*2.3. qRT-PCR*

RNA was isolated from the cells using the NucleoSpin® RNA isolation kit (Macherey-Nagel, Dueren, Germany). First strand cDNA was then synthesized using the cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The qPCR reaction was then carried out with the Taqman® Universal PCR Master Mix (Thermo Fisher Scientific) and performed on a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster city, CA, USA). Primers of the following target genes: *Pou5f1* (Mm00658129\_gH), *cTnnt2* (Mm01290256\_m1), *MesP1* (Mm00801883\_m1), and *Nkx2.5* (Mm01309813\_s1) were purchased from Thermo Fisher Scientific. Gene expression values of the target genes at day 6 were then normalized to the housekeeping gene *Hprt* (Mm00446968\_m1; Thermo Fisher Scientific) and compared relative to the expression values at day 0 using the ΔΔCt method for relative quantifications.

#### *2.4. Beating Foci Analysis*

The number of beating foci per EB was analyzed from day 7 to day 30 of differentiation. The EB were observed under a microscope (Carl Zeiss, Oberkochen, Germany) and the beating foci per each EB were then visually analyzed using the ZEN2011 software (Carl Zeiss).

#### *2.5. Flow Cytometry*

Single cell cardiac monocyte suspensions were prepared for flow cytometry, as previously described [11] Briefly, the remote and infarct tissue of the heart was dissected and enzymatically digested separately in HBSS with Ca2<sup>+</sup> and Mg2<sup>+</sup>(450 U/mL collagenase type I, 125 U/mL collagenase type XI, 120 U/mL DNase I, 60 U/mL hyaluronidase, all Sigma-Aldrich) for 30 min at 37 ◦C. The digested samples were then passed through a 100 μm filter and centrifuged to enrich for mononuclear cells. Red blood cells were then lysed using erythrocytes lysis buffer (eBioscience, San Diego, CA, USA) and the digest was then washed and suspended in MACS® buffer (PBS, 2 mM EDTA, 0.5% BSA). Samples were then labeled using Zombie Aqua dye (BioLegend, San Diego, CA, USA.), washed, resuspended in MACS buffer containing FCR Block (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and stained (see Table 1 for antibody list). Stained samples were then analyzed on a BD FACS LSR II® running BD FACS Diva software (version 6.1.2, Franklin Lakes, NJ, USA). The various immune cell populations in the heart tissue were then assessed, as described in Figure 1.


**Table 1.** Antibodies used for flow cytometry.

**Figure 1.** Gating strategy for identifying the different immune populations in the heart. Mononuclear cells expressing CD45 were gated and doublets (FSC-W vs. FSC-A) were excluded. Dead cells were excluded by Zombie aqua. The live single CD45<sup>+</sup> cells were then grouped into R1, CD11b+ myeloid cells (CD45+/CD11b+/CD11c−); R2, dendritic cells (CD45+/CD11b+/CD11c+); and R3, NK cells (CD45+/CD11b−/CD11c−/NK1.1+) based on their relative expression of CD11b and CD11c. R5, neutrophils (CD45+/CD11b+/CD11c- /Ly6Ghi) were then excluded from R1 based on their Ly6G expression. The remaining R4 monocytic cells were then further characterized into R6, Ly6Chi or commonly known as M1 cells (CD45+/CD11b+/CD11c−/Ly6Glo/Ly6Chi); R7, Ly6Clo or commonly known as M2 cells (CD45+/CD11b+/CD11c−/Ly6Glo/Ly6Clo) based on their Ly6C expression; and into R8, fetal liver HSC-derived resident macrophages (CD45+/CD11b+/CD11c- /Ly6Glo/CCR2−/MHC-IIhi); R9, monocyte derived macrophages (CD45+/CD11b+/CD11c- /Ly6Glo/CCR2+/MHC-IIhi); R10, monocytes (CD45+/CD11b+/CD11c−/Ly6Glo/CCR2+/MHC-IIlo); and R11, yolk sac-derived resident macrophages (CD45+/CD11b+/CD11c−/Ly6Glo/CCR2−/MHC-IIlo) based on their CCR2 and MHC-II expression. These CCR2 and MHC-II gated populations were then back gated on R6 and R7 and their relative contribution to the M1 (Ly6Chi) and M2 (Ly6Clo) cells was assessed.

#### *2.6. PET Imaging*

For the PET study, mice of the groups 1,3, 6, and 7 were anaesthetized by inhalation of isoflurane (4% for induction and 1% to 2.5% maintenance during preparation and scanning), whereas mice of the groups 2,4, and 5 were anaesthetized by i.p. injection of ketamine/xylazine (ketamine 84 mg/kg and xylazine 11.2 mg/kg) 20 min before tracer application. All PET/CT scans were performed on a small animal PET/CT scanner (Inveon MM-PET/CT, Siemens Medical Solutions, Knoxville, TN, USA) [12] according to a standard protocol: Mice were injected intravenously with a dose of approximately 10 MBq 18F-FDG via a custom-made micro catheter placed in a tail vein. After an uptake period of 60 min, mice were imaged in prone position for 20 min. During the imaging session, respiration of the mice was controled and core body temperature was constantly kept at 38 ◦C via a heating pad. For attenuation correction and anatomical reference, whole body CT scans were acquired. CT images were reconstructed with a Feldkamp algorithm. The PET images were reconstructed with the three-dimensional (3D) iterative ordered subset expectation maximization reconstruction algorithm (3D-OSEM/OP-MAP) with the following parameters: 4 iterations (OSEM), 32 iterations (MAP), 1.7 mm target resolution, and 128 × 128 matrix size. Reconstruction included corrections for random coincidences, dead time, attenuation, scatter, and decay.

#### *2.7. PET Image Analysis*

Image analyses were performed using an Inveon Research Workplace (Siemens, Knoxville, TN, USA), as described previously [13]. PET (Positron emission tomography) and CT (Computerized tomography) images were fused by the use of an automated volumetric fusion algorithm and then verified by an experienced reader for perfect alignment. Consecutively, standardized representative volumes of interest (VOI) were manually placed in the remote area and in the infarcted region as well as the whole heart guided by anatomical landmarks, as described in detail in Figure 2. Correct VOI positioning was visually verified in axial, coronal, and sagittal projection.

Carimas 2 software (Turku PET Centre, Turku, Finland) was used for generating polar maps of the left ventricle according to the manual provided by the developer. Results are presented using 17-segmental standardized myocardial segmentation.

**Figure 2.** Representative images visualizing our volumes of interest (VOI) positioning strategy: (**A**) Myocardium of healthy mice anaesthetized with isofluorane can be clearly delineated and served as a reference for VOI positioning. (**B** and **C**) Anaesthesia with ketamine/xylazine. A VOI of 5 μL was positioned in both anterolateral wall (infarct area, green) and remote area (inferobasal, blue). Thereby both anatomical landmarks form the CT scan and the image of healthy myocardium (**A**) was used.

#### *2.8. Cardiac Magnetic Resonance Imaging*

Cardiac magnetic resonance (CMR) measurements were performed on a 7 Tesla small animal MRI system (BioSpec 70/30, maximum gradient strength 440 mT/m, Bruker BioSpin Gmbh, Ettlingen, Germany) equipped with a 1H transmit volume coil (86 mm, volume resonator) and a 2-by-2 receive-only surface coil array (both Bruker BioSpin GmbH). After induction of anaesthesia using 2% to 3.5% isoflurane in oxygen, animals were placed in a supine position on a dedicated mouse bed and surface coil was placed on the chest of the mice. Respiration rate and body temperature were monitored

using an MR-compatible small animal monitoring and gating system (Model 1030, SA Instruments, Inc., Stony Brook, NY, USA), and stable body temperature was maintained by a warm water heating. Anaesthesia was maintained during the experiment with isoflurane oxygen (1.5% to 2%) to achieve a respiration rate of about 35 to 55 breaths.

After planning sequences, for the short axes view final images of the left ventricular ejection fraction (LVEF) measurements were acquired using a IntraGate gradient-echo cine sequences (IntraGate Cine-FLASH) in six short-axis planes completely covering the left ventricle. Acquisition parameters included: echo time (TE) 2.38 ms, repetition time (TR) 5.89 ms, flip angle 15◦, 14 frames per cardiac cycle, oversampling 140, averages 1, field of view (FOV) 29.4 × 25.2 mm, matrix size 211 × 180, resolution in-plane 0.14 × 0.14 mm, slice thickness 1 mm, and scan time per slice 2 min.
