1. Generation of human embryonic stem cells-derived cardiovascular progenitor cells (hESC-CPC)
Human embryonic stem cells (hESC), Royan H6 (RH6) line which was established and characterized as previously reported (17), was received from Royan Stem Cell Bank and expanded in adherent culture according to a previously described protocol (18). In order to prompt hESC for cardiogenic differentiation, the expansion system was changed to static suspension culture, where hESC were expanded as previously described (18). Briefly, the 5-day-old hESC spheroids were subjected to cardiogenic differentiation using a cocktail of small molecules including CHIR (Stemgent, 04-0004-10), IWP2 (Tocris Bioscience, 3533), SB-431542 (Cayman, 13031), and purmorphamine (Pur) (Stemgent, 04–0009), according to a previous study (18). A detailed description of the differentiation method can be found in the supplemental information.
2. Culture and expansion of hESC-CPC
The 4-day-old differentiated hESC-CPC spheroids were dissociated into single cells and cultured on Matrigel (Sigma-Aldrich, E1270)-coated plates with specific expansion medium (CPCxm) composed of a basal medium and fresh small molecules; bFGF, CHIR, and A83-01 (Stemgent, 04–0014). Furthermore, hESC-CPC was passaged every 72 hs and expanded for 8 passages. Characterization of hESC-CPC was performed at passages 0 (P0), P4, and P8 using flow cytometry and immunofluorescence staining against CPC-specific markers. For a detailed description, see supplemental information. Furthermore, the freeze-thaw procedure of hESC-CPC was assessed, and the resulting cells were characterized, for which details can be found in supplemental information. Population Doubling Time (PDT) and expansion fold assays were evaluated as described previously (15), whose details are described in supplemental information.
3. Differentiation of hESC-CPC towards cardiovascular lineage cells
3-1. Differentiation of hESC-CPC into cardiomyocytes, endothelial and smooth muscle cells
To generate cardiovascular lineage cells, hESC-CPCs (P0 and P8) were seeded on Matrigel-coated plates at a density of 3 × 104 cells/cm2. Induction towards cardiomyocytes (CM) was achieved by using CM differentiation medium (CMm) composed of RPMI1640 (Gibco, 52400-041) supplemented with B27 without insulin (Gibco, A18956-01), 2 mM L-glutamine, 1% NEAA, and 0.1 mM β-mercaptoethanol, as well as growth factor and small molecules including BMP4 (10 ng/ml) (314-BP-010/CF R&D) and IWP2 (5 µM) (Tocris). hESC-CPCs were differentiated into endothelial cells (EC) by using EC differentiation medium (ECm) composed of DMEM/F12 supplemented with 2% B-27 without vitamin A, 2 mM L-glutamine, 1% NEAA, and 0.1 mM β-mercaptoethanol, as well as growth factors VEGF (50 ng/ml) (Royan Biotech, PRP-1109) and bFGF (10 ng/ml). Smooth muscle cell differentiation required the culture of hESC-CPC in SMC differentiation medium (SMCm) composed of DMEM/F12 supplemented with 2% B-27 without vitamin A, 2 mM L-glutamine, 1% NEAA, and 0.1 mM β-mercaptoethanol, as well as growth factors of PDGF-BB (10 ng/ml) (Royan Biotech, RP-1111) and TGF-β (2 ng/ml) (Fitzgerald 30R-AT072). hESC-CPCs were differentiated into cardiac fibroblasts (CF) using a two-step protocol, which will be explained in a separate section.
For EC and SMC differentiation, hESC-CPCs were cultured in ECm and SMCm for 12 days, and the medium was renewed every 2 days. For differentiation to CM, hESC-CPCs were cultured in CMm for 3 days and subsequently in CMm without growth factors and with B27 complete (Thermo Fisher Scientific, 17504044) for 9 days.
3-2. Differentiation of hESC-CPC into cardiac fibroblasts
Regarding differentiation of CFs from hESC-derived CPCs (P0 and P8), we used the following protocol, which obtains CFs from epicardial origin. Initially, hESC-CPCs were seeded on Matrigel-coated plates at a density of 3 × 104 cells/cm2 and maintained in a basal differentiation medium (CFbm1) consisting of RPMI1640 supplemented with B27 without insulin, 2 mM L-glutamine, 1% NEAA, and 0.1 mM β-mercaptoethanol for 24 h in order to adapt to 2D culture. After this time, the CFbm1 was renewed and supplemented with a concentration of 3 µM of the small molecule CHIR for 48 h. Subsequently, the medium was replaced by CFbm1, and the culture medium was exchanged every 24 hs. After 48 h, epicardial-like cells were obtained and subjected to CF differentiation. To do so, the medium was replaced by DMEM/F12 supplemented with 2 mM L-glutamine, 1% NEAA, 0.1 mM β-mercaptoethanol, 15% FBS, and 10 ng/ml bFGF for a further 6 days.
4. Characterization of hESC-CPC and differentiated cardiovascular lineage cells
4 − 1. Gene expression analysis by real-time RT-qPCRFor detailed methods of gene expression analysis, see supplemental information.
4 − 2. Flow Cytometry
The methods of flow cytometry are fully described in supplemental information.
4 − 3. Immunofluorescence stainingA full description of the method can be found in the supplemental information.
4–4. Functional analysis
4-4-1. Field potential recording of CPC-derived cardiomyocytes
We evaluated the functional characteristics of CPC-derived CMs by conducting an extracellular field potential (FP) recording using a microelectrode array (MEA) data acquisition system (Multi Channel Systems in Reutlingen, Germany). The MEA plates had a grid of 60 titanium nitride electrodes (30 µm) with a 200 µm inter-electrode distance. Before the experiment, the MEA plates were sterilized, hydrophilized with FBS for 30 minutes, rinsed with sterile water, and coated with gelatin for 1 h. For the analysis, 4–5 × 105 CPCs were placed in the center of a sterilized MEA plate in a medium containing 20% FBS for 24 h. Subsequently, the MEAs were connected to a head-stage amplifier. The extracellular field potentials were recorded at a sampling rate of 10 kHz, and all the measurements were conducted at 37°C. Recordings lasted for 100 seconds at baseline. The FP signals were assessed for rhythmicity. The data were analyzed using Cardio2D+ software.
4-4-2. Tube formation assay of CPC-derived endothelial cells
To prepare for the experiment, a 96-well cell culture plate and P100 (t100 LRS) tips were placed at -80°C for 10 min. After that, 50 µl of ice-cold Matrigel (Corning, USA) was coated in the wells. The plates were then incubated at 37°C for 30 minutes to allow the Matrigel to solidify. CPC-derived ECs (CPC-EC) were seeded on the Matrigel at a density of 5 × 104, and a mixture of ECm plus 10% FBS was added. For control, HUVECs were used at a density of 13 × 103 cells (19). After 2 hs, images were captured using a CKX41 inverted (OLYMPUS) microscope from five random fields per well. These images were analyzed with the Angiogenesis Analyzer macro for ImageJ (20), and the number of nodes, junctions, and branches were calculated and compared. Each experiment was repeated three times.
4-4-3. Contraction assay of CPC-derived smooth muscle cells
CPC-SMCs were cultured on Matrigel-coated 4-well plates. After 24 h, carbachol (Sigma-Aldrich, C4382) was added to the culture medium at a final concentration of 10 µM. Images were taken 10 minutes after the treatment (21). The cell surface area was measured using ImageJ 1.54h software, and the average reduction in cell area was calculated.
4-4-4. Activation of CPC-derived cardiac fibroblasts
In order to activate CPC-CFs, the cells were incubated in serum-free culture media for 24 h at 5% CO2 and 37°C, followed by treatment with doxorubicin (DOX) 0.5 µM for 48 h. Both DOX-treated and control CFs were collected and subjected to RT-qPCR analysis for the detection of αSMA expression level, as previously described. The expression levels were then compared between the two groups.
5. Cardiac microtissue formation by co-culture of CPC-CM, CPC-EC, CPC-SMC and CPC-CF
For microtissue formation, CM, EC, SMC, and CF, which were derived from CPC differentiation, were added to 2.5 mg/ml of collagen derived from rat tail at a ratio of 2:1:1:2 and a final density of 2 × 106 cells/ml and cultured in 24-well plates. The co-culture medium (MTm) consisted of two media in an equal proportion: (1) DMEM F12, 15% FBS, 2 mM L-glutamine, 1% NEAA, 1% penicillin/streptomycin, and 0.1 mM β-mercaptoethanol, and (2) RPMI1640 supplemented with complete B27, 2 mM L-glutamine, 1% NEAA, and 0.1 mM β-mercaptoethanol. The medium was refreshed every day for one week. The microtissue started to form after 1 h and continued to make a 3D-like structure, and the area-to-volume ratio decreased over the next 24 h. The cardiac microtissues were then harvested for histological analysis.
5 − 1. Histological analysis
For detailed methods of histological analysis, see supplemental information.
5 − 2. Confocal imaging:
To evaluate the cell distribution in the microtissue, rhodamine Rhodamine Phalloidin (Invitrogene, B7474) staining was used and visualized by confocal laser scanning microscopy (CLSM; LSM800, Carl Zeiss, Germany). The detailed descriptions can be found in the supplemental information.
6. Statistical Analysis:
Statistical analysis was performed using the GraphPad-Prism software (9.0.2), and all results were presented as mean ± standard deviation (SD). The data is obtained from a minimum of three independent replicates. Following the assessment of normal distribution, significant differences between groups were calculated using proper statistical tests, including an unpaired t-test or one-way and two-way ANOVA. A statistically significant level was considered as P < 0.05.