63 Massive Expansion of Functional Human iPSC-derived Cardiomyocytes 3 In vivo studies in mice. Pregnant mice at gestational day 16.5 and 4-day-old CD1 mice (Jackson Laboratory, Bar Harbor, ME) were given 4–6 consecutive daily intraperitoneally injections of CHIR at a dose of 50mg/kg. After 4 days (embryonic studies) or 6 days (neonatal studies) post-injection, euthanasia was performed by first sedating the mice via isoflurane (inhalant, 2% in 100% oxygen, neonate placed on a warm pad), followed by a secondary cervical dislocation. Death was verified after euthanasia and prior to disposal. All animal experiments were approved by the animal care and use committee (APLAC) at Stanford University. All experiments were performed in accordance with relevant guidelines and regulations of Stanford University. Body and heart weights were measured by a blinded observer. Freshly isolated adult hearts were dissected from mouse chest cavity and washed in PBS to remove excess blood. The postnatal hearts were incubated in 30% sucrose in phosphate buffered solution (PBS) overnight followed by stepwise incubation with a graded concentration of OCT in PBS for cryo-sectioning. Following cryopreservation, hearts were cut into 10 μm sections and lightly fixed in 4% paraformaldehyde in PBS prior to immunostaining. All quantitative analyses of the histological sections were performed on numerically coded animals in an observer-blinded fashion to prevent subjective bias in data analysis. Statistical analysis. Cell counts were performed with Luna-FL fluorescence cell counter, BD flow cytometry system, Image J software or manually. Numerical data are presented individually or as mean ± standard deviation (SD), standard error (SE) or standard error of the mean (SEM). Statistical significance was performed using Excel or Prism software. Values of p<0.05 were considered statistically significant.
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