title: Lack of cardiac differentiation in c-kit-enriched porcine bone marrow and spleen hematopoietic cell cultures using 5-azacytidine.
abstract: The adult spleen is a source of early hematopoietic stem cells (HSC). We therefore studied whether culturing spleen or bone marrow (BM) HSC in medium containing 5-azacytidine could induce a cardiac phenotype. c-kit enrichment and depletion of adult pig spleen and BM mononuclear cells were obtained by magnetic bead separation using biotinylated pig stem cell factor (c-kit ligand). Cells were incubated with 5-azacytidine for 24 h and refreshed with 5-azacytidine-free medium every 48 h. Western blot was used to detect cardiac troponin and myosin heavy chains. Although 5-azacytidine treatment led to the formation of ball-like cell clusters in both c-kit-enriched populations, these clusters showed no rhythmic contractions (beating), as observed by others. Furthermore, neither cardiac troponin nor myosin was detected in cells derived from either source. Our methodology and treatment with 5-azacytidine did not induce cardiac gene expression in porcine HSC derived from either pig spleen or BM.
authors.affiliation: Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Ramirez
authors.forename: I M
authors.initials: IM
authors.lastname: McMorrow
authors.forename: T M
authors.initials: TM
authors.lastname: Sanderson
authors.forename: C J
authors.initials: CJ
authors.lastname: Lancos
authors.forename: Y-L
authors.initials: YL
authors.lastname: Tseng
authors.forename: D K C
authors.initials: DK
authors.lastname: Cooper
authors.forename: F J M F
authors.initials: FJ
authors.lastname: Dor
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D006410:Hematopoiesis; D018189:Immunomagnetic Separation; D009206:Myocardium / Q000166:cytology*; D009218:Myosins / Q000172:deficiency; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D013154:Spleen / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D013552:Swine; D019210:Troponin I / Q000172:deficiency
chemical_list: D019210:Troponin I; D019009:Proto-Oncogene Proteins c-kit; D009218:Myosins; D001374:Azacitidine