59 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 minutes and immediately replated in 1:10 to 1:15 split ratios in RPMI 1640 + B27 1× with 10% Knock Out Serum Replacement (Gibco) and Thiazovivin 1.0 μM (Selleckchem). At day 12, hiPSCCMs were further expanded for downstream assays in RPMI 1640 + B27 1× differentiation media supplemented with 2.0 to 4.0 μM CHIR99021 (Selleckchem). For the first 24h after passaging, 10% Knock Out Replacement Serum and Thiozovivin 1.0 μM were added to the differentiation media. Generation of eGFP-anillin+ expressing hiPSCs. The previously described eGFP-anillin expression cassette (Hesse et al., 2012) was inserted into the AAV-CAGGS vector (Addgene #22212). hiPSCs (iLB-C1–30m-r12), kindly provided by Dr. O. Brüstle (Univ. of Bonn), were cotransfected (NEONTransfection, Invitrogen) with the AAV-CAGGS-eGFP-anillin vector, the hAAVS1 1L TALEN vector (Addgene #35431) and the hAAVS1 1R TALEN vector (Addgene, #35432) for targeted integration into the AAVS1 locus and several independent eGFP-anillin expressing hiPSC lines were generated. Targeted integration into the AAVS1 locus was proven by a PCR strategy, copy numbers were determined by qPCR. The eGFP-anillin+ expressing hiPSC line used was shown to have one specific AAVS1 integration and one random integration site without known functional consequence. Contact inhibition study. For high cell-cell contact condition (i.e. dense), hiPSC-CMs were either left unpassaged or passaged without splitting, while for low cell-cell contact condition (i.e. sparse), the cells were passaged at 1:10 – 1:15 splitting ratio, depending on cell confluency of the original well. To examine paracrine effect, the conditioned media was collected from densely plated hiPSC-CMs, concentrated using Pierce™ protein concentrator (3K MWCO; Thermoscientific 88512). The concentrated factors were diluted with fresh expansion media (B27 + CHIR) at different ratios (1×, 0.1×, 0.01×), then given to sparsely plated hiPSC-CMs. To examine direct cell-cell contact effect, 100,000 hiPSC-CMs were plated in small surface area (i.e. 48-well-plate) to induce dense culture, while the same number of cells were plated in large surface (i.e. 6-well-plate) for sparse condition. For immunostaining, cells were fixed at 4% paraformaldehyde for 15 min at room temperature, washed three times with washing buffer (0.1% Tween-20/PBS), permeabilized and blocked with blocking buffer (3% bovine serum albumin/2% goat serum/0.01% saponin/PBS for 30 min at room temperature. Corresponding primary antibodies were diluted at desired ratio in blocking buffer (TnT: 1:250, Ki67: 1:200, phH3: 1:400) and were incubated with cells overnight at 4 °C on shaker. Next day, the cells were washed three times with washing buffer, each time for 5 min at room temperature on shaker. Then, the appropriate secondary antibodies were diluted in blocking buffer at 1:300 dilution ratio and were incubated with cells for 1 hr at room temperature on shaker. After the incubation, the samples were washed three times to remove any unbound secondary antibodies. Nuclei were counterstained using Dapi.
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