We propose to continue our studies of the cell biology of hemopoietic stem cells. Recent progress in the molecular biology of colony-stimulating factors (CSFs) has not only made available most of the hemopoietic factors but has also revealed the presence of complex molecular and cellular mechanisms of hemopoiesis in culture. Therefore in order to elucidate the precise mechanisms of hemopoiesis, the development of a serum- free culture system for purified progenitors is necessary. We have partially succeeded in developing a serum-free culture system for enriched human progenitors based on modifications of alpha-medium. We propose to improve the technique and establish an optimal serum-free culture system for purified human hemopoietic progenitors.
Our second aim i s to continue to delineate the lineage specificities and targets of recombinant CSFs. Preliminary information on serum-free culture suggests that interleukin-3 (IL-3) is a stage-specific CSF and does not support the terminal process of hemopoietic differentiation. We recently discovered that B cell stimulatory factor (BSF-2) appears to possess synergistic activity with IL-3. We will characterize these factors in serum-free as well as serum-containing cultures of the purified hemopoietic progenitors. The third specific aim relates to the development of a primary clonal culture assay for B-lymphocyte progenitors. We have observed lymphocyte colonies containing mostly cells in the B cell lineage in high cell density culture of spleen cells obtained from 5-fluorouracil (5-FU)- treated mice in the presence of BSF-2. We will confirm the clonal nature of the colonies and develop a quantitative culture method for early lymphocyte progenitors. Finally, effects of recombinant CSFs will be tested in vivo in mice. We propose to test the hypothesis of stochastic stem cell differentiation in vivo and examine the mechanisms by which factors such as IL-1 alpha and BSF-2 interact with IL-3. The information to be obtained is likely to be pertinent to the understanding of the mechanisms of hemopoiesis in vivo and will provide basic information for the future use of CSFs in patients with hemopoietic disorders.
|Sato, T; Laver, J H; Ogawa, M (1999) Reversible expression of CD34 by murine hematopoietic stem cells. Blood 94:2548-54|
|Ogawa, M (1999) Stochastic model revisited. Int J Hematol 69:2-5|
|Ogawa, M; Matsunaga, T (1999) Humoral regulation of hematopoietic stem cells. Ann N Y Acad Sci 872:17-23;discussion 23-4|
|Hirayama, F; Aiba, Y; Ikebuchi, K et al. (1999) Differentiation in culture of murine primitive lymphohematopoietic progenitors toward T-cell lineage. Blood 93:4187-95|
|Matsunaga, T; Kato, T; Miyazaki, H et al. (1998) Thrombopoietin promotes the survival of murine hematopoietic long-term reconstituting cells: comparison with the effects of FLT3/FLK-2 ligand and interleukin-6. Blood 92:452-61|
|Matsunaga, T; Hirayama, F; Yonemura, Y et al. (1998) Negative regulation by interleukin-3 (IL-3) of mouse early B-cell progenitors and stem cells in culture: transduction of the negative signals by betac and betaIL-3 proteins of IL-3 receptor and absence of negative regulation by granulocyte-macrophage colo Blood 92:901-7|
|Shimizu, Y; Ogawa, M; Kobayashi, M et al. (1998) Engraftment of cultured human hematopoietic cells in sheep. Blood 91:3688-92|
|Ogawa, M; Yonemura, Y; Ku, H (1997) In vitro expansion of hematopoietic stem cells. Stem Cells 15 Suppl 1:7-11; discussion 12|
|Aiba, Y; Hirayama, F; Ogawa, M (1997) Clonal proliferation and cytokine requirement of murine progenitors for natural killer cells. Blood 89:4005-12|
|Aiba, Y; Ogawa, M (1997) Development of natural killer cells, B lymphocytes, macrophages, and mast cells from single hematopoietic progenitors in culture of murine fetal liver cells. Blood 90:3923-30|
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