In vitro quantitation of lethal and physiologic effects of total body irradiation on stromal and hematopoietic stem cells in continuous bone marrow cultures from Rf mice

Abstract

The role of stromal-supportive cells in hematopoietic stem cell responses to irradiation is poorly understood. The effects of in vivo total body irradiation (TBI) and interval from TBI to explant of marrow on: stromal cell proliferation in vitro; stromal cell support of hematopoiesis in continuous bone marrow culture; and generation of WEHI-3 growth factor (GF)-dependent lines of hematopoietic progenitor cells were evaluated. Continuous marrow cultures from non-irradiated control RfM/UN, C57BL/6J, C3H/HeJ, and N:NIH (Swiss) mice generated pluripotential hematopoietic stem cells (CFUs) and committed granulocyte-macrophage progenitor cells (GM-CFUc) for over 20 weeks. Explant of marrow at 2, 4, 5, or 6 months after single fraction TBI (300-800 rad) was associated with decreased longevity of hemopoiesis (2-12 weeks), and a decrease in the proliferative capacity of fibroblastic adherent-stromal colony forming cells (CFUf) as measured by colony size at 14 days and number of colonies per 10(6) cells plated. In contrast, explant of marrow 8 to 24 months after TBI produced cultures with longevity that was indistinguishable from age-matched control cultures (19-24 weeks). Marrow from irradiated first and second generation recipients of serially transferred marrow demonstrated a similar 7-month in vivo recovery period; however, the plateau maximum duration of hemopoiesis did not return to control levels. Purified stromal cell cultures were prepared by corticosteroid-deprivation of explanted marrow for 28 days and were then engrafted in vitro with marrow from C57BL/6J or RfM/UN mice that had been irradiated 1 month previously. Hemopoiesis in these cultures was restored, and they produced GM-CFUc and granulocytes for 15-24 weeks. Thus, healthy stroma supported growth of recently irradiated hemopoietic cells in vitro. Nonadherent cells removed from the above continuous marrow cultures generated clonal non-leukemogenic WEHI-3 GF-dependent hemopoietic progenitor cell lines with a frequency concordant with radiation effects on culture longevity, and this was increased by the presence of purified healthy stromal cultures. Indirect effects of x-irradiation on hemopoietic stem cells through damage and repair in the stromal cell compartment can be effectively studied with the present bone marrow culture system.