11 research outputs found
Role of splenic stroma in the action of bacterial lipopolysaccharides on radiation mortality: a study in mice carrying the Slj allele
Abstract. Slj/+ mice display a slight macrocytic anaemia due to a defect in their haemopoietic organ stroma. They have a deficient endogenous spleen colony (CFUâend) formation following sublethal doses of gammaâradiation compared with their normal +/+ littermates, which is likely to be due to the low preâirradiation CFUâS content of the Slj/+ spleen. CFUâS in these congenic mice do not differ in their sensitivity to gammaâirradiation or stem cellâactivating factor. While injection of +/+ mice with 10 ÎŒg of lipopolysaccharideâW (LPS) one day prior to irradiation led to a substantial increase in their survival, the survival of Slj/+ mice was only slightly increased. Irradiation induced a similar doseârelated reduction in the numbers of CFUâS in the spleen and femora of LPSâinjected Slj/+ mice compared to similarly treated +/+ mice when measured directly after irradiation. At Day 9 after irradiation, injection of LPS led to a significantly higher CFUâend formation and higher numbers of CFUâS and nucleated cells in the Slj/+ spleens compared to LPSâinjected +/+ mice. No such differences in the radioprotective effect of LPS were observed in the +/+ and Slj/+ mice with respect to the splenic and femoral 59Feâincorporation and the femoral CFUâS numbers at Day 9. These data strongly suggest a contribution by immigrating CFUâS to the CFUâS numbers and endogenous colony formation in at least the Slj/+ spleen after LPS injection and subsequent sublethal irradiation. The observations also imply that the splenic organ stroma may play a mediatory role in the radioprotective action of LPS. In addition, the data represent an extreme example of a lack of correlation between animal survival and haemopoietic parameters. Caution should be taken when applying endogenous colony counts as a means of screening potential antiâradiation drugs. Copyrigh
Characteristics of the CfuâS Population In Mice Carrying the SlJ Allele
Abstract Abstract. A tentative characterization of haemopoietic stem cells with respect to their organ distribution, seeding fraction and colony formation in the spleen, radiosenâsitivity and humoral regulation was attempted in mice heterozygous for the mutant allele SlJ and in their normal littermates. SlJ/+ mice were characterized by a deficient CFUâs content of the blood and spleen and had slightly lower femoral CFUâs numbers. This CFUâs distribution could not be explained by differences in seeding efficiency âfâ between CFUâs of SlJ/+ and +/+ origin in lethally irradiated recipients used in the CFUâs assay. the seeding fraction of CFUâs of +/+ origin did not differ in +/+ and SlJ/+ recipients. However, in irradiated SIJ/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFUâs of both +/+ and SlJ/+ origin. the serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in SlJ/+ mice, but significantly increased in Sl/Sld mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in SlJ/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFUâs from spleen and bone marrow had a similar survival following inâvitro y irradiation. Femurs and spleens of both SlJ/+ and +/+ origin were implanted into both SlJ/+ and +/+ hosts. Six weeks later the SlJ/+ grafts contained less CFUâs than the +/+ grafts. These data show that the splenic stroma of SlJ/+ mice is not defective in its capacity to lodge injected CFUâs but is deficient in its ability to maintain CFUâs under âsteadyâstateâ conditions and stimulate their colony formation in a âperturbed stateâ. Some of the characteristics of SlJ/+ mice segregate them from Sl/Sld mice, i.e. a deficient splenic CFUâs content, normal seeding fractions âfâ of CFUâs from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. the study of the SlJ trait may be a useful extension of the current Sl/Sld model for exploration of hereditary defects in haematopoietic stroma. Copyrigh
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 30â40 to about 300â400 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 2â3 days after injection and attained a peak on the 6thâ7th 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 âopen sitesâin the haemopoietic tissue for capturing CFUs was studied by emptying these sites through a lethal Xâirradiation 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
In vitro frequency analysis of spleen colony-forming and marrow-repopulating hemopoietic stem cells in the mouse
An assay is described for Day-12 spleen colony-forming cells (CFU-S-12) and hemopoietic stem cells with marrow-repopulating ability (MRA) in the mouse using a miniaturized stroma-dependent bone marrow culture assay in vitro. Bone marrow cells are grown in liquid culture in microtiter wells, and the resulting adherent stromal layers are depleted of all hemopoietic activity by 20 Gy gamma irradiation. Subsequently, single cell suspensions containing stem cells are overlaid in a range of concentrations, and the presence of one or more emerging phase nonrefractive cell clones (cobblestone areas) in a single well scored as positive. The frequencies of cobblestone area-forming cells (CAFC) are then calculated by employing Poisson statistics. It is shown that the CAFC Day-10 and CAFC Day-28 frequencies closely correlate with those of CFU-S-12 and MRA cells, respectively
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
An in vitro model for cytogenetic conversion in CML. Interferon-alpha preferentially inhibits the outgrowth of malignant stem cells preserved in long-term culture
IFN-alpha has been shown to prolong survival in chronic myeloid leukemia
patients, but its mechanism of action is still not understood. The human
cobblestone area-forming cell (CAFC) assay allows for the measurement of
the concentration of normal as well as malignant stem cells, while their
progeny can be measured in parallel long-term culture (LTC) in flasks
Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis
Throughout life, the hematopoietic system requires a supportive
microenvironment that allows for the maintenance and differentiation of
hematopoietic stem cells (HSC). To understand the cellular interactions
and molecules that provide these functions, investigators have previously
established stromal cell lines from the late gestational stage and adult
murine hematopoietic microenvironments. However, the stromal cell
microenvironment that supports the emergence, expansion and maintenance of
HSCs during mid-gestational stages has been largely unexplored. Since
several tissues within the mouse embryo are known to harbor HSCs (i.e.
aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal
cell clones from these mid-gestational sites. Owing to the limited cell
numbers, isolations were performed with tissues from transgenic embryos
containing the ts SV40 Tag gene (tsA58) under the transcriptional control
of constitutive and ubiquitously expressing promoters. We report here that
the growth and cloning efficiency of embryonic cells (with the exception
of the aorta) is increased in the presence of the tsA58 transgene.
Furthermore, our results show that the large panel of stromal clones
isolated from the different embryonal subregions exhibit heterogeneity in
their ability to promote murine and human hematopoietic differentiation.
Despite our findings of heterogeneity in hematopoietic growth factor gene
expression profiles, high-level expression of some factors may influence
hematopoietic differentiation. Interestingly, a few of these stromal
clones express a recently described chordin-like protein, which is an
inhibitor of bone morphogenic proteins and is preferentially expressed in
cells of the mesenchymal lineage
Seeding efficiency of primitive human hematopoietic cells in nonobese diabetic/severe combined immune deficiency mice: implications for stem cell frequency assessment
Nonobese diabetic/severe combined immune deficiency (NOD/SCID) mouse
repopulating cells (SRC) have been proposed to represent a more primitive
human stem cell subset than the cobblestone area-forming cell (CAFC) week
(wk) 6 or the long-term culture-initiating cell (LTC-IC) wk 5 on the basis
of their difference in frequency, phenotype, transfectibility, and
multilineage outgrowth potential in immunodeficient recipients. We have
assessed the percentage of various progenitor cell populations
(colony-forming cell [CFC] and CAFC subsets) contained in unsorted
NOD/SCID BM nucleated cells (nc), human umbilical cord blood (UCB) nc,
bone marrow (BM) nc, peripheral blood stem cells (PBSC), and CD34(+)
selected UCB nc, seeding in the BM and spleen of NOD/SCID mice within 24
hours after transplantation. The seeding efficiency of NOD/SCID BM CAFC wk
5 was median (range) in the spleen 2.9% (0.7% to 4.0%) and in the total BM
8.7% (2.0% to 9.2%). For human unsorted UCB nc, BM nc, PBSC, and CD34(+)
UCB cells, the seeding efficiency for CAFC wk 6 in the BM of NOD/SCID mice
was 4.4% (3.5% to 6.3%), 0.8% (0.3% to 1.7%), 5.3% (1. 4% to 13.6%), and
4.4% (3.5% to 6.3%), respectively. Using flow cytometry, the percentage
CD34(+) UCB cells retrieved from the BM of sublethally or supralethally
irradiated NOD/SCID mice was 2.3 (1.4 to 2.8) and 2.5 (1.6 to 2.7),
respectively. Because we did not observe any significant differences in
the seeding efficiencies of the various stem cell subsets, it may be
assumed that the SRC seeding efficiency in NOD/SCID mice is similarly low.
Our data indicate that the seeding efficiency of a graft can be of great
influence when assessing stem cell frequencies in in vivo repopulation
assays