2010 Fiscal Year Final Research Report
Identification of priming factors that determine cardiac lineage of cardiac stem cells
| Project/Area Number |
20249046
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Kyoto Prefectural University of Medicine |
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Principal Investigator |
MATSUBARA Hiroaki Kyoto Prefectural University of Medicine, 医学研究科, 教授 (10239072)
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| Co-Investigator(Kenkyū-buntansha) |
UEYAMA Tomomi 京都府立医科大学, 医学研究科, 講師 (80379388)
MATOBA Satoaki 京都府立医科大学, 医学研究科, 講師 (10305576)
AMANO Katsuya 京都府立医科大学, 医学研究科, 博士研究員 (70468263)
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| Project Period (FY) |
2008 – 2010
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| Keywords | 再生医学 / 循環器・高血圧 / 内科 / 臨床 / 心筋幹細胞 |
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| Research Abstract |
Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation ; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of down-stream targets Smadl/5/8 protein and Idl gene, whereas treatment with Cv2 conversely inhibited Smadl/5/8 activation and Idl expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smadl/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Idl and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.
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