The most profound effect was observed between days 28 and 42 after MI induction, highlighting the need for long-term evaluation of any cell-based therapy effects on regenerating myocardium. of the more receptive strain to compare the therapeutic effect of both cell types. Ultrasonography, performed on days 7, 14, 28 and 42, revealed a significant decrease of left ventricular ejection fraction (LVEF) in all MI-induced groups. No improvement of LVEF was observed in ADSC-treated and mice. In contrast, administration of hiPSC-CMs resulted in a substantial increase of LVEF, occurring between 28 and 42 days after MI, and decreased fibrosis, regardless of genetic modification. Importantly, bioluminescence analysis, as well as immunofluorescent staining, confirmed the presence of hiPSC-CMs in murine tissue. Interestingly, the luminescence signal was strongest in hearts treated with hiPSC-CMs overexpressing HO-1. Performed experiments demonstrate that hiPSC-CMs, unlike ADSCs, are effective in improving heart function after MI. Additionally, long-term evaluation of heart function seems to be crucial for proper assessment of the effect of cell administration. for 40 min at room temperature (RT). The layer of mononuclear cells was then transferred into a new conical tube, washed twice with PBS and resuspended in StemPro-34 medium supplemented with StemPro-34 Nutrient Supplement (ThermoFisher Scientific) and cytokines: 100 ng/mL SCF (Peprotech, Cranbury, NJ, USA), 100 ng/mL FLT3-Ligand (Peprotech), 20 ng/mL IL-3 (Peprotech) and 20 ng/mL IL-6 (PBMC medium). Cells were plated on a 24-well plate and cultured for an additional 6 days with medium changed every other day. Subsequently, 105 isolated PBMCs were reprogrammed using Cytotune-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific) according to the manufacturers protocol. Briefly, cells were suspended in PBMC medium made up of KOS (encoding KLF4, OCT4 and SOX2), hc-MYC and hKLF4 Sendai vectors (multiplicity of contamination (MOI) = 5, 5 and 3, respectively), centrifuged at 1000 for 30 min at RT and seeded on a 24-well plate. After 24 h, medium was replaced with fresh PBMC medium for an additional 2 days, after which cells were collected and seeded in 2 wells of a 6-well plate covered with Geltrex (ThermoFisher Scientific, diluted according to manufacturers protocol) in StemPro-34 medium supplemented with StemPro-34 Nutrient Supplement. On day 7, half of the medium was replaced with Essential 8 medium (E8, ThermoFisher Scientific), and cells were further cultured in E8 for additional 2 weeks. Then, the hiPSC colonies were picked and expanded. The hiPSC line used in this study was cultured in E8 on Geltrex-coated plates in standard culture Rabbit Polyclonal to CSE1L conditions (37 C, 5% CO2) and passaged upon reaching 80C90% confluency using 0.5 mM EDTA solution. Pluripotency of hiPSCs was confirmed with immunofluorescent analysis of OCT4, NANOG, SSEA4, TRA-1-60 and TRA-1-81 expression as well as in vitro spontaneous differentiation assay (as described in the Supplementary Methods). 2.3. Cardiac Differentiation of hiPSCs To obtain genetically modified cardiomyocytes, lentiviral vector-transduced and sorted hiPSCs were subjected to cardiac differentiation according to the protocol described by Lian et al. [16]. Briefly, 3 104 cells were seeded on Geltrex-coated 24-well plate and Acrizanib cultured in E8 for additional 4 days until they reached 100% confluency. Then, the Acrizanib medium was changed to RPMI1640 (Lonza) supplemented with 2% B27 without insulin (ThermoFisher Scientific, RMPI/B27-ins medium) and 12 M CHIR99021 (Sigma-Aldrich). After 24 h, the medium was replaced with RMPI/B27-ins for an additional 2 days. On day 3, cells were stimulated with 5 M IWR-1 (Sigma-Aldrich) in RPMI/B27-ins (collected from the cells and fresh mixed 1:1). After 48 h, the medium was replaced with fresh RPMI/B27-ins; starting from day 7, cells were cultured in RPMI1640 supplemented with 2% B27 (ThermoFisher Scientific, RMPI/B27), which was changed every third day. On day 20, the cells were collected and passaged using Multi Tissue Dissociation Kit 3 (Miltenyi Biotec, Bergisch Gladbach, Germany), collected in RPMI1640 supplemented with 20% FBS (EURx, Gdask, Poland), centrifuged (200 mice (strain [13], which on the other hand did not tolerate ketamineCxylazine well (unpublished). In that case, all animals anesthetized with 2,2,2-tribromoethanol survived the MI, and any observed demise occurred mainly in wild-type mice between the 3rd and 5th days after LAD ligation due to left ventricular free wall Acrizanib rupture [13]. It is known that.
