Chalcone‐Supported Cardiac Mesoderm Induction in Human Pluripotent Stem Cells for Heart Muscle Engineering

Abstract Human pluripotent stem cells (hPSCs) hold great promise for applications in cell therapy and drug screening in the cardiovascular field. Bone morphogenetic protein 4 (BMP4) is key for early cardiac mesoderm induction in hPSC and subsequent cardiomyocyte derivation. Small‐molecular BMP4 mimetics may help to standardize cardiomyocyte derivation from hPSCs. Based on observations that chalcones can stimulate BMP4 signaling pathways, we hypothesized their utility in cardiac mesoderm induction. To test this, we set up a two‐tiered screening strategy, (1) for directed differentiation of hPSCs with commercially available chalcones (4’‐hydroxychalcone [4’HC] and Isoliquiritigen) and 24 newly synthesized chalcone derivatives, and (2) a functional screen to assess the propensity of the obtained cardiomyocytes to self‐organize into contractile engineered human myocardium (EHM). We identified 4’HC, 4‐fluoro‐4’‐methoxychalcone, and 4‐fluoro‐4’‐hydroxychalcone as similarly effective in cardiac mesoderm induction, but only 4’HC as an effective replacement for BMP4 in the derivation of contractile EHM‐forming cardiomyocytes.


Experimental Section
Ethics. The import and use of human embryonic stem cells (HES2-ROSA26-RFP [HES2]) [1] ; Standard cardiac differentiation protocol. We applied the ABCF+I cardiac differentiation protocol ( Figure 1A), [2] which we introduced previously with robust cardiomyogenesisinducing activity in all so far tested hPSC lines, including several human embryonic and induced pluripotent stem cell lines. In brief, mesoderm induction was for 3 days with ABCF: Activin A (9 ng/ml), BMP4 (5 ng/ml), CHIR99021 (1 µmol/L) and FGF2 (5 ng/ml) in serum- Candidate compounds were ranked according to their cardiomyogenesis inducing activity using a combined differentiation score (DS) derived from an assessment of multiple parameters: (1) reproducibility of cardiomyocyte derivation, (2) cardiomyocyte beating rate, (3) total cell number as well as (4) percentage Figure 2B; synthesis of compounds 5e [3] , j [4] , k [5] , n [6] , and o [5a, 7] were described previously. A detailed compound structure analyses of all compounds and the synthesis procedures for compounds 6 a-i (summarized also in Figure 4A) are listed below.
NMR spectroscopy. NMR spectra were recorded on Varian "Mercury-300", "Unity-300", "Inova-500" and on a "AMX-300" spectrometer from Bruker. Chemical shifts are given in ppm IR spectroscopy. IR spectra were recorded with a FT/IR-4100 spectrometer from JASCO (substances were applied neat on an ATR unit).
UV spectroscopy. UV spectra were recorded on a JASCO V-630 spectrometer.
Mass spectrometry. ESI-MS and ESI-HRMS spectra were recorded on a "Apex IV" spectrometer from Bruker Daltronik.  Figure S2.
Immunofluorescence staining. Cells were plated on Matrigel™-coated glass coverslips for 24 h in SF-medium before fixation in 4% FA. After blocking for 30 minutes in blocking buffer, primary antibodies (Table S1) were added for 90 min followed by secondary antibodies (Table   S1) and Hoechst 33342 (10 µg/mL) for 60 min at room temperature. Coverslips were mounted onto glass slides (SuperFrost Plus, Thermo Fisher Scientific) using Fluoromount (Dako) and imaged using a Zeiss 710 NLO confocal microscope or a Zeiss AxioImager.M2 fluorescence microscope.
Polymerase chain reaction. Cells were harvested and RNA extraction was performed using Trizol™ following manufacturer's instructions (Ambion). RNA (1 µg) was treated with DNAse (Roche) followed by cDNA synthesis using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR (qPCR) was performed using the Fast SYBR Green Master Mix (Applied Biosystems) in a 384-well format AB7900 HT (Applied Biosystems).
Gene expression analyses were performed according to the standard curve method. [8] GAPDH transcript abundance was used for normalization. Primer details are given in Table S2.

C NMR
and pyridine (1 mL) in EtOH (10 mL The organic phase was dried (Na2SO4) and evaporated in vacuo. The obtained crude material was purified by column chromatography on silica gel using 5 -15% ethyl acetate in pet ether to get compound 6c (321 mg, Yield: 96%) as a white solid.
The reaction mixture was heated at 80 o C for 4 h, then cooled to room temperature, diluted with water (100 mL), and extracted with CH2Cl2 (3 x). The organic phase was dried (Na2SO4) and evaporated in vacuo. The obtained crude material was purified by column chromatography on silica gel using 5 -10% ethyl acetate in petroleum ether to afford compound 6d (206 mg, Yield: 16%) as a colourless thick liquid.

3-(4-Fluorophenyl)-1-(4-methoxyphenyl)
propan-1-one (7) [13] : To a solution of 5a (200 mg, 0.78 mmol) in EtOH (50 mL) was added a solution of ammonium acetate (6 g, 78 mmol) in water (8 mL). Then zinc powder (382 mg, 5.85 mmol, 7.5 eq) was put in at room temperature in 5 portions at intervals of 15 min. After completion of the reaction within 70 min (monitored by TLC), the mixture was filtered, the filter cake washed with EtOH (2 x 15 mL), and the combined filtrates concentrated in vacuo. The residue was diluted with water, extracted with ethyl acetate (3 x) and the organic phase was dried (Na2SO4) and concentrated in vacuo. Crude product was purified by column chromatography on silica gel using 5% ethyl acetate in pet ether to obtain compound 7 (133 mg, Yield: 73%) as a colourless thick liquid.   (4 x 7 mL) and allowed to dry at the air. The product was purified by column chromatography on silica gel using 5% ethyl acetate in petroleum ether to afford compound 6h (56 mg, Yield: 35%) as a white solid.