Self-renewal of hematopoietic progenitor cells under defined condition.

Intra-marrow hematopoiesis is regulated by a meshwork of stromal cell components consisting of histologically distinct cell types. The establishment of culture conditions that selectively support hematopoietic stem cells is an important goal of hematology. We established new culture condition without serum. It is necessary for long term bone marrow (BM) culture to use stroma cell, but in order to inhibit the dominant proliferation of macrophage in culture, we used the OP9 stromal cell line that lacks macrophage-colony stimulating factor (M-CSF) production. Moreover we added epidermal growth factor (EGF) for survival of OP9 cells under chemically defined medium, mSF02 and stem cell factor (SCF) for proliferation of BM cells. This condition (OP9/EGF/SCF) provides a microenvironment wherein c-Kit + Thy-1 +/10 Mac-1 + / 10 B220 - TER119 - AIC2B 4 IL-2Rγ + gp130 + gp130 + (c-Kit + Mac-1 dull ) cells are selectively propagated from normal unfractionated bone marrow cells. This cell population produced an in vitro colony used with methylcellulose semi-solid culture containing IL3 and erythropoietin at 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 CFU in culture with short term reconstituting hematopoietic stem cells. Transferring this cell population into medium containing differentiation signals resulted in the rapid production of mature myelo-monocytic and B cell lineages in vitro and in vivo. Next, such c-Kit + Mac-1 dull cells don't bind to EGF directly, then this selective proliferation of progenitor cell was thought to be mediated by a factor from OP9 stimulated with EGF. Then we transduced v-erbB2 containing constitutively active site in the cytoplasmic domain into OP9 cells and established OP9/erbB2 transfectant which can survive under serum free medium without exogenous factor, such as EGF. On this OP9/erbB2 cells under mSFO2 medium with SCF, progenitor cells which are phenotypically similar to those on OP9/EGF/ SCF are selectively propagated from normal unfractionated bone marrow cells. Cells that were c-Kit + Mac-1 dull did not selectively proliferate on the M-CSF-expressing stromal cell lines, PA6 or ST2. Moreover, medium containing serum provided a microenvironment that favored hematopoietic differentiation, even if EGF was added to the culture. The addition of 2-ME in mSF02 promoted the proliferation of pre-B cells (c-Kit + B220 + Mac-1 - ) even in the presence of EGF and SCF. Therefore, the 2-ME free chemically defined medium and the M-CSF-deficient OP9 stromal cell line are essential components of these culture conditions. The addition of bFGF instead of EGF however, produced mature hematopoietic cells. This suggested that an in vitro microenvironment formed by a uniform stromal cell line in the absence of serum could be varied according to the stimulation given to the stromal cells. Thus, the selective proliferation of c-Kit + Mac-1 dull cells described here appeared to be an outcome of a particular combination of stromal cell molecules expressed by EGF-stimulated or v-erbB2-transduced OP9. Nevertheless, these results indicated that hematopoiesis is regulated not only by molecules directly acting on hematopoietic progenitors but also by those that stimulate stromal cell components.