A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells


Most of the mammalian heart is formed from mesodermal progenitors in the first and second heart fields (FHF and SHF), whereby the FHF gives rise to the left ventricle and parts of the atria and the SHF to the right ventricle, outflow tract and parts of the atria1,2,3. Whereas SHF progenitors have been characterized in detail, using specific molecular markers4,5,6,7,8, comprehensive studies on the FHF have been hampered by the lack of exclusive markers. Here, we present Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) as an FHF marker. Lineage-traced Hcn4+/FHF cells delineate FHF-derived structures in the heart and primarily contribute to cardiomyogenic cell lineages, thereby identifying an early cardiomyogenic progenitor pool. As a surface marker, HCN4 also allowed the isolation of cardiomyogenic Hcn4+/FHF progenitors from human embryonic stem cells. We conclude that a primary purpose of the FHF is to generate cardiac muscle and support the contractile activity of the primitive heart tube, whereas SHF-derived progenitors contribute to heart cell lineage diversification.

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Figure 1: Hcn4 specifically marks the FHF at the earliest stages of mouse cardiogenesis.
Figure 2: Hcn4+/FHF lineage tracing in the embryonic heart.
Figure 3: Hcn4+/FHF lineage contribution to the heart.
Figure 4: Clonal analysis of single Hcn4+/FHF cells (derived) from mouse embryos.
Figure 5: Isolation and differentiation of HCN4+ cells from differentiated hESCs.


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We would like to thank C. Cowan for his support, L. Prickett-Rice, K. Folz-Donahue and M. Weglarz of the Harvard Stem Cell Institute Flow Cytometry Core Facility for assistance with FACS analysis, C. Du of the Tufts Electrophysiology Core for assistance with electrophysiology recordings, L. Bu for technical advice, K. Buac, E. Hansson, C. Riedel and L. Bu for critical reading of the manuscript and discussions, and C. Hartmann for advice on double-fluorescence in situ hybridizations. D.S. has received a D.F.G. (German Research Foundation) postdoctoral fellowship. K.B. was supported by a NHLBI T32HL007208 grant. This work is financially supported by the NIH U01 HL098 166 and NIH U01H100408 research grants.

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D.S. and K.R.C. designed the experiments and wrote the manuscript. D.S. carried out most of the experiments and analysed most of the data. M.K.A. helped to perform and analyse clonal analysis experiments. K.B. contributed PCRs with reverse transcription of in vivo clonal analysis. L.Z. performed some immunohistochemical staining, M.W.S. helped with some hESC differentiation assays and RNA isolations, J.C. performed acetylcholinesterase staining, M.S. helped with some mESC clonal analysis cultures and A.L. provided the Hcn4CreErt2 mouse line.

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Correspondence to Kenneth R. Chien.

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Integrated supplementary information

Supplementary Figure 1 Section in situ hybridisation analysis of crescent stage mouse embryos (E7.5).

(a) More anterior section level of the same embryo as shown in Fig. 1h ( 4ss). Expression domains of Hcn4, Tbx5 and Mlc2a (all shown in green) are predominantly overlapping, but not with that of Isl1(shown in red). (b) Corresponding section levels through an E7.5 embryo as shown below in (c+d). (c-d) Double in situ hybridisations. Blue staining: NBT/BCIP (BM Purple) and brown staining: INT/BCIP. (c) Hcn4 (blue) and Isl1 (brown) are expressed in predominantly non-overlapping expression domains. (d) Tbx5 (blue) and Isl1 (brown) are expressed in predominantly non-overlapping expression domains. Expression domains of Hcn4 and Tbx5 are mostly overlapping, when comparing their expression on adjacent sections. (e) Single fluorescence in situ hybridisations on adjacent sections of an E7.5 ( 4ss) embryo. (e-f) Expression of Mlc2a, Hcn4 and Tbx5 (all in green) are predominantly expressed within the presumptive FHF, in contrary to Isl1 expression primarily in the SHF (red). (e’-f’) Section levels corresponding to (e) and (f), respectively. Scale bar: 50μm.

Supplementary Figure 2 Section in situ hybridisation and IHC analysis of a crescent stage embryo (E7.5).

(a) Single in situ hybridisations (NBT/BCIP, BM Purple) on adjacent sections of an E7.5 ( 2–4ss) mouse embryo. Corresponding section levels are shown in panel on left side. Expression domains of Hcn4 and Tbx5 are mostly overlapping in presumptive FHF, but not with Isl1. However, in the posterior area of the FHF and SHF domains there is an overlapping expression domain between Tbx5 and Isl1 visible (orange arrows), but not with Hcn4 (black arrow), suggesting that Hcn4 is more exclusive to the FHF than Tbx5. (b) Co-staining showing single fluorescent in situ hybridisation of Tbx5(green) and ISL1 protein location (red) on adjacent sections of an E7.5 ( 2–4ss) mouse embryo. The two domains are largely non-overlapping; however, there is some co-localization visible in the most lateral sides of the splanchnic mesoderm (arrows). There are also some areas, where there is weak staining for ISL1 protein visible (asterix), overlapping with Tbx5 mRNA, but the ISL1 protein does not co-localize with the nucleus but seems to be present within the cytoplasma, indicating that it may not be functional in these areas. Scale bar: 50μm.

Supplementary Figure 3 Section in situ hybridisation analysis of an early heart tube stage embryo (E8.0).

(a) Single colour in situ hybridisations on adjacent sections. Corresponding section level as shown in drawing. Hcn4, Tbx5, Mlc2a and Nkx2.5 are expressed within the primitive heart tube, unlike Isl1, which is expressed in the pharyngeal mesoderm. At this developmental stage, Nkx2.5 is mostly non-overlapping with Isl1 expression. (b) Single fluorescent in situ hybridisations on adjacent sections. Corresponding section levels are shown in drawings above images. Expression domains of Hcn4, Tbx5 and Mlc2a (all green) are mostly overlapping, but predominantly non-overlapping with Isl1 expression (red). However, posterior to the primitive heart tube, there is an area of overlap between Tbx5 and Isl1 expression (arrow), but not with Hcn4 and Mlc2a (most likely no overlap with Isl1, as the section level is a bit more anterior compared to Isl1 (adjacent sections) and it seems more lateral compared to Isl1). Scale bar: 50μm.

Supplementary Figure 4 Hcn4+/FHF lineage tracing in the embryonic heart and Hcn4+/ FHF cell lineage contribution to atria.

(a) Whole-mount X-gal staining of Hcn4CreErt2/+;R26RlacZ hearts at E19.5. Tamoxifen induced Hcn4CreErt2/+ activation at E8.0 labels the Hcn4+/FHF derivatives, such as the left ventricle (LV) and parts of the atria. At this stage, also the sinus venosus (SV) and sinoatrial node (SAN) are stained positive. (b) Section through an E19.5 Hcn4CreErt2/+;R26ReYFP heart; Hcn4CreErt2/+ activation by tamoxifen at E7.0. Within the left ventricle, there is Hcn4+/FHF lineage contribution to the trabecular and compact layer of the left ventricle, and some minor contribution to the septum. (c-d) Whole-mount X-gal staining of a Hcn4CreErt2/+;R26RlacZ heart at E12.5 (c), and embryos at E9.5 (d), and E10.5 (e). Tamoxifen induced Hcn4CreErt2/+ activation at E7.0 labels the Hcn4+/FHF derivatives, such as the left ventricle (LV) and parts of the atria. No staining is visible within the pharyngeal mesoderm (arrows, d+e). In situ hybridisation for Isl1 of E9.5 embryo marks the pharyngeal mesoderm (arrow, f). Hcn4+/FHF cells also contribute to the AV canal (red asterix, d+e). (g) Section through an E19.5 atria of Hcn4CreErt2/+;R26ReYFP mice; Hcn4CreErt2/+ activation by tamoxifen at E7.0. Co-immunostaining for YFP as a marker for Hcn4+/FHF lineage traced cells and TNNT2 shows contribution to cardiomyocytes. Yellow box represents magnified area shown in merge image (h) FACS quantification of the cellular contribution of Hcn4+/FHF lineage traced cells to an Hcn4CreErt2/+;R26RtdTomato E19.5 atria (Tamoxifen E7.0). FACS analysis of tomato+ Hcn4+/FHF lineage traced cells co-stained for TNNT2, PECAM-1 (CD31) or smMHC (MYH11) showed a predominant contribution to cardiomyocytes, but no double positive cells for endothelial/endocardial-, and vascular smooth muscle markers, suggesting no contribution to these cell lineages within atria. Representative FACS dot-plots are shown. Secondary antibody controls are shown in Supplementary Fig. S6b. Scale bar: (a+c) 0.5mm, (b) 100μm, (d-f) 0.2mm, (g) 50μm.

Supplementary Figure 5 C lonal analysis of Hcn4+/FHF cells isolated from mouse ESCs and TgMef2c-AHF-eGFP/SHF cell lineage contribution in vivo.

(a) Embryonic stem cells (ECSs) were derived from Hcn4CreErt2/+;R26ReYFP mice. Mouse ESCs were differentiated using the embryoid body technique. Hcn4CreErt2/+ activity was induced by administration of 4-OH-Tamoxifen on day 4 and 6 of differentiation. eYFP positive Hcn4+/FHF cells were FACS sorted (representative FACS dot-plots are shown). (b) Single eYFP positive Hcn4+/FHF cells were sorted as 1 cell/well into 96-well plates. After 2-3 weeks single cell derived clones showed limited expansion and efficient differentiation into cardiomyogenic clones, visualized by direct fluorescence of eYFP and immunostaining for the cardiac muscle marker TNNT2 (cTnT). (c) RT-PCR analysis, showing expression of cardiac marker genes in single Hcn4+/FHF cell derived cardiomyogenic clones. (d) Single cell patch clamp analysis (n = 39). Most clamped cells showed a ventricular-like action potential (AP) (64%), while some showed a more atrial-like AP (16%), or nodal-like AP (20%). Representative action potentials are shown. (e) FACS quantification of the cellular contribution of TgMef2c-AHF-eGFP/SHF lineage traced cells to the right ventricle and outflowtract (OFT). Fixed single cells were co-stained for TNNT2, demonstrating contribution to cardiomyogenic cell lineages, particularly within the RV. Co-staining for smMHC (MYH11) showed a minor contribution to vascular smooth muscle cells in RV and contribution in the OFT. Co-staining for PECAM-1 (CD31) showed a minor contribution to endothelial cell lineage in RV and contribution in the OFT. Representative FACS dot-plots are shown. Scale bar: 50μm.

Supplementary Figure 6 Uncropped key electrophoresis data and secondary antibody controls of FACS analyses.

(a) Uncropped electrophoresis data of RT-PCR shown in Fig. 4c. (b) Background signal of secondary antibodies used for FACS sorting as shown in Fig. 3f and Supplementary Fig. S4h.

Supplementary Table 1 Forward and reverse PCR primer sequences utilised for generation of RNA probes used in whole-mount in situ hybridisation.
Supplementary Table 2 Forward and reverse PCR primer sequences used for clonal analysis from mouse embryos and mouse ESCs, and qPCR analysis of human HCN4+/FHF cells.

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Clonal analysis of single Hcn4+/FHF cells isolated from mouse embryos.

Representative example of a beating cardiomyogenic clone derived from a single Hcn4+/FHF cell from mouse embryos. (AVI 7234 kb)

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Später, D., Abramczuk, M., Buac, K. et al. A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nat Cell Biol 15, 1098–1106 (2013). https://doi.org/10.1038/ncb2824

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