Bone Marrow Microenvironments for Hematopoiesis

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1)Analysis of osteoclastogenesis for bone marrow formation :Osteoclasts are hematopoietic cells, which participate in bone resorption and remodeling. Receptor activator of NF-κB ligand(RANKL) and macrophage colony-stimulating factor(M-CSF) are critical for development of osteoclasts. The Toll-like receptor(TLR) family shares some of the downstream signaling with RANK, however, the signaling via TLRs has never been reported to induce osteoclastogenesis without RANKL. We showed that significant numbers of mature osteoclasts were generated from protein tyrosine phosphatase SHP-1-defective me^v/me^v bone marrow cells in the presence of M-CSF and LPS, a TLR4 ligand without addition of RANKL in culture. The osteoclast precursors were present in the Kit-positive cell enriched fraction of BM cells. Although me^v/me^v bone marrow cells required a comparable concentration of RANKL or TNF-α as wild-type cells for the initiation of osteoclastogenesis, numbers of multinucleated osteoclasts in me^v/ … More me^v bone marrow cultures were significantly increased by the equivalent dose of RANKL or TNF-α in the presence of M-CSF. These results indicate that a defect of SHP-1 function not only accelerates physiological osteoclast development by RANKL/RANK, but also acquires an aberrant pathway for osteoclastogenesis by LPS.2)Induction of hematopoiesis from blood-less embryonic stem (ES) cell line :Transcription factor Tal-1 is essential for the specification of hematopoietic development. Mice lacking Tall fail to generate any hematopoietic precursors. Using our co-culture system with stromal cells, we demonstrate that enforced expression of the transcription factor PU.1 under tetracycline control in Tall-null ES cells rescues the development of osteoclasts and macrophage-like phagocytes expressing a macrophage restricted marker. Other hematopoietic lineage cells were not generated. Their development was dependent on M-CSF and RANKL. These results suggest that the expression of PU.1 is a critical event for osteoclastogenesis and that Tal-1 may lie upstream of PU.1 in a regulatory hierarchy during osteoclastogenesis. Less