Long‐term stromal cultures produce dendritic‐like cells

Abstract

Summary.A stromal cell-dependent long-term culture (LTC) system has been developed from spleen which continuously generates non-adherent cells with dendritic cell (DC) characteristics. Bioassays using factor-dependent cell lines have revealed that both spleen and thymic stromal cultures secrete interleukin (IL)-3 and IL-6-like growth factors. Conditioned medium from LTC also contains factors which appear to be unrelated to IL-3 and induces growth of stromal cells from bone marrow. Non-adherent cells generated in LTC were not T or B lymphoid cells or granulocytes, nor were mast cells detectable. Morphological and electron microscopic examination has also excluded the presence of macrophages (Mø). Cells with DC morphology have been detected by both light and electron microscopy. The majority of cells in the non-adherent population were found to have multiple membrane pseudopodia, with a small acentric nucleus. These appear to be the precursors of DC. They expressed cell surface markers detectable with DC-specific antibodies and antibody specific for major histocompatibility complex Class II molecules. A proportion of cells also expressed myeloid markers, but since this expression was not supported by histochemical staining for myeloperoxidase or non-specific esterase, it was concluded that the cells produced are not typical of the myeloid lineage. Cells generated in LTC were shown to be potent stimulators for allogeneic and syngeneic MLR and for antigen-specific T-cell proliferation.

Identification of progenitor cells in long-term spleen stromal cultures that produce immature dendritic cells

“…It has remained constant after establishment of multiple LTC by passage of a mixture of stromal and nonadherent cells. Previous studies have shown the importance of maintaining small hemopoietic cells in LTC to perpetuate LTC-DC production (11,12). The constant presence of these two subsets within LTC suggests that they are integral to the structure and continuation of the LTC system and that the small subset may contain progenitors of the large-cell subset.…”

“…The antigenpresenting capacity of cells produced in LTC has been confirmed at numerous time points (14). LTC-DC can stimulate both allogeneic and syngeneic naive T cells as well as present antigen to antigen-specific T helper cells (11,15). In this report, surface marker expression, function, and differentiative capacity have been used to characterize two major DC subsets produced in LTC.…”

“…Established LTC do not produce lymphoid cells, granulocytes, or monocytes͞macrophages (11)(12)(13). Production of cells expressing markers associated with DC, including CD11c, CD11b, DEC-205, and 33D1, has been shown to continue for up to 7 years for some LTC (14).…”

“…A long-term culture (LTC) system that supports hemopoiesis has been developed from murine spleen that continuously produces nonadherent DC (LTC-DC), in the absence of exogenous growth factors, including granulocyte͞ macrophage colony-stimulating factor (11)(12)(13). The production of LTC-DC appears to depend on the maintenance of small progenitor cells carried through many passages of cultures.…”

Development of Dendritic Cells from GM‐CSF-/-Mice invitro: GM‐CSF Enhances Productionand Survival of Cells

“…A constant population of cells is released into the culture supernatent and can be readily collected for analysis. Cells produced in LTC have a cell surface phenotype resembling DC with high levels of CD 1 lb, CDllc, 33D1 and CD80/CD86 (Ni & O'Neill, 1997;. Their cell surface phenotype is more consistent with myeloid DC since the great majority of cells are LTC-DC maintain the functional characteristics of DC, being endocytic (Ni & O'Neill, 1997) and highly potent APC (O'Neill et al, 1999a).…”