Ere kindly provided by Prof. Duanqing Pei. Cells were cultured on 0.2 gelatin-coated 15900046 plastic petri dishes without feeder cells in Dulbecco’s modified Eagle’s minimal essential medium (DMEM, Gibco, Invitrogen Corporation, Grand Island, NY, USA) supplemented with 15 fetal bovine serum (Gibco), 0.1 mmol/L nonessential amino acids (Sigma, St. Louis, MO, USA), 0.1 mmol/L b-mercaptoethanol, penicillin (100 U/mL), streptomycin (100 mg/mL), and 100 U/ mL leukemia inhibitory factor (LIF) (Chemicon International Inc., Temecula, CA).Isolation and Culture of NCMs and EKsNCMs were obtained from enzymatically isolated crude cellular fractions from neonate mouse ventricle as described previously [31]. Animal experiments were approved by the Fourth Military Medical University on the Use and Care of Animals. Myocyte isolation was conducted in accordance to Institutional Animal Care and Use Committee Guidelines. 1-day-old aMHC-GFP transgenic mice, identified by genotype PCR, were euthanized by injection of pentobarbital (80 mg/kg). The hearts were quickly excised, and washed with normal Tyrode solution. Ventricles were trimmed free of atria and major blood vessels, minced and placed in 0.1 collagenase (Sigma) solution. After 20 min enzyme digestions, the released cells were filtered, centrifuged and resuspended. Only cardiomyocytes, which expressed GFP, were sorted from the mixed cells by reporter-based NHS-Biotin price fluorescenceactivated cell sorting (FACS). The sorted NCMs were co-cultured with EBs in DMEM supplemented with 20 ES cell-qualified FBS(Gibco), 2 mM GlutaMAX (Invitrogen), 0.1 mM nonessential amino acid(Invitrogen) at a density of 26104 cells/cm2. EKs were obtained from the skin of newborn (2?-day old) mice. The detached 
epidermal sheets from newborn (2?-day old) mice were cut roughly into 1-mm-diameter pieces, and shaken in a flask with 0.1 trypsin-EDTA solution for 6? min at 37uC. The suspension was then filtered through 15755315 a mesh (74 m pore size) and centrifuged at 400 g for 5 min. EKs were obtained as sediment, which predominantly consisted of basal cells, intermingled with stratum spinosum cells. Keratinocytes were cultured in keratinocyte serum-free medium (Gibco) with 25 g/ml bovine pituitary extract (Gibco). These EKs were used for reconstruction culture after subculturing 2 or 3 times for 2 weeks. EKs were observed and photographed under a phase-contrast inverted microscopy (Olympus Optical Co. Ltd.) to evaluate their appearances.Semi-quantitative Reverse Transcription-PCRSemi-quantitative reverse transcription (RT)-PCR for MLC2v, MLC2a, a-MHC, ANF, Nkx2.5, GATA-4 and GAPDH was performed using standard procedures. Briefly, total RNA was prepared using Trizol reagent 
(Invitrogen). First strand cDNA was synthesized from 1 mg of total RNA, in a total volume of 20 mL, using oligo (dT)18 primer and a RevetAidTM First Strand cDNA Synthesis Kit. The RT-PCR was performed with GAPDH mRNA as a normalizing internal PD-1/PD-L1 inhibitor 1 custom synthesis control. The resulting cDNA (50 ng) was amplified by PCR using specific primers. Primer sequences and PCR conditions are detailed in Table S1. Thermal cycling (in 20 mL) was performed as follows: a 3 min denaturation at 94uC, 30 cycles of 94uC for 30 sec, 60uC for 30 sec and 72uC for 1 min, and a final extension for 6 min at 72uC. PCR products were resolved by electrophoresis on 1.5 agarose gels. They were visualized by UV transillumination and photographed. Semiquantitative analysis was done by Alphaview 1.3 software (Alpha Lnnotech Inc.).Rea.Ere kindly provided by Prof. Duanqing Pei. Cells were cultured on 0.2 gelatin-coated 15900046 plastic petri dishes without feeder cells in Dulbecco’s modified Eagle’s minimal essential medium (DMEM, Gibco, Invitrogen Corporation, Grand Island, NY, USA) supplemented with 15 fetal bovine serum (Gibco), 0.1 mmol/L nonessential amino acids (Sigma, St. Louis, MO, USA), 0.1 mmol/L b-mercaptoethanol, penicillin (100 U/mL), streptomycin (100 mg/mL), and 100 U/ mL leukemia inhibitory factor (LIF) (Chemicon International Inc., Temecula, CA).Isolation and Culture of NCMs and EKsNCMs were obtained from enzymatically isolated crude cellular fractions from neonate mouse ventricle as described previously [31]. Animal experiments were approved by the Fourth Military Medical University on the Use and Care of Animals. Myocyte isolation was conducted in accordance to Institutional Animal Care and Use Committee Guidelines. 1-day-old aMHC-GFP transgenic mice, identified by genotype PCR, were euthanized by injection of pentobarbital (80 mg/kg). The hearts were quickly excised, and washed with normal Tyrode solution. Ventricles were trimmed free of atria and major blood vessels, minced and placed in 0.1 collagenase (Sigma) solution. After 20 min enzyme digestions, the released cells were filtered, centrifuged and resuspended. Only cardiomyocytes, which expressed GFP, were sorted from the mixed cells by reporter-based fluorescenceactivated cell sorting (FACS). The sorted NCMs were co-cultured with EBs in DMEM supplemented with 20 ES cell-qualified FBS(Gibco), 2 mM GlutaMAX (Invitrogen), 0.1 mM nonessential amino acid(Invitrogen) at a density of 26104 cells/cm2. EKs were obtained from the skin of newborn (2?-day old) mice. The detached epidermal sheets from newborn (2?-day old) mice were cut roughly into 1-mm-diameter pieces, and shaken in a flask with 0.1 trypsin-EDTA solution for 6? min at 37uC. The suspension was then filtered through 15755315 a mesh (74 m pore size) and centrifuged at 400 g for 5 min. EKs were obtained as sediment, which predominantly consisted of basal cells, intermingled with stratum spinosum cells. Keratinocytes were cultured in keratinocyte serum-free medium (Gibco) with 25 g/ml bovine pituitary extract (Gibco). These EKs were used for reconstruction culture after subculturing 2 or 3 times for 2 weeks. EKs were observed and photographed under a phase-contrast inverted microscopy (Olympus Optical Co. Ltd.) to evaluate their appearances.Semi-quantitative Reverse Transcription-PCRSemi-quantitative reverse transcription (RT)-PCR for MLC2v, MLC2a, a-MHC, ANF, Nkx2.5, GATA-4 and GAPDH was performed using standard procedures. Briefly, total RNA was prepared using Trizol reagent (Invitrogen). First strand cDNA was synthesized from 1 mg of total RNA, in a total volume of 20 mL, using oligo (dT)18 primer and a RevetAidTM First Strand cDNA Synthesis Kit. The RT-PCR was performed with GAPDH mRNA as a normalizing internal control. The resulting cDNA (50 ng) was amplified by PCR using specific primers. Primer sequences and PCR conditions are detailed in Table S1. Thermal cycling (in 20 mL) was performed as follows: a 3 min denaturation at 94uC, 30 cycles of 94uC for 30 sec, 60uC for 30 sec and 72uC for 1 min, and a final extension for 6 min at 72uC. PCR products were resolved by electrophoresis on 1.5 agarose gels. They were visualized by UV transillumination and photographed. Semiquantitative analysis was done by Alphaview 1.3 software (Alpha Lnnotech Inc.).Rea.
