Supplementary MaterialsVideo 1: Supplementary Video 1: Time lapse imaging of primary mouse cardiomyocytes (related to Figure S1A) Day 7 primary mouse cardiomyocytes infected with CDK1:CCNB:AURKB adenoviruses. Damage Response, Related to Figures 1C2 and Supplementary Videos 1C2)(A) Time lapse imaging of cell division in P7 mouse cardiomyocytes isolated from -MHC-GFP transgenic mice overexpressing CDK1, CCNB and AURKB (3F). Panels are representative of images recorded every hour for 4 days and demonstrate cell division of β-Secretase Inhibitor IV a cardiomyocyte, followed by rapid cell death seen in last panel (see Supplementary Video 1 and 2). (B) Time lapse imaging of cell division in 60-day-old hiPS-derived cardiomyocytes overexpressing 3F. Panels are representative of images collected every hour for 2 days. Last panel represents immunocytochemistry for cardiac Troponin T (cTnT) in the 36-hour cells. Arrows denote dividing cells and their progeny. (C) Representative western blots and β-Secretase Inhibitor IV quantification for the indicated DNA damage response markers (p-ATM, p-Chk1 and p-Chk2) in response to virus encoding 4F, 3F or LacZ (control) in human iPS-CMs (n=3 independent experiments with two replicates in each; *p 0.05, bars indicate means with SEM). Figure S2. Validation of the Mosaic Analysis with Double Markers (MADM) System to Detect 4F-Induced Cardiomyocyte Proliferation Related to Figure 3 (A) Schematic diagram showing the principle behind the lineage tracing of proliferating cells in MADM mice (adapted from (Gitig, 2010)). (B, C) Representative histologic images of cardiomyocyte-specific -MHC-Cre MADM hearts infected with 4F at the time of infarct and sectioned 4 days later. Single-colored cardiomyocytes stained positive for PHH3 (B) and EDU incorporation (C). Low and high magnification of indicated areas are shown, Figure S3. Validation of -MHC-Cre MADM Fluorescent Reporter and Examples of Single-Colored Cells in Infarct and Peri-Infarct Regions, Related to Figure 3 (A) Representative GFP- or RFP-immunostained and unstained adjacent heart sections from -MHC-Cre MADM mice showing that the signal intensity was similar in immunostained sections compared to sections visualized by fluorescence, validating use of the fluorescent reporter in this system. Arrows are pointing to Rabbit Polyclonal to SLC15A1 two single-colored cells showing similar signal intensities in the two adjacent sections. (B) Representative images from -MHC-Cre MADM mouse heart sections treated with 4F showing single-colored cardiomyocytes at the infarct zone (top two panels). Bottom panel shows a representative peri-infarct region without scar where there are many events of recombination including a single-colored cardiomyocyte. Figure S4. Spatial Location and Nucleation of Divided Cardiomyocytes or Related to Figure 4 (A) Quantification of isolated Thy1+ cells from ubiquitous -Actin-Cre-MADM mice by using a Langendorff preparation, digesting the heart, and sorting a cardiac fibroblast-enriched population marked with the APC-conjugated-Thy1 antibody. FACS was used to quantify the number of single-colored fibroblasts and revealed no difference between animals treated with 4F or LacZ control virus (n=4 animals in each group). (B) Representative FACS plots showing infection efficiency of GFP adenovirus in Thy1+ cardiac fibroblasts infected with 10 or 100 MOI, compared to iPS-CMs infected with 10 MOI of the virus. (C) Representative FACS plots (left panels) and immunostaining (right panels) of EDU incorporation in DDR2+ cells (pre-sorted for Thy1+) infected with either LacZ control virus, or CDK1-CDK4-CCNB-CCND (4F) for 48 hours (n=3 independent experiments and 3 technical replicates in each). (D) Quantification of FACS analysis (C) from pre-sorted Thy1+cardiac fibroblasts co-stained with DDR2 (fibroblast marker) and EDU and infected with either LacZ control virus, or 4F viruses for 48 hours (n=3 independent experiments with 3 replicates in each). Bars indicate means and SEM. NIHMS966576-supplement-supplement_1.pdf (1.4M) GUID:?3BB7CF35-BDBB-4B01-89A1-DB907A4E8003 SUMMARY Human diseases are often caused by loss of somatic cells incapable of re-entering the cell cycle for regenerative repair. Here, we report a combination of cell-cycle regulators that induce stable cytokinesis in β-Secretase Inhibitor IV adult post-mitotic cells. We screened cell-cycle regulators expressed in proliferating fetal cardiomyocytes and found overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 efficiently induced cell division in post-mitotic mouse, rat and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome-mediated degradation of the protein products. with the Cre-recombinase dependent Mosaic Analysis with Double Markers (MADM) lineage tracing system revealed similar efficiency in mouse hearts, with cardiac regeneration upon delivery of cell-cycle regulators immediately after myocardial infarction and even 1 week after injury. RESULTS Screening β-Secretase Inhibitor IV for Cell-Cycle Genes That Promote Cardiomyocyte Proliferation To identify factors that influence cardiomyocyte proliferation, we performed transcriptome analyses on embryonic day 10.5 (E10.5, fetal), 1-day-old (P1, neonatal), and 8-week-old (adult) C57/Bl6 mouse hearts and compared the expression levels of the major cell-cycle regulators. Most cell-cycle genes in adult hearts were significantly downregulated, compared to neonatal β-Secretase Inhibitor IV and fetal hearts (Figure 1A). We cloned 15 of the top differentially regulated genes between proliferative (fetal/neonatal) and non-proliferative (adult).