| Budget Amount *help |
¥19,000,000 (Direct Cost: ¥19,000,000)
Fiscal Year 1985: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1984: ¥17,000,000 (Direct Cost: ¥17,000,000)
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| Research Abstract |
The conventional culture condition for colony formation of hemopoietic cells has problems that serum and crude stimulator (CSF) are added to the medium. This makes it impossible to analyze fine requirements necessary for differentiation and proliferation of hemopoietic stem cells. It was our aim of this project to compose a culture system without the addition of serum, with highly purified CSF, and the isolated hemopoietic stem cells picked up by a micromanipulator.
We completed serum-free culture system of GM-, erythroid and mixed colony formation by murine as well as human hemopoietic cells. To concentrate stem cells we treated mice with 5-fluorouracil, which eliminated mature hemopoietic cells. Bone marrow cells from treated mice made colonies consisting of undifferentiated stem cells, serving for the single cell culture material.
Highly purified interleukin 3 supported mixed colony formation, in which erythroid cells proliferated only when highly purified erythropoietin was added. There seemed two step requirement of hemopoietic factors during the stem cell maturation.
Simultaneous analysis of chromosomes and morphology for single colonies of hemopoietic cells were performed on various hemopoietic disorders. We confirmed that CMMoL, juvenile type CML, erythroleukemia, myelofibrosis and CML are clonal disorders of multipotent stem cells. Of the first two diseases, such clonality has first been reported in this project.
We also found that the formation of human megakaryocyte colonies were specifically inhibited by platelet lysate, one of which the platelet derived growth factor is a candidate.
In conclusion we achieved a more than expected saticefactory results first proposed for this project.
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