Spleen stromal cells support haemopoiesis and in vitro growth of dendritic cells from bone marrow

Abstract

Summary. Long-term stroma-dependent cultures from murine spleen have been previously shown to support dendritic cell (DC) development in vitro. Secondary cultures have now been established using a splenic stromal cell layer overlaid with cells from different lymphoid sites. Cells resembling DCs can be generated in vitro from unfractionated murine lymphoid cells in the absence of added growth factors. Bone marrow (BM) cultures are the most successful but some cultures have been derived from spleen and thymus. Large numbers of mononuclear cells with dendritic morphology can be generated from BM within 2 weeks and cell production in many cultures has been maintained for at least 6 months. A significant proportion of cells binds antibodies specific for DC markers. No lymphoid cells, mast cells or granulocytes are detectable in culture by antibody and histochemical staining and light and electron microscopy. As with cells generated in primary cultures, cells grown in secondary cultures are equally potent stimulators of both syngeneic and allogeneic mixed lymphocyte reactions, confirming their function as antigen-presenting cells. They are also capable of endocytosing and presenting protein antigen to the D10.G4.1 Th2 clone and to unprimed T cells.This study confirmed the presence of DC precursors in multiple lymphoid sites which can be induced to proliferate in the presence of a spleen stromal cell monolayer. The secondary culture system provides an ideal in vitro model for investigation of the development of DC from different tissue sites. It also provides a stable and continuous resource of cells for further studies on DC development.

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

“…Previously it was shown that cells resembling DC both functionally and phenotypically could be produced in LTC without the addition of an exogenous source of GM-CSF ,1999). …”

“…Another consideration is whether LTC-DC are more closely related to the apparent myeloid or lymphoid DC lineages (Shortman & Caux, 1997 (BM) as previously described (Ni & O'Neill, 1997;1999 In vitro agar colony assay…”

“…A long term culture (LTC) system for production of murine DC has been developed which generates a continuous supply of immature DC from haemopoeitic precursors maintained ,within the culture without the addition of GM-CSF ,1999O'Neill et al, 1999a). Continuous production of cells depends on the presenc e of a stromal cell layer of endothelial and fibroblastic cells.…”

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

“…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.…”