Expression of congenital defects in the haemopoietic micro-environment. Erythroid and granulocyte-macrophage progenitor cells in pre-natal 'steel' (Slj/Slj) anaemic mice.

The erythropietin sensitivities of dissociated cell cultures and explanted fragments of fetal livers of congenitally anaemic Slj/Slj mice, and their normal littermates, have been compared. The erythropoietin responsiveness of Slj/Slj foetal liver cells is deficient in both types of culture. The maximum liver complement of erythroid colony forming cells (CFUe) occurs on the 16th day of development when 'normal' livers contain approximately 6 X 10(5) erythroid colony forming cells/liver. In Slj/Slj fetuses the maximum reached is only 1 X 10(5). Granulocyte-macrophage colony forming cells (CFUc) in Slj/Slj fetal livers are also reduced to approximately 60% of normal numbers. Erythroid colony forming cells are also reduced in the spleen and femoral bone marrow of Slj/Slj mice in the 2-3 days preceding birth. Granulocyte-macrophage colony forming cells are rare in the femoral marrow of pre-natal Slj/Slj mice, but their production in the Slj/Slj pre-natal spleen appears unaffected.     

Characteristics of the Cfu-S 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, radiosen-sitivity and humoral regulation was attempted in mice heterozygous for the mutant allele Sl^J and in their normal littermates. Sl^J/+ mice were characterized by a deficient CFU-s content of the blood and spleen and had slightly lower femoral CFU-s numbers. This CFU-s distribution could not be explained by differences in seeding efficiency ‘f’ between CFU-s of Sl^J/+ and +/+ origin in lethally irradiated recipients used in the CFU-s assay. the seeding fraction of CFU-s of +/+ origin did not differ in +/+ and Sl^J/+ recipients. However, in irradiated SI^J/+ recipient mice a 30% decrease was observed in the number of the colonies derived from splenic and femoral CFU-s of both +/+ and Sl^J/+ origin. the serum level of SHSF (splenic haemopoiesis stimulating factor) was decreased in Sl^J/+ mice, but significantly increased in Sl/Sl^d mice, as compared to their respective normal +/+ littermates. Endogenous colony formation in Sl^J/+ spleens was deficient in comparison to that observed in +/+ spleens, and distinct sex differences were observed. However, mutant and normal CFU-s from spleen and bone marrow had a similar survival following in-vitro y irradiation. Femurs and spleens of both Sl^J/+ and +/+ origin were implanted into both Sl^J/+ and +/+ hosts. Six weeks later the Sl^J/+ grafts contained less CFU-s than the +/+ grafts. These data show that the splenic stroma of Sl^J/+ mice is not defective in its capacity to lodge injected CFU-s but is deficient in its ability to maintain CFU-s under ‘steady-state’ conditions and stimulate their colony formation in a ‘perturbed state’. Some of the characteristics of Sl^J/+ mice segregate them from Sl/Sl^d mice, i.e. a deficient splenic CFU-s content, normal seeding fractions ‘f’ of CFU-s from spleen and bone marrow in the presence of an almost compensated anemia, and decreased serum levels of SHSF. the study of the Sl^J trait may be a useful extension of the current Sl/Sl^d model for exploration of hereditary defects in haematopoietic stroma.     

A DEFICIENCY OF HEMATOPOIETIC STEM CELLS IN STEEL MICE

In the present study, population sizes of high self-renewal potential stem cells, i.e. colony forming units (CFU), and low self-renewal potential stem cells, i.e. transient endogenous colony forming units (TE-CFU) in Sl/Sl^d mice and their normal congenic littermates were measured and compared. By correcting for differences in the seeding efficiency ‘f’, it was possible to demonstrate that Sl/Sl^d mice suffer a deficiency of both stem cell populations. Therefore, it is concluded that the defective stromal tissue of the Sl/Sl^d mouse does not support normal size stem cell populations. However, as noted in the discussion, it remains an open question as to whether the defective stromal tissue supports normal erythroid differentiation at the stem cell level.     

EXPRESSION OF CONGENITAL DEFECTS IN THE HAEMOPOIETIC MICRO-ENVIRONMENT: PRE-NATAL ERYTHROPOIESIS IN ANAEMIC 'STEEL'(SljSlj) MICE

The production of red cells by the livers of congenitally anaemic Sl^jSl^j foetal mice is reduced, so that the red cell concentration reaches only 0.4 × 10⁹/ml, compared to 2.3 × 10⁹/ml in normal littermates. The deficiency in haemoglobin concentration is not so severe (3 g/100 ml compared to 10 g/100 ml), because Sl^jSl^j red cells are macrocytic and normochromic. The number of erythroblasts in the livers of Sl^jSl^j foetuses is considerably less than in normal littermates, but the ratio of early erythroblasts to late erythroblasts is not altered. The cell cycles of Sl^jSl^j and normal early erythroblasts are similar, but the cycle time of Sl^jSl^j late erythroblasts is longer than that of the corresponding normal cells. Sl^jSl^j foetal liver cells have a restricted response to erythropoietin in vitro in terms of haem synthesis, whereas foetal liver cells of normal littermates are highly responsive until the 16th day of gestation. Mixtures of late 16 day Sl^jSl^j and normal foetal liver cells (both non-responsive alone) respond to erythropoietin in vitro.     

Role of splenic stroma in the action of bacterial lipopolysaccharides on radiation mortality: a study in mice carrying the Slj allele

Abstract. Sl_j/+ mice display a slight macrocytic anaemia due to a defect in their haemopoietic organ stroma. They have a deficient endogenous spleen colony (CFU-end) formation following sublethal doses of gamma-radiation compared with their normal +/+ littermates, which is likely to be due to the low pre-irradiation CFU-S content of the Sl_j/+ spleen. CFU-S in these congenic mice do not differ in their sensitivity to gamma-irradiation or stem cell-activating factor. While injection of +/+ mice with 10 μg of lipopolysaccharide-W (LPS) one day prior to irradiation led to a substantial increase in their survival, the survival of Sl_j/+ mice was only slightly increased. Irradiation induced a similar dose-related reduction in the numbers of CFU-S in the spleen and femora of LPS-injected Sl_j/+ mice compared to similarly treated +/+ mice when measured directly after irradiation. At Day 9 after irradiation, injection of LPS led to a significantly higher CFU-end formation and higher numbers of CFU-S and nucleated cells in the Sl_j/+ spleens compared to LPS-injected +/+ mice. No such differences in the radioprotective effect of LPS were observed in the +/+ and Sl_j/+ mice with respect to the splenic and femoral ⁵⁹Fe-incorporation and the femoral CFU-S numbers at Day 9. These data strongly suggest a contribution by immigrating CFU-S to the CFU-S numbers and endogenous colony formation in at least the Sl_j/+ 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 anti-radiation drugs.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

A kinetic study of the genetic control of hemopoietic progenitor cells assayed in culture and in vivo

The kinetics of growth of bone marrow cells from normal or genetically anemic mice (Sl/Sl^d and W/W^v) were studied in irradiated normal and genetically anemic hosts. The parameters followed included total cellularity, the number of peroxidase positive cells, and the number of cells capable of forming colonies in vivo (CFU-S) or in culture (CFU-C). The results of these experiments demonstrate that W and Sl defects alter the growth of CFU-C and peroxidase-positive cells to a modest degree; that the defects are more obvious when studied in spleen rather than in bone marrow; and that there is no additivity of W and Sl defects. Nineteen irradiated recipients of marrow from W/W^v mice were studied after three to six months. Of these, 18 showed host-type erythrocytes, while in one mouse the erythrocytes had the size distribution of W/W^v cells. This finding indicated that occasionally genetically defective stem cells may repopulate irradiated hosts.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium

Erythroid colony formation in agar cultures of CBA cells was stimulated by the addition of pokeweed mitogen-stimulated C57BL spleen conditioned medium. Both 48-hour colonies ("48-hour benzidine-positive aggregates") and day 7 large burst or unicentric erythroid colonies ("erythroid colonies") developed, together with many neutrophil and/or macrophage colonies. In CBA mice, the cells forming erythroid colonies occurred with maximum frequency (650/10(5) cells) in 10- to 11-day-old yolk sac and fetal liver but were present also in fetal blood, spleen and bone marrow. The frequency of these cells fell sharply with increasing age and only occasional cells (2/10(5) cells) were present in adult marrow. A marked strain variation was noted, CBA mice having the highest levels of erythroid colony-forming cells. The erythroid colony-forming cells in 12-day CBA fetal liver were radiosensitive (DO 110-125 rads), mainly in cycle and were non-adherent, light density, cells sedimenting with a peak velocity of 6-9 mm/hr. These properties are similar to those of other hemopoietic progenitor cells in fetal tissues. The relationship of these apparently erythropoietin-independent erythroid colony-forming cells to those forming similar colonies after stimulation by erythropoietin remains to be determined.     

Colony formation in agar by multipotential hemopoietic cells.

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.     

Cellular uptake of 3H-actinomycin D in the hematological tissues and liver of the mouse

Actinomycin D(AM), an inhibitor of DNA-dependent RNA synthesis, produces a reversible cessation of red blood cell production. This study examines the in vivo cellular uptake of ³H-AM in the hematological tissues and livers of B6D2F₁ mice. ³H-Am (sp. act. = 2.97 to 4.20 C/mmole) was given IV at a dose of 4.0 to 5.7 μg (14 μc) per mouse. Spleen, bone marrow, blood, and liver samples were taken for autoradiography at post-injection times of five minutes to 67 hours. We have confirmed the rapid in vivo cellular uptake of AM; substantial quantities of the drug were in the nuclei within five minutes of IV administration. Not all cell types became labeled. Erythroid, hepatic, lymphoid, and reticulo-endothelial (RE) cells and monocytes took up the label, whereas labeling of granulocytic elements was doubtful. Most heavily labeled were liver cells (highest mean grain count = 110.1) and splenic RE(19.1) and erythroid (16.1) cells. Erythroid cells in the spleen were more heavily and more rapidly labeled than those in the bone marrow. All nucleated erythroid maturational stages, in both the spleen and the bone marrow, were labeled, even at five minutes. The time course of erythroid and hepatic labeling was quite different. Whereas early erythroid cells required six hours to become 100% labeled, liver cells were 100% labeled at five minutes and loss of hepatic labeling began as early as 15 to 30 minutes.     

Microenvironment created by stromal cells is essential for a rapid expansion of erythroid cells in mouse fetal liver

Mouse stromal cell lines (FLS lines), established from the livers of 13-day gestation mouse fetus, supported the proliferation and differentiation of the erythroid progenitor cells from mouse fetal livers and bone marrow in a semisolid medium in the presence of erythropoietin. A large erythroid colony of over 1000 benzidine-positive erythroid cells was developed from a single erythroid progenitor cell on the FLS cell layer after 4 days of culture. When in close contact with the layer, the erythroid progenitor cells divided rapidly with an average generation time of 9.6 h and mature erythroid cells, including enucleated erythrocytes, were produced. The present studies demonstrate that the microenvironment created by the stromal cells can support the rapid expansion of erythropoietic cell population in the fetal liver of mice.     

On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

Fetal erythropoiesis in steel mutant mice : I. A morphological study of erythroid cell development in fetal liver

A method of definitive identification of mutant (S1/S1^d) and wild-type (+/+) mouse embryos in segregating litters is described, based on the total number of circulating erythrocytes in a unit volume of embryonic blood and the relative proportion of nonnucleated vs. nucleated red blood cells. Evidence is presented that from days 13–17 of gestation, S1/S1^d embryos have many fewer fetal liver derived nonnucleated erythrocytes whereas the number of yolk sac-derived nucleated red blood cells is similar between S1/S1^d and +/+. Erythroid precursor cells at various stages of maturation in mutant fetal livers are studied by light and electron microscopy, and their fine structure is found to be identical to those present in normal embryos. The number of hemoglobin-containing mature erythroblasts in mutant fetal livers is far fewer than that of the normal, whereas the number of immature erythroid precursors present in a unit area of fetal liver is not significantly different between S1/S1^d and +/+. It is suggested that the mutant S1 gene product(s) interferes with or fails to support the differentiation of immature erythroid precursors into hemoglobin synthesizing cells.     

Interactions between purified GM-CSF, purified erythropoietin and spleen conditioned medium on hemopoietic colony formation in vitro.

Preincubation of C57BL adult marrow cells or CBA fetal liver cells with a 250-fold excess concentration of purified GM-CSF failed to reduce the frequency of cells forming eosinophil, megakaryocyte or erythroid colonies in subsequent agar cultures. When excess concentrations of purified GM-CSF were added to agar cultures stimulated by pokeweed mitogen-stimulated spleen conditioned medium (SCM), no reduction was observed in the frequency of eosinophil, megakaryocyte or erythroid colonies. Addition of 4 units of purified erythropoietin (EPO) to cultures of fetal liver or adult marrow cells stimulated by SCM increased the number of erythroid colonies but did not reduce the number of non-erythroid colonies or the non-erythroid content of mixed erythroid colonies. Although neither GM-CSF nor EPO alone was able to stimulate erythroid colony formation in agar cultures of fetal liver cells, small numbers of large erythroid colonies were stimulated to develop in cultures containing both purified regulators. Purified GM-CSF was also able to support the survival in vitro of a small proportion of erythroid colony-forming cells in fetal liver populations cultured initially in the absence of SCM and the survival of some eosinophil and megakaryocyte colony-forming cells in similar cultures of adult marrow cells. The results do not support the hypothesis that GM-CSF and EPO compete for a common pool of uncommitted progenitor cells. On the contrary, the data indicate that GM-CSF und EPO are able to collaborate in stimulating the proliferation of some erythropoietic cells. Furthermore, purified GM-CSF appears to be able to support temporarily the survival and/or initial proliferation of at least some cells forming erythroid, eosinophil and megakaryocyte colonies, even though GM-CSF is unable to stimulate the formation of colonies of these types.     

Regulation of haemopoietic stem-cell proliferation in mice carrying the Slj allele

We investigated a haemopoietic stromal defect, in mice heterozygous for the Sl^j allele, during haemopoietic stress induced by treatment with bacterial lipopolysaccharides (LPS) or lethal total body irradiation (TBI) and bone-marrow cell (BMC) reconstitution. Both treatments resulted in a comparable haemopoietic stem cell (CFU-s) proliferation in Sl^j/+ and +/+ haemopoietic organs. There was no difference in committed haemopoietic progenitor cell (BFU-e and CFU-G/M) kinetics after TBI and +/+ bone-marrow transplantation in Sl^j/+ and +/+ mice. the Sl^j/+ mice were deficient in their ability to support macroscopic spleen colony formation (65% of +/+ controls) as measured at 7 and 10 days after BMC transplantation. However, the Sl^j/+ spleen colonies contained the same number of BFU-E and CFU-G/M as colonies from +/+ spleens, while their CFU-s content was increased. On day 10 post-transplantation, the macroscopic ‘missing’ colonies could be detected at the microscopic level. These small colonies contained far fewer CFU-s than the macroscopic detectable colonies. Analysis of CFU-s proliferation-inducing activities in control and post-LPS sera revealed that Sl^j/+ mice are normal in their ability to produce and to respond to humoral stem-cell regulators. We postulate that Sl^j/+ mice have a normal number of splenic stromal ‘niches’ for colony formation. However, 35% of these niches is defective in its proliferative support.     

DISSECTING THE HEMATOPOIETIC MICROENVIRONMENT. I. STEM CELL LODGMENT AND COMMITMENT, AND THE PROLIFERATION AND DIFFERENTIATION OF ERYTHROPOIETIC DESCENDANTS IN THE Sl/Sld MOUSE

The anemic Sl/Sl^d mouse and its normal (+/+) congenic control were used to explore the possibility of stromal control over four phases of erythropoiesis: CFU lodgment, commitment of multipotent stem cells to the erythropoietic line, proliferation of stem cells and their descendants, and the differentiation of those descendants into successively more mature forms. Lodgment was found to be the same in the Sl/Sl^d as in the normal mouse, but commitment, although characteristically different for spleen compared to the bone marrow, was subnormal. The stimulus to proliferate, as measured by spleen colony size and cell type content, was even more reduced. It is suggested that the direct control of differentiation into more mature cells may not be under stromal control.     

B lymphocyte differentiation in lethally irradiated and reconstituted mice. I. The effect of Strontium-89 induced bone marrow aplasia on the recovery of the B cell compartment in the spleen.

The influence of ⁸⁹Sr-treatment on the recovery of the B cell compartment in lethally irradiated, fetal liver reconstituted mice was studied by means of membrane fluorescence. ⁸⁹Sr is a bone-seeking radio-isotope which causes in a dose of 3 μCi ⁸⁹Sr/g body weight a depletion of all nucleated cells, including immunoglobulin-bearing (B) cells, of the bone marrow.Treatment of irradiated and fetal liver reconstituted mice with 3 μCi ⁸⁹Sr/g body weight immediately and at 17 days after irradiation and reconstitution prevented recovery of the nucleated cell population, including B cells, in the bone marrow. In the spleen of such mice both nucleated cells and B cells reappeared at day 7 and 14 respectively. The B cell population in the spleen did not recover up to normal values during the experimental period of 45 days. It is concluded that B cell differentiation in lethally irradiated, fetal liver reconstituted mice can take place outside the bone marrow. The efficiency of this extra-medullary differentiation is discussed. The conclusion was drawn that mice with a ⁸⁹Sr-induced bone marrow aplasia are able to generate B lymphocytes. Consequently the bone marrow microenvironment seems not to be obligate to the differentiation of B lymphocytes. The peripheral lymphoid organs of such mice were found to be unable to compensate completely for the absence of B lymphocyte production in the bone marrow.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFU_c-GM compartment followed by an increase in the number of femoral CFU_s. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFU_e) compartment expanded to 168% of control, while the more differentiated (CFU_e) compartment was reduced (45% of control at day 28). Reductions in both ⁵⁹Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr × 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. the data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.