Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells

Large ex vivo expansion of hematopoietic stem cells (HSCs) sufficient for use in clinical applications has not been achieved, although the influence of some cytokines including SCF, IL-11, Flt3-L, and TPO for this purpose has been reported. We present evidence for an indirect effect of macrophage colony-stimulating factor (M-CSF) on expansion of murine HSCs. Fresh Lin(-/low) cells were isolated from Ly5.1 mouse bone marrow and cultured with or without M-CSF in the presence of SCF + IL-11 + Flt3-L or SCF + IL-11 + TPO for 6 days. The expanded cells were harvested and transplanted into lethally irradiated Ly5.2 recipients with competitor cells. Culture of Lin(-/low) cells with M-CSF significantly enhanced long-term engraftment. When the more enriched HSC populations of Lin(-/low) c-Kit(+) Sca-1(+) cells were used as a source of HSCs, such a promotive effect was not observed, in agreement with negative expression of the M-CSF receptor (c-Fms). However, co-culture with Lin(-/low) c-Fms(+) resulted in a significant increase of long-term engraftment. These results suggested that M-CSF is an indirect stimulator for ex vivo expansion of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. These observations provide new directions for ex vivo expansion and insight into new engraftment regulation through M-CSF signaling.     

IL-1 alpha stimulates the formation of osteoclast-like cells by increasing M-CSF and PGE2 production and decreasing OPG production by osteoblasts

Interleukin-1alpha (IL-1alpha) is one of the most potent bone-resorbing factors involved in the bone loss that is associated with inflammation. We examined the effect of the inflammatory mediator IL-1alpha on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E2 (PGE2) in rat osteoblasts, and the indirect effect of IL-1alpha on the formation of osteoclast-like cells. Osteoblasts were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with or without 100 units/ml of IL-1alpha for up to 14 days. The gene and protein expression of M-CSF and OPG were estimated by determining mRNA levels using the real-time polymerase chain reaction and protein levels using Western blot analysis. PGE2 expression was determined using an enzyme-linked immunosorbent assay. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from IL-1alpha-treated osteoblasts and the soluble receptor activator of NF-kappaB ligand (RANKL). M-CSF and PGE2 expression in osteoblasts increased markedly in cells cultured with IL-1alpha, whereas OPG expression decreased. The conditioned medium containing M-CSF and PGE2 produced by IL-1alpha-treated osteoblasts and soluble RANKL increased the TRAP staining of osteoclast precursors. These results suggest that IL-1alpha stimulated the formation of osteoclast-like cells via an increase in M-CSF and PGE2 production, and a decrease in OPG production by osteoblasts.     

Role of macrophage colony-stimulating factor in the differentiation and expansion of monocytes and dendritic cells from CD34+ progenitor cells

The present study focused on whether it is possible to expand monocytic cells from CD34⁺ progenitor cells by using macrophage colony-stimulating factor (M-CSF) in the absence and presence of mast cell growth factor (MGF) and IL-6. It was demonstrated that CD34⁺ cells differentiate without expansion to functional mature monocytic cells in the presence of M-CSF or combinations of M-CSF plus IL-6 and MGF. A different response pattern was observed for the number of clonogenic cells. The addition of IL-6 or both IL-6 and MGF to M-CSF containing cultures resulted in significant higher numbers of colony-forming unit-macrophage (CFU-M) as tested in clonogenic and³H-thymidine assays. Furthermore, M-CSF plus both IL-6 and MGF appeared to be the most potent combination to preserve the monocytic precursor in cell suspension culture assays. These results indicate that IL-6 and MGF in conjunction with M-CSF affect CD34⁺ cells especially at precursor level without distinct effect on the more mature stages. Secondly we studied whether M-CSF is only critical for the monocytic lineage or also affects dendritic cell (DC) development. Indeed, we were able to culture CD83⁺ DC from CD34⁺ progenitor cells in the presence of M-CSF in conjunction with TNF-α, IL-4, and MGF although their absolute number is almost threefold lower than the number of CD83⁺ cells yielded from GM-CSF plus TNF-α, IL-4, and MGF stimulated CD34⁺ cells.     

Cellular and molecular properties associated with osteosarcoma cells.

Osteosarcoma cells are recognized by abnormal function that causes a primary bone tumor. Osteosarcoma cells U(2)OS and SAOS-2 were analyzed for the expression of cell surface markers. High expression was quantified for hyaloronidase receptor (CD-44) > moderate for integrins (CD-51 and -61), > and lower for selectins (CD-62). High mitotic capacity were demonstrated by gene expression (measured by RT-PCR) and the protein level (measured by FACS) for cFOS, cMYC, and cJUN. The basic definition of osteosarcoma is excessive production of pathological osteoid. Expression of mRNA for matrix genes osteocalcin, osteonectin, and biglycan was studied. Osteocalcin and osteonectin were detected in RNA from primary cultured marrow stromal, trabecular bone cells, and osteosarcoma cell lines (U(2)OS, SAOS-2). mRNA for biglycan was detected only in primary cells and MG-63 cell line and was undetectable in RNA from U(2)OS, SAOS-2 osteosarcoma cell lines and by RNA extracted from bone biopsies of osteosarcoma patients. The absence of biglycan message observed in osteosarcoma samples provides evidence for the alterations in the extra cellular matrix which result with non-mineralized osteoid produced by the osteosarcoma cells.