B27 without insulin

Preparation and culturing of dissociated cells from postnatal mouse cortices was similar to as described previously (Beaudoin et al., 2012 (link)) using the following materials: Neurobasal-A Medium (10888-022, Thermo Fisher Scientific), Neurobasal Plus Medium (A3582901, Thermo), Neurobasal-A without D-glucose or sodium pyruvate (A2477501, Thermo), B-27 Supplement (17504-044, Thermo), B-27 Plus Supplement (A3582801, Thermo), B-27 without insulin (A1895601, Thermo), GlutaMAX Supplement (35050061, Thermo), sodium pyruvate (11360070, Thermo), Hibernate-A Medium (A1247501, Thermo), gentamycin (G1272, Sigma-Aldrich), papain (PDS kit: LK003178, Worthington Biochemical Corporation), DNase I (90083, Thermo), fetal bovine serum (FBS; 26140-087, Thermo), poly-L-lysine (P2636, Sigma), coverslips precoated with poly-L-lysine (12 mm #1.5 Biocoat 354085, Corning; or GG-12-1.5-PLL, Neuvitro Corporation), Biosafety Cabinet Class II A2 (NU-540-500, Nuaire), CO₂ incubators (NU-5710, Nuaire).

For cardiac reprogramming, 1 × 10⁵ cells/well were seeded on 0.1% gelatin-coated 6 well plates and cultured to 80% confluency. We transfected 40 pmol of each miRNA (Pre-miR™ hsa-miR-1, Pre-miR™hsa-miR-499a-5p, Pre-miR™hsa-miR-208a-3p, Pre-miR™hsa-miR-133a-3p, all Thermo Fisher) with Lipofectamine^® 2000 according to the manufacturer’s instructions (Thermo Fisher). Transfection of custom-made mRNA (Trilink) was performed with Viromer Red^® transfection reagent (Lipocalyx, Halle, Germany). Cells were either transfected with 2 µg MESP1 or with a combination of 1 µg GATA4, 1 µg MEF2C and 1 µg TBX5. One day after transfection of miRNA or mRNA, cells were subjected to two different medium conditions. For cardiac induction medium I (card ind. I), cells were incubated in RPMI, supplemented with B27 without insulin (Thermo fisher) for 7 days, followed by incubation in RPMI containing B27 +insulin/- vitamin A (Thermo Fisher) for another 21 days. Additionally, culture medium was supplemented with ascorbic acid (Sigma Aldrich, St. Louis, USA) and Wnt pathway targeting small molecules, including 6 µM CHIR99021 (days 1–2), and 5 µM IWP-2 (days 4–5) (both Stemcell Technologies). For cardiac induction II (card ind II), a commercially available cardiomyocyte differentiation kit was used according to the instructions given by the manufacturer (Thermo Fisher, A2921201).

EHM was prepared as described previously (Figure 2).^{23 (link),30 (link)} In brief: A mixture of iPSC-CM (70%), human fibroblasts (30%) and collagen was cast into a custom made 48-well plate (myrPlate, myriamed GmbH, Germany). EHM was constructed using iPSC-CM expressing f-Chrimson (see Supplementary materialonline, Figure S1) unless otherwise indicated. Spontaneous contractions were observed 3–5 days after the casting procedure. EHM was maintained in a serum-free medium (SFMM) containing IMDM with Glutamax, MEM Non-Essential Amino Acids Solution, 4% B27 without insulin (all Thermo Fisher Scientific), 200 µmol/L ascorbic acid 2-phosphate (Sigma-Aldrich), 100 ng/mL recombinant human IGF-1, 5 ng/mL recombinant human VEGF, 10 ng/mL animal-free recombinant human FGF-basic (all PeproTech). To allow for continuous real-time monitoring of force of contraction, EHM was prepared in an alternative engineering format embedded within custom-made culture vessels each directly coupled to an isometric force sensing device as previously described.^{31 (link)} All experiments were performed after a tissue culture period between 28 and 38 days.

hESCs and hiPSCs were differentiated in either RPMI+B27-ins medium or CDM3 medium. RPMI+B27-ins medium consists of RPMI 1640 medium supplemented with 2% B27 without insulin (Life Technologies). CDM3 consists of RPMI 1640 medium (Life Technologies), 500 μgmL⁻¹O. sativa-derived recombinant human albumin (Sigma-Aldrich), and 213 μg L⁻¹ L-ascorbic acid 2-phosphate (Sigma-Aldrich). From day 0 to day 2, the medium was supplemented with 6 μM CHIR99021 (LC Laboratories, Woburn, MA, USA). On day 2, the medium was changed to one that was supplemented with 2 μM Wnt-C59 (Selleck Chemicals). From day 4 on, the medium was changed to one without any supplements. On days 15–20, the cells were plated on the nanopillar devices with the same medium. During the first 24 h after plating on the devices, the human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were incubated with 20% fetal bovine serum, while the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were incubated with 10 μM ROCK inhibitor (Y27632). The medium was changed daily.