1995 Fiscal Year Final Research Report Summary
PURIFICATION OF HEMATOPOIETIC STEM CELLS BY A CD117 MONOCLONAL ANTIBODY,MTK1, IN ORDER TO TRANSFECT HUMAN GENES-A PRELIMINARY STUDY
| Project/Area Number |
06454295
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pediatrics
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| Research Institution | INSTITUTE FOR DEVELOPMENT,AGING AND CANCER (IDAC) Tohoku University |
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Principal Investigator |
TSUCHIYA Shigeru Tohoku University Idac, Pediatric Oncology (Po) , Assistant Professor, 加齢医学研究所, 助教授 (30124605)
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| Co-Investigator(Kenkyū-buntansha) |
OHASHI Yoshiyuki Tohoku University Idac, Po, Lecturer, 加齢医学研究所, 助手 (60250825)
KONNO Tasuke Tohoku University Idac, Po, Professor, 加齢医学研究所, 教授 (00004846)
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| Project Period (FY) |
1994 – 1995
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| Keywords | HEMATOPOIETIC STEM CELLS / C-KIT / IL-2 RECEPTOR gamma CHAIN / ELECTROPORATION / GENE TRANSFECTION / BCR / ABL |
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| Research Abstract |
By immunizing an acute megakaryoblastic leukemia cell line (M-MOK) cells to Balb/c mice we have produced 2 monoclonal antibodies, MTK1 and MTK2, with the specificity to CD117 (c-kit). We found that treatment of bone marrow mononuclear cells with the MTK1 and immunomagnetic beads, as well as a CD34 antibody and the beads, was able to purify hematopoietic stem cells (CFU-GM and BUFU-E). In ordor to know stage and lineage specific expression of CD117 we examined leukemic cells and leukemia cell lines. CD117 expressed approximately 70% of myeloid leukemia cells. During in vitro adapation of leukemic cells dominant expression of CD117 on the cell lines with megakaryo/erythromegakaryocytic lineage was noticed, but the mechanism was still under investigation.
To characterize bone marrow hematopoietic cells in a different way we examined the expression of the IL-2 receptor gamma c chains on CD34 positive cells. We found that approximately 40% of CD34 positive cells expressed gamma c chains. Then we examined the number of CFU-GM and BFU-E in CD34 (+) gamma c chain (+) and CD34 (+) gamma c chain (-) cells. CD34 (+) gamma c chain (-) cells contained more BFU-E as compared with those of CD34 (+) gamma c chain (+) cells. No differences were observed for CFU-GM in these 2 fractions.
We tried to introduce bcr/abl cDNA in GM-CSF dependent M-MOK cells by an electroporation method as a preliminary experiment of transfection of genes to hematopoietic cells. Presence of Bcr/abl cDNA in M-MOK cells after selection by G418 was confirmed by a RT-PCR method. However, GM-CSF dependent M-MOK cells were not converted to cytokine independent one under the influence of bcr/abl cDNA.
In order to introduce genes to hematopoietic stem cells there seemed to be many things concerning vectors and target cells which should be solved soon.
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