cTNT+ cells and NKX2-5-cTNT+ SANLPCs in day 20 HES2-hPSC-derived populations generated from mesoderm specified with either 20 ng/ml bFGF, or no additional FGF (endogenous) or FGFi (480 nM) between days 3-6 in the background of BMP4+SB and RA signaling (days 3-6). t-test: **P < 0.01 vs Nkx2-5-cTNT+ cells at endogenous (E) FGF levels, ##P < 0.01 vs Nkx2-5+cTNT+ cells at endogenous (E) FGF levels (n = 5). (d) Flow cytometric analyses of the proportion of NKX2-5+cTNT+ VLCMs and NKX2-5-cTNT+ cells in day 20 HES2-hPSC-derived populations that were specified under VLCM differentiation concentrations (n = 5). (e-g) Generation of SANLPCs and VLCMs from the MSC-iPS1 hPSC line. (e) Flow cytometric analyses of the proportion of NKX2-5+cTNT+ cells and NKX2-5-cTNT+ SANLPCs in day 20 MSC-iPS1-derived populations generated from mesoderm specified with either 20 ng/ml bFGF, or no additional FGF (endogenous) or FGFi (480 nM) between days 3-6 in the background of BMP4+SB and RA signaling (days 3-6). t-test: *P < 0.05, **P < 0.01 vs Nkx2-5-cTNT+ cells at endogenous (E) FGF levels, ##P < 0.01 vs Nkx2-5+cTNT+ cells at endogenous (E) FGF levels (n = 4). (f) Flow cytometric analyses of the proportion of NKX2-5+cTNT+ VLCMs and NKX2-5-cTNT+ cells in day 20 MSC-iPS1-derived populations that were specified under VLCM differentiation conditions (n = 4). (g) Beating rate of MSC-iPS1-derived cell aggregates in day 20 populations specified with VLCM or SANLPC (FGFi+BMP+SB+RA) differentiation condition. **P < 0.01 vs VLCM (n = 10). All error bars represent s.e.m. AB, antibody d, day; E, endogenous; FGFi, FGF inhibitor (PD173074)

Supplementary Figure 10 Expression analysis of SANLPCs generate with and without FGF inhibition from different hPSC lines.

Graphs of the qRT-PCR analysis presented as a heat map in Fig. 6h showing the expression levels of: pan-cardiomyocyte (a), ventricular cardiomyocyte (b), SAN pacemaker (c), AVN pacemaker (d), atrial/posterior cardiomyocyte (e), cardiac ion channel (f) and connexin channel (g) genes in NKX2-5+SIRPA+CD90- VLCMs generated from the HES3 line (VLCM HES3 NKX2-5gfp/w), SIRPA+CD90- VLCMs generated from the HES2 line (VLCM HES2), NKX2-5-SIRPA+CD90- SANLPCs generated from the HES3 line (SANLPC HES3 NKX2-5gfp/w), SIRPA+CD90- SANLPCs generated from the HES3 line using FGF inhibition (FGFi SANLPC HES3 NKX2-5gfp/w) and SIRPA+CD90- SANLPCs generated from the HES2 line using FGF inhibition (FGFi SANLPC HES2). All populations were isolated at day 20 of differentiation. Values represent expression levels relative to housekeeping gene TBP. Error bars represent s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test: *P < 0.05, **P < 0.01 vs VLCM HES3 NKX2-5gfp/w, or indicated sample. #P < 0.05, ##P < 0.01 vs VLCM HES2.

Supplementary Figure 11 Functional characterization of SANLPCs generated from the HES2 hPSC line.

(a) Representative recordings of spontaneous action potentials in a HES2 hPSC-derived (HES2) SANLPC and VLCM. (b) Histogram plot showing the distribution of the maximum upstroke velocities (dV/dtmax) recorded in HES2 SANLPCs and VLCMs. (c) Current-voltage relationship for pacemaker funny current (If) densities and summary of maximum If densities recorded at -120 mV in HES2 VLCMs and SANLPCs. t-test: **P < 0.01 vs VLCMs. (d,e) ECG recordings and optical mapping in the Langendorff isolated rat heart model harvested 14 days after transplantation with HES2 SANLPCs (1.25x106 cells). The SANLPC graft evoked isolated ectopic beats during induction of pharmacological AV block. Original ECG traces (d) and optical mapping derived activation maps (e) are shown after the application of Adenosine (0.6 mg). The orange circle in the far left image indicates the site of the HES2 SANLPC transplant (TP). Scale bar represents 5 mm. Note, the presence of premature beats (indicated by * in the ECG trace) between the sinus rhythm beats. These premature beats map to the HES2 SANLPC transplantation site. (f,g) ECG recordings and optical mapping in the Langendorff isolated rat heart model, harvested 14 days after transplantation with HES2 VLCMs (1.5x106 cells). Original ECG traces (f) and optical mapping derived activation maps (g) are shown before and after the application of 0.1 ml Methacholine (1 μM) + Lidocaine (0.005%) for induction of transient AV block. The black circle in the far left image indicates the site of the HES2 VLCM transplant (TP). Scale bar represents 5 mm. (h-i) VLCM and SANLPC graft size determined by qPCR using primers specific to human ALU repeat elements at 2 weeks after transplantation presented as percentage of graft cell survival (h) and total number of engrafted cells (i). Error bars represent s.e.m.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Protze, S., Liu, J., Nussinovitch, U. et al. Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker. Nat Biotechnol 35, 56–68 (2017). https://doi.org/10.1038/nbt.3745

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3745

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing