Maintenance and differentiation of hemopoietic progenitor and stem cells in long-term bone marrow culture (LTBMC) critically depends on the presence of a functional layer of adherent stromal cells [1-6]. The precise role of stromal cells in hemopoiesis has not yet been fully elucidated. Stromal cells, however, are an important source of mediators required for the controlled differentiation and proliferation of progenitor cells [7-9]. In addition stromal cells also provide a complex functional extracellular matrix supporting direct cell-to-cell contacts between stromal and progenitor cells. The heterogeneous cellular composition of this stromal layer including macrophages, fibroblasts, adipocytes and endothelial cells [1-3], makes it extremly difficult to analyze the role of each cell type in hemopoietic development.
Established bone marrow stromal cell lines provide a useful tool for the analysis of discrete stromal functions. While a number of spontaneously immortalized murine stromal cell lines have been described [13-15] attempts to establish corresponding human lines have failed [16]. Human bone marrow stromal cell lines are also described in K. Thalmeier et al. [41]. However, no cell lines which remain adherent after irradiation are described in this publication.
Some of the problems associated with the establishment of human stromal cell lines have been solved by introducting DNA into the cellular genome encoding the SV40 large T-Ag [17-21]. This has been accomplished by a variety of gene transfer methods including Ca-phosphate precipitation [17], electroporation of recombinant SV40 constructs [18, 20, 21], and infection with SV40 wild-type viruses [19, 20]. These stromal cell lines have been used as model systems for analyzing stromal cell-progenitor cell interactions [22-26]. Nevertheless the use of SV40-immortalized stromal cell lines as supportive feeder layers in LTBMCs still has two important drawbacks. Firstly, SV40-immortalized cells grow very rapidly for up to 100 cell generations [27] and then enter a characteristic crisis leading to the death of the cells [19]. Secondly, growth of SV40-immortalized stromal cells cannot be inhibited by irradiation or mitomycin C without detachment from the culture flasks [21].
In this invention there are described new human bone marrow stromal cell lines and their use. These cell lines proliferate at a high rate and can be growth-arrested by irradiation without detachment. The functional capacity of the cell lines according to the invention as feeder cells is exemplified by their ability to support the long-term proliferation of e.g. CD34.sup.+ enriched human cord blood progenitor cells and clonogenic growth of the feeder-dependent cell line BL70.
USPTO Backgrounds
USPTO Backgrounds