Cardiomyocyte differentiation of rat bone marrow multipotent progenitor cells is associated with downregulation of Oct-4 expression.
This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.
Lu
Tiewei
T
Department of Cardiology, Children's Hospital of Chongqing Medical University , Chongqing, PR China.
Pelacho
Beatriz
B
Hao
Hong
H
Luo
Min
M
Zhu
Jing
J
Verfaillie
Catherine M
CM
Tian
Jie
J
Liu
Zhenguo
Z
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D018031:Connexin 43 / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D050991:GATA4 Transcription Factor / Q000378:metabolism; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism
D018031:Connexin 43; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C524161:Nkx2-5 protein, rat; D050814:Octamer Transcription Factor-3; D014157:Transcription Factors; D019210:Troponin I; D001374:Azacitidine