1999 Fiscal Year Final Research Report Summary
Effects of inhibition of gap junctional intercellular communication on neural differentiation of mouse embryonic stem cells in vitro
| Project/Area Number |
09670233
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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 |
Experimental pathology
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| Research Institution | Kyoto Prefectural University of Medicine |
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Principal Investigator |
OYAMADA Yumiko Kyoto Prefectural University of Medicine, Department of Pathology and Cell Regulation, Instructor, 医学部, 助手 (40231245)
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| Co-Investigator(Kenkyū-buntansha) |
OYAMADA Masahito Kyoto Prefectural University of Medicine, Department of Pathology and Cell Regulation, Associate Professor, 医学部, 助教授 (30183255)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Intercellular communication / Gap junctions / Connexin / Neural differentiation / Embryonic stem cells |
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| Research Abstract |
1. Establishment of an in vitro system for neural differentiation using mouse embryonic stem (ES) cells in vitro
We established an in vitro system for neural differentiation using mouse ES cells. Undifferentiated ES cells were cultured in suspension for 8 days and treated with all-trans retinoic acid during the latter half of the period. Thereafter, cell were transferred to adhesion culture. Neurites were observed in more than 90% of embryoid bodies. We dispersed embryoid bodies by treatment with trypsin and developed a dispersed cell culture system for early neuronal cells. In this system, cells positive for neuronal markers formed small colonies after one day of culture.
2. Changes in gap junctional intercellular communication during neural differentiation of ES cells in vitro
We measured gap junctional intercellular communication by microinjection tracer coupling with neurobiotin. Microinjected neurobiotin was found to spread into all cells constituting colonies that expressed A2B5 at day 1 of adhesion culture, but the dye did not spread into flat cells when there was no A2B5 expression around the colony. After day 3 of adhesion culture, neurobiotin injected into a single cell in the colony was restricted to small portions of the colony. These results suggest that compartmentalization of gap junctional communication within neural cells decreases rapidly during the process of neural differentiation.
3. Preparation of a dominant-negative Cx43 expression vector
We prepared a dominant-negative Cx43-green fluorescent protein (GFP) expression vector and transfected into a cell line expressing wild-type Cx43. This vector showed dominant negative effects on wild-type Cx43.
4. In vitro differentiation of ES cells deficient in Cx43
We found that Cx43 -/- ES cells differentiated into spontaneously contracting cardiomyocytes in vitro similar to the wild-type cells although they showed a very small extent of Lucifer Yellow dye coupling at all stages of in vitro differentiation.
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