| Project/Area Number |
13480298
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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 |
Biomedical engineering/Biological material science
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| Research Institution | Tokyo women's Medical university |
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Principal Investigator |
KAWAUCHI Kiyotaka Tokyo women's Medical university, Department of Medicine, Assistant professor, 医学部, 助教授 (00214594)
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| Co-Investigator(Kenkyū-buntansha) |
YAMADA Osamu Tokyo women's Medical university, Medical Research Institute, Assistant professor, 医学部, 助教授 (30167712)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Cord blood / Hematopoietic stem cell / Angioendothel / Tissue-engineering / Megakaryopoiesis |
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| Research Abstract |
Human umbilical cord blood was obtained during normal full-term deliveries after obtaining informed consent. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation and CD34^+ cells were purified by a magnetic bead separation method. More than 95% of recovered cell was CD34^+.Purified CD34^+ cells were incubated in serum-free suspension culture in the presence of various combination of cytokines and analyzed by flowcytometry using a panel of monoclonal antibodies at day 14 and 21. A combination of IL6, SCF, TPO, IL3, EPO, FLT3 and G-CSF was used for the determination of various progenitors generated in suspension culture at each time points. In some experiments, the plasmid containing the full-length cDNA of telomerase(hTERT) and mock vector were transfected to K562 cells with the characteristics of leukemic stem cells in order to provide data on a possible approach to telomerase gene therapy.
The megakaryocytic fraction was determined as the percentage of CD41^+ and/or CD42^+ cells. TPO always resulted in growth advantage for megakaryocytic differentiation, whereas SCF suppressed the differentiation. In cultures containing TPO as a single growth factor, maximal expansion of CD41^+ cells was achieved at day 14 and total percentage of CD41^+ cells steadily increased when ingenol was added in culture medium. Ectopic expression of hTERT in k562 cells showed a survival advantage in the absence of serum. Transduced cells retained phenotypic characteristics, differentiation ability, and the signal transduction response to TPA. These data suggest that ectopic expression of hTERT by normal hematopoietic stem cells may confer a survival advantage without changing innate biological characteristics.
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