| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We successfully identified human cord blood-derived CD34-negative (CD34) severe combined immunodeficiency (SCID)-repopulating cells (SRCs), which could not home into the bone marrow (BM) niche by conventional tail vein injection (TVI) method, using newly developed intra-bone marrow injection (IBMI) method. Moreover, in vitro transwell migration assay clearly identified hitherto unidentified CD34-positive (CD34^+) SRCs, which do not respond to the gradient of SDF-1. Both CD34^- and CD34^+ SRCs expressed lower levels of important homing receptors, such as CXCR4. Therefore, they could not efficiently home into the BM niche by conventional TVI. These results strongly suggest that the application of IBMI method in the clinical hematopoietic stem cell transplantation (HSCT) may make it possible to develop a very effective HSCT in the near future.
Our identified CD34^- SRCs had a potential to differentate into both myeloid and lymphoid lineages, as did CD34^+ SRCs. The coculture of these CD34^
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-SRCs and mouse stromal cell line, HESS-5 cells in vitro, and the secondary transplantation experiments, demonstrated that CD34^- SRCs are very primitive hematopoietic stem cells (HSCs). Until now, CD34^+CD38^- SRCs are believed to be most primitive HSCs, however, CD34^- SRCs had more immature characteristics of HSCs, including more potent proliferative potential and self-renewal ability. Interestingly, there was a clear difference between the pattern and timing of mobilization of these SRCs in vivo. Especially, CD34^- SRCs seemed to be retained much longer time in the niche of the injected site of bone.
To highly purify the CD34^- SRCs, it is necessary to more precisely clarify the immunophenotypes of these CD34^- SRCs. Our studies clearly demonstrated that CD34^-SRCs did not express tyrosine kinase receptors, such as c-kit and flt3. According to our recent data, it is suggested that the imuunophenotype of the most primitive human hematopoietic stem cells is Lin^-CD45^<low>CD34^-CD38^-c-kit^-flt3^-. Less
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