In vitro hemopoiesis within a microenvironment created by MC3T3‐G2/PA6 preadipocytes

Abstract

The clonal preadipose cell line, MC3T3-G2/PA6, has the capacity to differentiate into adipocytes in response to glucocorticoids and to support in vitro growth of hemopoietic stem cells (CFU-S). To study the relationship between these capacities, we precultured the MC3T3-G2/PA6 cells for varying days in the presence or absence of dexamethasone and then cocultured them with mouse bone marrow cells. Logarithmically growing cultures contained no detectable adipocytes and showed the highest growth-supporting activity for CFU-S, whereas cultures containing the largest number of adipocytes showed the lowest activity. When bone marrow cells were seeded onto 3-day-old MC3T3-G2/PA6 preadipocyte layers at 1 X 10(5) cells/35-mm dish, day 12 CFU-S grew with a population doubling time of about 37 hr, and at least 75% of them were associated with the cell layer between days 2 and 7. In the absence of the preadipocytes, CFU-S were not detected in the adherent cell fraction and decreased with a half-life of about 18 hr. More than 80% of CFU-C were also found to be associated with the preadipocyte layer, and they increased about 24-fold in number during 7 days in culture. Morphologically, hemopoietic cells developing into mature granulocytes and macrophages were distributed between the layers of preadipocytes. Dendritic processes of preadipocytes were frequently in close alignment with the hemopoietic cells. However, adipocytes failed to show such an intimate association with hemopoietic cells. These results indicate that MC3T3-G2/PA6 cells in the preadipocyte stage, but not in the adipocyte stage, have the capacity to support CFU-S growth, and that hemopoiesis in our cocultivation system proceed within the microenvironmental milieu provided by MC3T3-G2/PA6 preadipocytes.

Molecular Characterization of the MouseTem1/endosialin Gene Regulated by Cell Density in Vitro and Expressed in Normal Tissues in Vivo

“…SR-4987 cells were kept in McCoy's 5a medium with 10% FCS. Stromal cell line MC3T3-G2/PA6 (PA6) (13) was provided by Kevin Holmes and maintained in ␣-minimal essential medium supplemented with 10% FCS. A primary culture of rat brain capillary endothelial cells was prepared and kept as described previously (14).…”

Absence of p53 permits propagation of mutant cells following genotoxic damage

“…B cell precursors from bone marrow were expanded for 7 days in suspension culture with IL7 as previously described. Immediately after radiation at 4 Gy, the cells were plated onto a bone marrow stromal cell line PA6 (irradiated at 30 Gy) which supports B cell precursor proliferation (Winkler et al, 1995;Kodama et al, 1984). Low dose (1 m/ml) IL7 was also added along with IL3 and concentrated supernatant from non-irradiated PA6 cultures (Nagasawa et al, 1994).…”

A Novel Gene (drad‐1) Expressed in Hematopoiesis‐Supporting Stromal Cell Lines, ST2, PA6 and A54 Preadipocytes: Use of mRNA Differential Display

“…However, it is possible to maintain for long term or to support the growth of immature hematopoietic progenitor cells by coculture with stromal cells [5][6][7][8][9][10][11]. The cited studies suggest that the stromal cells succeed in constructing some kind of hematopoiesis-inductive environment in vitro by producing hematopoiesis-regulating factors.…”