Sensitivity of murine haemopoietic stem cell populations to X-rays and I MeV fission neutrons in vitro and in vivo under hypoxic I. Conditions

The radiosensitivity of primitive haemopoietic stem cells that repopulate the bone marrow with precursors of granulocytes and macrophages (MRA[CFU-C]), mature stem cells capable of forming spleen colonies in lethally irradiated recipients (CFU-S-7) and colony-forming units in culture (CFU-C) were determined in vitro and under hypoxic conditions in vivo for 1 MeV fission neutrons and 300 kV X-rays. The obtained D0's were compared with previously observed D0's after irradiation in vivo under normal oxic conditions. With 1 MeV fission neutron irradiation no significant difference in radiosensitivity of the cell populations was observed between normal in vivo irradiation and in vitro irradiation. With 300 kV X-rays a lower radiosensitivity for all three cell populations was observed after in vitro compared to in vivo irradiation. In vivo irradiation with fission neutrons under hypoxic conditions led to a small decrease in radiosensitivity. The obtained oxygen enhancement ratio (OER) for fission neutrons varied from 1.2 for MRA[CFU-C] to 1.5 for CFU-C. After in vivo irradiation with 300 kV X-rays under hypoxic conditions much higher OERs were observed. An OER= 1.8 was obtained for CFU-S and for MRA[CFU-C] and for CFU-C OER 3.0 and 2.9 were observed. These results indicate that the radioresistance of primitive haemopietic stem cells (MRA[CFU-C]) compared to mature stem cells (CFU-S-7) is mainly due to intrinsic factors and not to differences in localization or oxygenation between primitive and mature stem cells

Bulk enrichment of transplantable hemopoietic stem cell subsets from lipopolysaccharide-stimulated murine spleen

Counterflow centrifugal elutriation (CCE) in combination with density flotation centrifugation and fluorescence-activated cell sorting on wheat-germ agglutinin-FITC(WGA)-binding cells within the light-scatter 'blast window' were used consecutively to enrich pluripotent hemopoietic stem cells (HSC) in bulk from lipopolysaccharide-stimulated mouse spleen. The medium-to-strong WGA+ve fraction contained 3.106 cells isolated from 3-4 x 109 spleen cells, with an average of 126% day-12 CFU-S and 65% day-8 CFU-S as calculated on the basis of their seeding fraction, suggesting that virtually all cells represented in vivo macroscopic colony formers. In view of the large differences reported elsewhere between stem cell subsets differing in reconstitutive capacity and secondary stem cell generation ability, we also studied various isolated cell fractions with respect to spleen colony formation, radioprotective ability, and spleen- and marrow-repopulating ability. Day-8 and day-12 CFU-S copurified when isolated by CCE. Cells from a fraction with high affinity for WGA were most highly enriched for their radioprotective ability (RPA) and their ability to repopulate the cellularity of the spleen and femur of irradiated recipients. This fraction contained virtually pure day-12 CFU-S. However, the ability to generate secondary day-12 CFU-S and CFU-GM in irradiated organs was enriched most in the medium WGA+ve cell fraction. MRA and SRA, according to the latter criteria, could therefore be partly separated from day-12 CFU-S and RPA on the basis of affinity for WGA. The data strongly suggest that at least part of all day-12 CFU-S have a high potential to proliferate and differentiate into mature progeny, but a relatively low self-renewal ability, and may therefore not be representative of the genuine stem cell

Mobilization of haemopoietic stem cells (cfu) into the peripheral blood of the mouse, effects of endotoxin and other compounds.

Factors affecting the circulation of haemopoietic stem cells (CFU) in the peripheral blood of mice were investigated. I.v. injection of sublethal doses of endotoxin, trypsin and proteinase appeared to raise the number of CFU per ml blood from about 30–40 to about 300–400 or more within 10 min. The effect was smaller when smaller doses of the substances were injected. After this initial rise the number of circulating cells returned to normal in a few hours. Following endotoxin there was a second rise which started 2–3 days after injection and attained a peak on the 6th–7th day. The first rise is explained as a mobilization of stem cells from their normal microenvironments into the blood stream; the second rise is considered to reflect proliferation of CFUs in the haemopoietic tissues. The spleen seems to be acting as an organ capturing CFUs from the blood and not as a source adding stem cells to the blood. The early mobilization of CFU after endotoxin injection did not coincide with a mobilization of neutrophils. The number of circulating band cells was increased during the first hours. The importance of ‘open sites’in the haemopoietic tissue for capturing CFUs was studied by emptying these sites through a lethal X‐irradiation and injecting normal bone marrow cells. When a greater number of syngeneic bone marrow cells was injected intravenously, the level of circulating CFU in irradiated mice was slightly lower than the level in unirradiated mice during the first hours. Copyrigh