2007 Fiscal Year Final Research Report Summary
Standardization of embryoid body formation from embryonic stem cellsand quality estimation of embryoid bodies
| Project/Area Number |
18560751
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | University of Yamanashi |
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Principal Investigator |
KUROSAWA Hiroshi University of Yamanashi, Department of Research Interdisciplinary Graduate School of Medicine and Engineering, Associate Professor (10225295)
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| Project Period (FY) |
2006 – 2007
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| Keywords | ES cell / embryoid body / quantitative PCR / EB formation method / deifferentiation / gene expression / standardization |
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| Research Abstract |
The gene expression of EBs was quantitatively analyzed by real-time PCR and the EBs were standardized based on the data of real-time PCR.
1. Formation of various types of EBs Various types of EBs were famed in different culture conditions of culture vessel, seeding cell density, oxygen, and nutrient. The EBs were famed by hanging drop method or in low-adherence 96-well multi-plate.
2. Gene analysis of EBs by real-time PCR
Marker genes (Oct3/4, Rex1, GATA4, GATA6, TTR, AFP, α-MHC, Nkx2.5), which represent differentiation status of EBs, were quantitatively analyzed by real-time PCR.
3. Graphical presentation of gene expression pattern of EBs The gene expression pattern of EBs was graphically shown to characterize the differentiation status of EBs.
4. Making sure of the relationship between gene expression pattern and actual differentiation observed in the EB differentiation culture
The EBs highly expressed of a-MHC and Nkx2.5 generate cardiomyocytes with high efficiency. The EBs highly expressed of FIR and AFP have a trend to generate visceral yolk-sac-like structure. It was found that the gene egression pattern in EBs directly reflected the differentiation tendency to be observed in the following culture of EBs.
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