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Nobuyuki Takakura^*, Hiroaki Kodama, Satomi Nishikawa^*, and Shin-Ichi Nishikawa^*

From the ^* Department of Molecular Genetics, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan; and the Institute of Allergy, Research Center Kyoto, Bayer Yakuhin, Ltd., Kyoto 619-02, Japan

The establishment of culture conditions that selectively support hematopoietic stem cells is an important goal of hematology. In this study, we investigated the possibility of using for this purpose a defined medium, mSFO2, which was developed for stromal cell–dependent bone marrow cultures. We found that a combination of epidermal growth factor (EGF), the OP9 stromal cell line, which lacks macrophage colony-stimulating factor, recombinant stem cell factor, and the chemically defined medium mSFO2 provides a microenvironment where c-Kit⁺ Thy-1^+/lo Mac-1^+/lo B220^– TER119^– commonβ⁺ IL-2R⁺ gp130⁺ cells are selectively propagated from normal, unfractionated bone marrow cells. This cell population produced an in vitro colony at a very high efficiency (50%), whereas it has only limited proliferative ability in the irradiated recipient. Thus, the cells selected in this culture condition might represent colony-forming units in culture (CFU-c) with short-term reconstituting ability. Transferring this cell population into medium containing differentiation signals resulted in the rapid production of mature myelomonocytic and B cell lineages in vitro and in vivo. The fact that a similar culture condition was created by erb-B2–transduced OP9 in the absence of EGF indicated that EGF exerts its effect by acting on OP9 rather than directly on CFU-c. These results suggested that the balance between self-renewal and differentiation of CFU-c can be regulated by extracellular signals.

This study was supported by grants from the Japanese Ministry of Education, Science and Culture (Nos. 07CE2005, 07457085, and 06277103), a grant from the Ministry of Science and Technology (No. 130732125-14), and a grant from RIKEN.

¹Abbreviations used in this paper: bFGF, basic fibroblast growth factor; BM, bone marrow; CFU-c, CFU in culture; CFU-s; CFU in spleen; EGF, epidermal growth factor; Epo, erythropoietin; G-CSF, granulocyte CSF; M-CSF, macrophage CSF; SCF, stem cell factor.

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