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

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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 × 105 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.