Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Induced Cardiac Progenitor Cells Repopulate Decellularized Mouse Heart Scaffolds and Differentiate to Generate Cardiac Tissue

doi: 10.1016/j.bbamcr.2019.118559

Figure Lengend Snippet: (A) Schematic timeline of experimental design for reprogramming of adult wild type lung fibroblasts (derived from C57BL/6J mouse) and cardiac fibroblasts homozygous for the rtTA transgene (derived from R26-M2rtTA knock-in mouse). (B) Adult mouse lung and rtTA-cardiac fibroblasts were infected with a doxycycline inducible GFP expressing lentivirus that also contained an IRES-mCherry cassette, and greater than 90% of infected fibroblasts showed GFP/mCherry expression after doxycycline induction indicating a robustly functional expression system. (C) rtTA-cardiac fibroblasts were infected with 5 cardiac factors (MTGNB) and cultured in iCPC induction medium containing doxycycline, LIF and BIO. Cells undergoing reprogramming appeared morphologically different from fibroblasts and could be identified as small proliferative clusters after 2 weeks. (D) 4 weeks after induction of reprogramming of rtTA-cardiac fibroblasts, proliferative reporterless iCPC colonies were observed. (E) Number of reporterless iCPC colonies, derived from lung fibroblasts or rtTA-cardiac fibroblasts, observed 4 weeks after infection with 5 cardiac factors (50,000 starting fibroblasts) (n=3, SEM). (F) Reporterless iCPCs, derived from wild type lung fibroblasts and rtTA-cardiac fibroblasts, could be stably expanded without exogenous expression of cardiac factors. (G) Reporterless iCPCs expressed transcription factors Gata4, Nkx 2.5, and Irx4 which are associated with cardiac progenitor cells. (H) Flow cytometry revealed reporterless iCPC generated from rtTA-cardiac fibroblasts expressed Cxcr4, showed dim expression for Flk1, but only a small fraction was ckit positive.

Article Snippet: Flow cytometry analysis was done using cell surface markers, including Cxcr4(BioLegend 647/APC conjugate – 1:50), Flk1 (BD Pharmingen PE conjugate – 1:20), cKit (BD Pharmingen PE/Cy7 conjugate – 1:50), and PDGFRα (Santacruz PEconjugate – 1:20).

Techniques: Derivative Assay, Knock-In, Infection, Expressing, Functional Assay, Cell Culture, Stable Transfection, Flow Cytometry, Generated