The use of cationized ferritin to measure cell surface charge of mouse bone marrow cells by flow cytometry

Summary We have prepared fluorescein isothiocyanate (FITC) conjugates of cationised ferritin (CF) and have investigated the usefulness of this CF-FITC to measure the negative cell surface charge of mouse bone marrow cells by flow cytometry. CF-FITC conjugates of low fluorochrome to protein ratios (F/P ratio) gave insufficient fluorescence and/or formed large aggregates when stored. CF-FITC conjugates of high F/P ratios (above 25) bound specifically to bone marrow cells, giving sufficient fluorescence, the intensity of which differed for the different cell types. When stored at –20° C the CF-FITC was stable and could be used over prolonged periods. CF-FITC could be used to selectively enrich for pluripotent stem cells (CFU-S) and granulocyte/macrophage progenitors (CFU-C) by fluorescence activated cell sorting (FACS), although the CF-FITC binding to CFU-S and CFU-C was unexpectedly low. No correlation between CF-FITC fluorescence, cell size and electrophoretic mobility (EMP) was observed of bone marrow cells fractionated by free flow electrophoresis. Neuraminidase treatment to remove negatively charged sialic acid groups from the cell surface resulted in an increased binding of CF-FITC, although the EPM was decreased. The biotin conjugate of CF bound to bone marrow cells and could be visualised by avidin-FITC. The relative fluorescence intensity for the individual cell types showed a good correlation with the cell surface charge as determined by the EPM of the different cell types.The mechanism of binding CF-FITC to the cell surface was not by electrostatic interaction of the negative cell surface and positively charged CF because CF-FITC of F/P ratios of above 20 was negatively charged. This has been shown by theoretical calculations and determination of the pI of CF-FITC by iso-electric focussing. Binding of CF-FITC to the cell surfaces was probably caused by hydrophobic interaction between bound fluorescein molecules and lipid domains in the cell surface membrane aided by some ionic interaction. CF-biotin is still positively charged and is probably bound through electrostatic interactions with negatively charged cell surface groups. The indirect detection of bound CF-biotin with avidin-FITC of high F/P ratio results in a high fluorescence signal, which is a measure of the negative cell surface charge density, in the FACS.In honour of Prof. P. van Duijn     

A fractionation procedure of mouse bone marrow cells yielding exclusively pluripotent stem cells and committed progenitors

The cell surface phenotype of pluripotent hemopoietic stem cells (CFU-S) and committed progenitors (CFU-C1, CFU-C2, BFU-E) of mouse bone marrow was analyzed with respect to their binding of wheat germ agglutinin (WGA) and two monoclonal antibodies, anti-GM-1.2 and anti-PGP-1. Stained cells were fractionated on the basis of differences in fluorescence and light scatter intensity using a light-activated cell sorter. The 6% of the cells that bound most WGA and that also had a relatively high forward light scatter (FLS) and low perpendicular light scatter (PLS) contained nearly all stem cells (CFU-S) and progenitors. Anti-GM-1.2 stained only mature myeloid cells, not CFU-S or the in vitro colony-forming cells. Anti-PGP-1 stained all bone marrow cells in varying intensities: lymphoid cells were dull, CFU-S were intermediate, CFU-C2 were brighter, and mature myeloid cells very bright. Enrichment of progenitor cells was performed by a two-step sorting procedure. First, the 6% most WGA-binding cells with high FLS and low PLS were sorted out. A 10-15-fold enrichment of progenitors and CFU-S was obtained. Next, these cells were restained with anti-GM-1.2 or anti-PGP-1 and again fractionated on the FACS. The GM-1.2-negative cells were then another four- to sevenfold more enriched for stem cells and progenitors. Of the cells in this fraction, 95% could be assigned to a colony-forming unit. With anti-PGP-1, CFU-C2 could be partly separated from more early cells such as CFU-S and BFU-E.     

唾液酸对正常造血细胞及白血病细胞增殖分化的影响

本实验以测定细胞电泳率(EPM)反映细胞表面唾液酸相对含量。发现胎肝细胞电泳率明显高于成年骨髓细胞,经神经氨酸酶(NA)处理胎肝细胞、骨髓细胞、L_(801)急性白血病细胞和KCL-22慢性白血病细胞后,细胞EPM均明显下降。细胞集落数及细胞形态学检测表明,NA处理后,多向造血干细胞(CFU-S)增殖能力减弱,向粒系的分化增强;KCL-22细胞生成的集落数明显减少,成熟细胞的比例明显增加。     

Effect of Myleran On Murine Hemopoiesis

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. the severity and duration was greatest at high dose levels of MY (800 μ). the action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. the peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Effect of myleran on murine hemopoiesis. I. Granulocytic cell line specificity of action on progenitor cells.

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. The severity and duration was greatest at high dose levels of MY (800 microgram). The action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. The peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Cytofluorometric analysis of R-Thy-1. antigens in various rat lymphocytes with different electrophoretic mobility and organ distribution.

Small bone marrow lymphocytes, which had been previously enriched by velocity sedimentation, thymocytes, lymph node cells and spleen cells were electrophoretically separated, stained with fluorescein conjugated rabbit a-rat-Thy-1. globulin and their fluorescence intensities analyzed with a flow cytophotometer. Thy-1. antigens were found in 80% of the bone marrow small lymphocytes showing low electrophoretic mobility (EPM), in all thymocytes, about 80% of which show low and the rest medium to high EPM, and in a few lymph node cells of high EPM. Thy-1. positive cells were not observed in the spleen. All fluorescence intensity histograms obtained were modal and could be properly fitted with normal curves showing coefficients of variation (C.V.) in the range of 20% to 30%. It was observed that the thymocytes of low EPM had an antibody binding affinity significantly different from that of the other stained lymphocytes. Moreover the surface antigen density decreased in the sequence: thymocytes of low EPM, bone marrow lymphocytes of low EPM and thymocytes of high EPM. The fluorescence intensity of stained lymph node cells of high EPM appeared similar to that of thymocytes of high EPM but was not evaluated precisely. Thus the two dimensional cell analysis provided by a combination of EPM and surface fluorescence of Thy-1.+ cells, allows the characterization of different lymphocyte populations which cannot be clearly identified with normal one dimensional techniques. The biological significance of the results is discussed briefly.     

Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation,differentiation and leukemogenic capacity

Long-term cultures of proliferating hematopoietic stem cells derived from bone marrow permit the study of the interaction between murine leukemia virus (MuLV) infection and the proliferation and differentiation of stem cells. We have used this system to analyze the replication of different biological variants of MuLV in bone marrow cells; the effect of MuLV infection upon pluripotent stem cell (CFU-S) proliferation; and the effect of MuLV on differentiation of CFU-S along different hematopoietic pathways. Two MuLV variants were studied in detail: the Moloney strain of lymphatic leukemia virus (Mol-MuLV) and the erythroleukemic Friend virus complex (FLV) consisting of the lymphoid leukemia helper virus and the defective spleen focus-forming virus (SFFV). Mol-MuLV and its sarcoma virus pseudotype, MSV(Mol-MuLV), replicate efficiently in the bone marrow cultures; however, CFU-S are lost more readily than in uninfected cultures, and the cultures are soon represented by a majority population of mononuclear macrophages. On the other hand, infection with FLV produces a prolonged survival of the spleen colony-forming cells, CFU-S, and CFU-C (the committed granulocytic precursor cells). Production of erythroleukemogenic SFFV is maintained in these cultures for more than 40 weeks. No erythroblastic differentiation was observed in vitro, however, neither erythroblast precursor cells (CFU-E) nor hemoglobin-producing cells could be detected. This suggests that the target cell for FLV is an earlier precursor cell.     

The kinetics of hematopoietic stem cells during and after hypoxia. A model analysis

A previously described mathematical model of the hematopoietic stem cell system has been extended to permit a detailed understanding of the data during and after hypoxia. The model includes stem cells, erythroid and granuloid progenitors and precursors. Concerning the intramedullary feedback mechanisms two basic assumptions are made: 1) The fraction "a" of CFU-S in active cell cycle is regulated. Reduced cell densities of CFU-S, progenitors or precursors lead to an accelerated stem cell cycling. Enlarged cell densities suppress cycling. 2) The self renewal probability "p" of CFU-S is also regulated. The normal steady state is described by p = 0.5, indicating that on statistical average each dividing mother stem cell is replaced by one daughter stem cell, while the second differentiates. Diminished cell densities of CFU-S or enlarged densities of progenitors and precursors induce a more intensive self renewal (p greater than 0.5), such that the stem cell number increases. The self renewal probability declines (p less than 0.5) if too many CFU-S or too few progenitors and precursors are present. The model reproduces bone marrow data for CFU-S, BFU-E, CFU-C, CFU-E, 59 Fe-uptake and nucleated cells in hypoxia and posthypoxia. Although the ratio of differentiation into the erythroid and granuloid cell lines is kept constant in the model, a changing ratio of CFU-E and CFU-C results. The model suggests that stem cells and progenitor cells are regulated by a regulatory interference of erythropoiesis and granulopoiesis.     

Surface differentiation of hemopoietic cells demonstrated ultrastructurally with cationized ferritin

The ultrastructural cationized ferritin (CF) technique was employed as a probe of the surface binding characteristics of the various cell types present in normal human bone marrow. The number of CF particles per micron length of cell surface were counted and data subjected to statistical analysis. All cells of the bone marrow exhibited CF reactivity. The extent of labeling was cell specific and could be related to the stage of maturation of the cells in a given lineage. In the neutrophilic series, myeloblasts showed moderate labeling while promyelocytes and myelocytes revealed only minimal binding; CF binding increased sequentially in metamyelocytes, band and segmented neutrophils. Eosinophils and eosinophilic myelocytes showed similar membrane differnetiation patterns while basophils exhibited stronger CF labeling that other granulocytic cells. Lymphocytes were strongly reactive while monocytes and their precursors were moderately labeled with CF. Surface reactivity of developing nucleated erythrocytic cells was similar to that of the lymphocytes. Surface labeling from the proerythroblasts to early normoblasts stage was identical, CF binding increased in the late normoblasts stage and then decreased in the reticulocyte and mature erythrocyte stages. The extent of surface CF reactivity of the marrow cells was markedly different from that obtained with Thorotrast and colloidal iron. Thorotrast and colloidal iron stained the surface of all marrow cell intensely but failed to yield distinctive surface labeling patterns for the differing cell population in bone marrow.     

Proliferation and splenic migration of mouse hematopoietic stem cells following a single injection of Mycoplasma arthritidis

The effect of a single injection of live M. arthritidis microorganisms on the bone marrow and spleen CFU-S populations was assessed. One of the principal findings is that marrow CFU-S are recruited into cell cycle (as determined by hydroxyurea kill) early after M. arthritidis administration and stay in the cycle for at least 2 weeks thereafter. The peak level of cycling value (47%) was observed on day 4. The duration of increased CFU-S cycling activity was shown to coincide with a time period during which a significant rise in the number of endogenous CFU-S was maintained. The other important finding is that splenic seeding efficiency ("f-factor") declines by 56% one day following M. arthritidis administration. The latter effect could be attributed to the binding of mycoplasmas to the surface of CFU-S as specific rabbit antiserum against M. arthritidis incubated in vitro with the bone marrow cells of infected donor mice caused an up to 48% reduction in the in vivo colony-forming ability of CFU-S.     

人骨髓细胞培养液中干细胞刺激物的分析研究

人骨髓细胞体外培养液中含有高活力的 CSF,在长期培养过程中,CSF 活力的变化,与 CFU-C 数量的变化有大致平行的趋势。这种 CSF 对狗和小鼠也同样有效。人骨體条件液中的 CSF 对培养中的 CFU-S 也有明显的激发作用。这一结论可以从几个方面获得证据:第一,小鼠骨髓细胞与人骨髓条件液保温六小时后,再测定其中 CFU-S 数,结果是增加了。第二,经亚致死剂量照射的小鼠,腹腔注射适量的人骨髓条件液,其内源性脾结节也明显增多。第三,采用阿糖胞苷自杀的方法,测定小鼠骨髓经与人骨髓条件液保温后,其中 CFU-S 的自杀率也有增高的趋势。上述几方面的实验,说明人骨髓长期培养中存在着某种活性物质,调节体外造血。至于这种物质的来源,以及在体外造血中所起的作用,还需要做很多工作,逐步予以澄清。     

Isolation of murine pluripotent hemopoietic stem cells in the Go phase

A method to purify pluripotent hemopoietic stem cells in the Go phase from mouse bone marrow was established. Bone marrow cells from 5-fluorouracil (5-FU)-treated mice were fractionated by Percoll density gradient. The cells with density between 1.063 and 1.075 were further separated into wheat germ agglutinin (WGA)-positive and -negative cells using fluorescent-activated cell sorter (FACS) after staining with fluorescein isothiocyanate-conjugated WGA (FITC-WGA). An assay for spleen colony-forming units (CFU-S) revealed that the WGA-positive cells (1 X 10(6)) produced 1380 CFU-S (about 150 times of the number in the original bone marrow cells) on day 12 (but no CFU-S on day 8), whereas the WGA-negative cells produced no CFU-S. Thus, the stem cells in the Go phase are found to be enriched 150 times in 5-FU-treated WGA-positive cells.     

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.     

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

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.     

Pluripotent (Cfu-S) and Granulocyte-Committed (Cfu-C) Stem Cells In Intact and 89Sr Marrow-Ablated S1/S1D Mice

Peripheral blood values, femur cell counts, spleen weights, pluripotent (CFU-S) and granulocyte progenitor cell (CFU-C) concentrations and total content of spleens and femurs have been evaluated in intact (non-marrow-ablated) and ⁸⁹Sr marrow-ablated S1/S1^d and +/+ mice. ⁸⁹Sr-irradiated mice were studied 6 and 11 days after the administration of ⁸⁹Sr. In intact S1/S1^d mice the femur CFU-S concentration, total femur CFU-S, femur CFU-C concentration and total femur CFU-C were 84, 54, 105 and 68% that of +/+ mice femurs respectively; the respective values for the spleens of S1/S1^d mice were 40,46,61 and 69%. These are the first simultaneous determinations of CFU-S and CFU-C concentrations, and content of spleens and marrows, of S1/S1^d and +/+ mice. In ⁸⁹Sr marrow-ablated mice, 11 days after injection of the radionuclide: (a) the total content of marrow CFU-C and CFU-S was about 1% of that found in the marrows of intact mice for both +/+ and S1/S1^d groups; (b) the spleens of +/+ mice increased in weight to 162% of the control, but the spleens of S1/S1^d mice did not increase in weight; and (c) the spleens of +/+ mice had a total content of CFU-C and CFU-S of 800% and 260% of the control, respectively, whereas the respective values for the S1/S1^d mice were 120% and 76% of the control. Thus the S1/S1^d spleen fails to compensate for marrow ablation by housing additional CFU-S and has an impaired ability to compensate by housing additional CFU-C.     

RECOVERY OF PROLIFERATING HAEMOPOIETIC PROGENITOR CELLS AFTER KILLING BY HYDROXYUREA

Two doses of 1 mg/g of hydroxyurea (HU), injected 7 hr apart into irradiated mice in which CFU-S were proliferating during marrow regeneration, killed about 90% of CFU-S. This same dose regime injected into normal female mice, with non-proliferating CFU-S killed 92 % of CFU-C, 99 % of ESC and only 30 % of CFU-S. One day after the treatment CFU-S had decreased to 50 % and remained at about this level for a further day then returned to normal values. In spleen the increase in CFU-S was delayed by a day and showed a marked overshoot. During the period that CFU-S were decreased in number they were actively proliferating. Marrow CFU-C recovered in an exponential manner with a doubling time of 16 hr. Spleen CFU-C recovered 1 day later than marrow and showed a pronounced overshoot. ESC recovered very rapidly with doubling time of 5 hr. The changes in ⁵⁹Fe incorporation into RBC, and the peripheral blood picture, were a delayed reflection of the changes in ESC and CFU-C.     

Erythropoietic progenitors capable of colony formation in culture: state of differentiation

The differentiated state of mouse erythropoietic progenitor cells (CFU-E), detected by their ability to form erythropoietin-dependent colonies in vitro, has been investigated. Transfusion-induced plethora was found to reduce the population size of CFU-E in both spleen and femoral marrow, which indicates that a significant number of CFU-E arise by differentiation processes that are themselves erythropoietin-dependent. Individual spleen colonies were found to be heterogeneous in their content of CFU-E, and the numbers of CFU-E per colony were not correlated either positively or negatively with the numbers of granulocyte-macrophage progenitors (CFU-C) present in the same colonies. The absence of a negative correlation between CFU-E and CFU-C indicates that the erythropoietic and granulopoietic pathways of differentiation are not mutually exclusive within individual spleen colonies. The numbers of CFU-E per spleen colony were also found to vary independently of the numbers of pluripotent stem cells (CFU-S) per colony; in contrast, as found previously, the numbers of CFU-C and CFU-S per colony were positively correlated. These results indicate that more randomizing events separate CFU-E from CFU-S than separate CFU-C from CFU-S, and are consistent with the view that CFU-E occupy a position on the erythropoietic pathway of differentiation that is more remote from the pluripotent stem cells than is the corresponding position of CFU-C on the granulopoietic pathway.     

Stimulator of proliferation of spleen colony-forming cells in T-cell deprived mice treated with cyclophosphamide or irradiation

A role for T-cells in the regulation of CFU-S proliferation was investigated by determining the presence and activity of CFU-S proliferation stimulator (CFU-S stimulator) in adult mouse bone marrow after irradiation or cyclophosphamide (Cy) treatment. CBA mice previously deprived of T-cells by thymectomy, irradiation and bone marrow reconstitution (TIR) were thereafter treated with 4.5 Gy irradiation or 200 mg/kg Cy. Regenerating bone marrow cells of TIR and corresponding control mice after irradiation or Cy treatment produced CFU-S stimulator. The dose dependent increase in cytosine arabinoside cell death of normal bone marrow day 8 CFU-S was found when both CFU-S stimulators obtained after irradiation of TIR or corresponding control animals were tested. CFU-S stimulator activity in the bone marrow of TIR-Cy treated mice was also detected, but the effect was not dose-dependent. This was not related to the presence of an inhibitor of CFU-S proliferation. It appears that the CFU-S stimulator activity is not related to IL-6, IL-1 or IL-2, or to an inhibitor of IL-6 or IL-1 activity. The results demonstrate the existence of CFU-S proliferation stimulator unrelated to the two major monokines in the bone marrow of immunosuppressed mice.     

Effects of plutonium-239 on haemopoiesis. I. Quantitative and qualitative changes in CFU-S in different regions of the mouse femur and vertebrae

Mice were injected with plutonium-239 (960 Bq/mouse) and, over a period of four months, the response of haemopoietic tissue and the self-renewal capacity of its stem cells was monitored. Cellularity, CFU-S concentration and self-renewal capacity were measured in five different regions of bone and marrow--axial and marginal marrow of the femoral shaft, femur shaft, proximal and distal ends of the femur shaft and vertebrae. Cellularities were little affected by plutonium but CFU-S were reduced in all regions, most severely in the bone shaft and marginal marrow due to the initial deposition of plutonium on the bone surface, by four days. The reduction in axial CFU-S, however, was due probably to a relatively long plasma half-life resulting from the tendency of plutonium to combine with plasma proteins. The capacity of CFU-S for self-renewal was reduced and remained low in all zones. Thus, although the highly self-renewing axial CFU-S were depleted, and remained so, due probably to a longer term redistribution of plutonium throughout the marrow, additional proliferation of the more mature CFU-S in the other zones kept their self-renewal low while replenishing their numbers and maintaining a normal cell output.     

Myeloproliferative sarcoma virus (MPSV), a new tool for the study of hematopoiesis

MPSV induces a myeloproliferative syndrome in susceptible mice associated with an invasion of hematopoietic and nonhematopoietic organs with tumor nodules. The effect of the virus on the various hematopoietic precursors (CFU-S, CFU-C, CFU-E, BFU-E) was studied in vivo in the spleen, blood, and bone marrow, and in vitro, using colony assays in semisolid medium. After in vivo and in vitro infection MPSV induces the appearance of CFU-C, independent of added colony-stimulating activity and of pure and mixed BFU-E, independent of burst-promoting activity. MPSV also induces in vivo an amplification of the size and concentration of the hematopoietic system, including hematopoietic stem cells. MPSV infection may also alter the hemapoietic microenvironment. Modification of the disease by total body irradiation followed by bone marrow stem cell reconstitution or by splenectomy is compatible with mediation of the virus effect at the level of hematopoietic microenvironment. MPSV may constitute a new tool to study the regulation of murine hematopoiesis and viral genetic information, which can specifically induce characteristic disturbances of this system.