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Low-adhesion culture selection for human iPS cell-derived cardiomyocytes – Scientific Reports

Reagents

The suppliers and catalog numbers of reagents used in this study are available in Supplementary Table S1 online.

Cell culture media

The serum-free medium for cardiomyocyte selection was prepared by adding 0.01% ascorbic acid phosphate magnesium salt n-hydrate and 1% penicillin–streptomycin solution to Dulbecco’s Modified Eagle’s Medium (DMEM). The medium for glucose-free culture was prepared adding 4 mM sodium l-lactate, 0.01% ascorbic acid phosphate magnesium salt n-hydrate, and 1% penicillin–streptomycin solution to glucose-free DMEM.

Preparation of cells

Cardiomyocytes were derived from hiPSC line 201B7 obtained from RIKEN (Tsukuba, Japan) using a previously reported method22,27. Initially, hiPSCs were genetically modified to express puromycin-resistance gene under the control of the α-myosin heavy chain promoter, enabling the subsequent selection of cardiomyocytes using puromycin. The hiPSCs were cultured on a feeder-layer of mitomycin C-treated mouse embryonic fibroblasts. Differentiation into cardiomyocytes was achieved using a designated suspended aggregation culture device. At the end of differentiation culture, the cells were seeded at a density of 1 × 107 cell/dish in 100 mm tissue culture dishes containing DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution. The cells were incubated in a humidified 37 ℃ incubator with 5% CO2.

Cardiomyocyte selection

The differentiated cells in tissue culture dishes were harvested using Trypsin–EDTA (37 ℃, 10 min), washed three times with serum-free DMEM, and then plated in 100 mm tissue culture dishes at a density of 3 × 106 to 1 × 107 cells/dish. Cardiomyocytes were treated with three main techniques. The first technique involved controlling cell attachment either by using media supplemented with BSA and high molecular weight DS (BSA/DS culture) or by using commercially available low cell attachment dishes (Corning Inc., C/N #3262). The second technique utilized glucose-free culture with lactate-supplemented glucose-free medium. The third technique involved puromycin treatment using the genetically introduced puromycin-resistance gene mentioned earlier. For puromycin treatment, 1.5 μg/mL puromycin dihydrochloride was added to the medium 24 h before harvest. Throughout the selection process, the medium was not replaced except during passaging.

Cell count and flow cytometry

To determine cell numbers and cardiomyocyte purities, the cells, including floating non-attached cells, were harvested using Trypsin–EDTA (37 ℃, 10 min). Cell counting was performed using a disposable cell counter (Waken B Tech, Kyoto, Japan) with Trypan Blue dye to exclude dead cells. Based on the calculated cell numbers, a sample of 5 × 105 cells or less was collected for flow cytometry analysis. If necessary, the remaining cells were plated in new dishes after three washes with serum-free DMEM. The sampled cells were immediately stained by LIVE/DEAD fixable green dead cell stain kit according to the manufacturer’s instructions (1 μL in 1 mL, 30 min, RT). Subsequently, the cells were centrifuged, resuspended in a 1% paraformaldehyde solution, and stored at 4 ℃. After 30 min or more, the cells were centrifuged and resuspended in phosphate buffered saline (PBS) containing 0.1% Triton X-100, 5% blocking one solution, and a 1/200 dilution of anti-cTnT antibody. After 30 min, the cells were centrifuged and resuspended in PBS with 0.1% Triton X-100, 5% blocking one solution, a 1/200 dilution of Alexa Fluor 647 anti-mouse IgG, and 2.5 μg/mL 4ʹ,6-diamidino-2-phenylindole (DAPI). Following another 30 min, the cells were centrifuged and resuspended in PBS. The cell suspension samples were analyzed using a Gallios flow cytometer (Beckman Coulter life sciences). The live cell population was gated using the fluorescence of DAPI and LIVE/DEAD fixable green dead cell stain kit. The ratio of cTnT-positive cells was determined using the fluorescence intensity threshold set at the geometric average between the low intensity peak position and the high intensity peak position.

Analysis of cardiomyocyte selection efficiency

The relative cell number, R, for each selection procedure was determined by comparing the number of cells plated and harvested. The accumulated relative cell number for repeated procedures (such as passaging) was calculated by multiplying the R values of each step. Cardiomyocyte purity, α, in a cell suspension sample was determined using flow cytometry as described above.

Immunofluorescence

The cells on the substrates were fixed using a paraformaldehyde aqueous solution. After rinsing with PBS, the cells were treated with a mouse monoclonal antibody for human cTnT. Subsequently, they were treated with Alexa Fluor 647 anti-mouse IgG secondary antibody, Alexa Fluor 568 conjugated phalloidin, and DAPI.

Measuring beating rates

The cells were seeded in five wells of an EZ-BindShut SP non-adherent U-bottom 96-well plate (AGC Techno Glass) at 1 × 104 cells/well in serum-free medium. After 5 or 6 days, microscopic videos (> 30 s) were captured both before and 30 min after the administration of 100 nM isoproterenol. Beating rates were measured from the captured videos.

Gene expression analysis

The cells were cultured in a 3.5 cm tissue culture dish with serum-free medium for 5 days to allow recovery from treatment stresses. Total RNA was extracted using the RNeasy plus mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized from the RNA using a RT-RamDA cDNA synthesis kit (Toyobo). Subsequently, gene expression was analyzed through TaqMan RT-PCR assay on a ViiA 7 real-time PCR system (Thermo Fisher Scientific). TaqMan probes for the target genes can be found in Supplementary Table S2 online. For comparison of the expression levels of each gene, we calculated − ΔCt as Ctref–Ctgoi where Ctref is the Ct value of reference gene (GAPDH) and Ctgoi is the Ct value of the gene of interest. Higher − ΔCt indicates higher expression of the gene of interest. Additionally, we computed − ΔCt of three cardiomyocyte-specific gene pairs, RYR2/TNNT2, MYL2/MYL7, and MYH7/MYL6. In this case, the gene after the slash is the reference gene.

Statistical analysis

Statistical analysis was conducted using paired Student’s t-test, two-way ANOVA, Dunnett’s test or one-way ANOVA with Greenhouse–Geisser correction. The results are presented on each figure.