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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.

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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.

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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.

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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.

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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.

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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

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