Shin-Ichi Hayashi

Conditions required for myelopoiesis in murine spleen

While the spleen is an active site for myelopoiesis during the late embryonal and perinatal stages, the activity is gradually lost later. However, myelopoiesis in the adult spleen can be reactivated by irradiation or various stimulants. In this study we investigated factors which determine the myelopoiesis-supporting activity in the adult spleen. To address this question, we used scid mouse because virtually no lymphocytes, which might compete in the splenic microenvironment with hematopoietic progenitors, are present there. The results demonstrated: 1. Even in scid mouse, the myelopoiesis-supporting activity in the spleen is lost within a week after birth as in normal mice. 2. While myelopoiesis does not occur in the spleen of unstimulated scid mouse by bone marrow transfer alone, myelopoiesis in the spleen is reactivated by irradiation or lipopolysaccharide (LPS) application. 3. Myelopoiesis in the spleen induced by irradiation is dependent on c-kit and its ligand steel factor (SLF), because it was suppressed completely by the monoclonal antibody (mAb) against c-kit. 4. The expression of SLF transcripts in the spleen was enhanced after irradiation. These results suggest that the factor which determines myelopoietic activity in the spleen resides primarily in the status of the splenic microenvironment.

Control of Intramarrow B-Cell Genesis by Stromal Cell-Derived Molecules

A remarkable progress has been made in the investigation of B cell-genesis since Whitlock and Witte have reported a long-term culture system of bone marrow B cells (Whitlock and Witte 1982). Although some more stromal cell molecules are yet to be cloned before concluding that we know all molecules involved in the intramarrow B cellgenesis, the basic framework of the molecular requirements for B cell-genesis has been understood to a considerable extent. As a result of this progress, a numbers of methods which use stromal cell lines and recombinant cytokines to control B cell-genesis in vitro are presently available (for review see Kincade et al. 1989; Rolink and Melchers 1991, Kunisada et al. 1992). It is also true, however, that some inconsistency is still present among the previous results on the growth signal requirement of B precursors. Given that control of cell proliferation in higher organisms might be achieved in a redundant and failsafe manner, this redundancy could be a source of the inconsistency among the previous results. Thus, in this article, we would like to re-examine three key propositions which we have been stated in the previous studies and discuss them in detail in light of the previous results of ours and other groups.

27 – Osteoclast Lineage

This chapter reviews the biological features of osteoclast development. Osteoclasts are hematopoietic cells that have bone-resorbing activity, and participate in bone remodeling and bone marrow formation. Mature functional osteoclasts are large multinuclear cells consisting of multiple osteoclasts fused with each other. Since embryonic stem (ES) cells have the potential to differentiate into all cell lineages, it should be possible to derive any cell lineage by appropriate induction of ES cells in culture. Osteoclasts, derived from hematopoietic stem cells, participate in bone remodeling and form bone marrow cavities through their bone resorbing activity. The precursors share their characteristics with the precursors of monocytic lineage cells, such as macrophages and dendritic cells. In one-step culture, a wider range of cell lineages could be observed in a dish compared with cloned stromal cell (OP9) cultures. In addition to hematopoietic lineages, at least endothelial cells, osteoblasts, myocardial cells, melanocytes, and pigmented epithelial cells are observed. Osteoblasts build the bone, endothelial cells invade the bone, and osteoclasts resorb the bone and make bone marrow cavity. These two lineages of cells are closely associated and located as concentric circles.

Defect of Scid Mouse Revealed in In Vitro Culture Systems

Since a series of analyses by Bosma’s group on the scid mouse demonstrated that there are virtually no lymphocytes in this mutant strain (Bosma et al 1983), it has been attracting those investigating the development of lymphocytes from hemopoietic stem cells. A pioneering study by Whitlock et al. enabled us to follow a considerable part, if not all, of the process during intramarrow B cell development reproducibly in an in vitro culture system (Whitlock and Witte 1982; Whitlock et al. 1984). In this situation, expecting that scid mouse might contribute to the understanding of B cell development, we started to analyze the defect of scid mouse in long-term bone marrow culture. The question which was addressed at the beginning by using the Whitlock-Witte type long-term bone marrow culture (W-LTBC) was whether or not B lineage cells were generated in the scid mouse culture. If the defect of scid mouse affects the commitment process of pluripotent stem cells into lymphoid cells, B lineage cells would not appear in the W-LTBC of scid bone marrow. On the other hand, if the defect affects the later differentiation stage, B lineage cells whose differentiation is arrested would be generated in this culture. In this article, we first briefly describe our previous results on scid mouse which utilized classical long-term bone marrow culture methods, then describe the system which we are currently using for the analysis of B cell differentiation, and finally our analysis on the defect of scid mouse in this culture system.