| Project/Area Number |
18390365
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Digestive surgery
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| Research Institution | Kyoto University |
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Principal Investigator |
IKAI Iwao Kyoto University, school of Medicine, Associate Professor (60263084)
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| Co-Investigator(Kenkyū-buntansha) |
NAKATSUJI Norio Kyoto University, Institute for Frontier Medical Sciences, Professor (80237312)
YASUCHIKA Kentaro Kyoto University, School of Medicine, Instructor (00378895)
HATANO Etsuro Kyoto University, School of Medicine, Instructor (80359801)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥17,500,000 (Direct Cost: ¥15,400,000、Indirect Cost: ¥2,100,000)
Fiscal Year 2007: ¥9,100,000 (Direct Cost: ¥7,000,000、Indirect Cost: ¥2,100,000)
Fiscal Year 2006: ¥8,400,000 (Direct Cost: ¥8,400,000)
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| Keywords | cell transplantation / human ES cells / hepatocytes / hepatic surgery |
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| Research Abstract |
1. Establishment of transgenic human embryonic stem cell line (hESCs)
The hybrid enhancer/promoter of human alpha-fetoprotein (AFP) was produced after cloning from the genome of human embryonic stem cells. The sequence of enhanced green fluorescent protein (EGFP) was ligated under the hybrid promoter of human AFP to construct the expression plasmid, which express EGFP corresponding to the expression of human AFP in the cells. This plasmid was transfected using lipofection to produce transgenic human embryonic stem cell lines, which express EGFP as a marker of AFP expression.
2. Isolation of hESCs derived endodermal cells and differentiation to hepatocytes
The expression of EGFP in the transgenic hESCs described previously was compared using flow cytometry (FACS Vantage SE) after the differentiation among several conditions to determine the ideal differentiation protocol. As a result, we identified the improvement of the efficacy (approx. 21%) and the yields of endodermal differentiation f
… More
rom embryonic stem cells when they were cultured on Matrigel with culture media containing activin and hepatocyte growth factor. The EGFP-expressing cells corresponding to the AFP-expressing cells differentiated from the transgenic hESCs were isolated using flow cytometry and investigated of the gene expression by RT-PCR. The EGFP-expressing endodermal cells expressed the marker genes of mesoendodermal cells, which indicated the fact that definitive endodermal cells were contained in the cell population differentiated and isolated from hESCs in our system. To promote the differentiation of hESC-derived endodermal cells into hepatocytes, the isolated EGFP-positive AFP-expressing endodermal cells derived from hESCs were co-cultured with mesenchymal cells derived from mouse embryonic liver tissue according to the evidence of our previous research. The expression of hepatic marker gene was identified only when hESC-derived endodermal cells were co-cultured with mesenchymal cells. These results suggested that we could produce hepatocytes differentiated from hESCs with enough function to be used for cell transplantation for severe liver diseases in the foreseeable future. Less
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