| Budget Amount *help |
¥3,920,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
(1) Establishment of regulatory system for the expression of transcription factors in mouse ES cells
We produced regulatory plasmid designed to express transcription factors specific for mesoendodermal cells such as Mix and Hex, which were subcloned from embryoid bodies. These transcription factors were expressed under the control of Tet-ON system. The target cells in which transcription factors were preferentially expressed could be determined as green fluorescent protein (GFP) expressing cells because GFP coding sequence was ligated after the sequence of internal ribosomal entry site (IRES). Transgenic mouse ES cells were produced after the transfection of this plasmid.
(2) In vitro differentiation of mouse ES cells into mature hepatocytes and insulin producing islet β cells
After the isolation of GFP positive cells derived from transgenic mouse ES cells produced as previously described, which express Mix by the administration of Doxcycline in the culture medium, GFP positive cells were
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cultured under several conditions identified by the combination of several growth factors (HGF, FGF2, ATRA) and also several extra-cellular matrices (Collagen type I, IV, Laminin, Matrigel). RT-PCR was performed to identify the expression of mesodermal and endodermal marker genes in the cells during the differentiation culture. Although the expression of several mesodermal markers such as Gooscoid and brachury and also several endodermal marker such as GATA4 and FoxA2 were identified in the isolated cells, the expression of hepatocyte marker such as alfa-fetoprotein, albumin were not identified. In addition, the expression of Glut-2 and Pdx-1 were not determined whereas the expression of insulin was detected in the isolated Mix expressing cells. Therefore, the sufficient differentiation findings for hepatocytes and also insulin producing islet β cells were not comfirmed in our experiments. On the other hand, we identified the expression of hepatocyte markers in the cells derived from mouse ES cells co-cultured with Thy-1 positive mesenchymal cells originated from mouse fetal liver. We also identified morphological features as hepatocytes in these differentiated cells using phase contrast microscopy and also electron microscopy.
(3) The transplantation of hepatocyte like cells derived from mouse ES cells
We performed transplantation experiments of hepatocyte-like cells differentiated from mouse ES cells marked with LacZ gene into transgenic mice in which lethal liver damage occurred by the administration of diphtheria toxin. We identified not only the incorporation of transplanted hepatocyte like cells into recipient liver but also the improvement of mortality rate ofrecipient mice suffered from lethal liver damage. Less
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