Pre CFU-F in bone marrow cultures from children with hematopoietic disease including acute leukemia

Stromal precursor cells from bone marrow aspirates of children have been studied in culture. In 7 day liquid cultures normal individuals and patients with acute leukemia in remission grew 110 +/- 50 CFU-F and 100 +/- 40 CFU-F (colony forming unit--fibroblasts) respectively, per 6 X 10(5) buffy coat mononuclear cells. Staining with monoclonal antibodies suggests that stromal cells from CFU-F colonies are fibroblasts. CFU-F colony growth from the bone marrow of patients with active leukemia was low. After cultivation periods of more than 21 days, we observed, in addition, still more immature, clonogenic fibroblast precursor cells, "pre CFU-F", and round cells attached to stromal cells from pre CFU-F colonies. From the round cells, we have passaged pre CFU-F and CFU-GM (colony forming unit--granulocytic, monocytic) in secondary cultures. Our observations are in agreement with the concept that the bone marrow stromal cell matrix serves as a sanctuary for reversibly attached clonogenic cells of both the hematopoietic and fibroblast lineages.     

Origin of stromal bone marrow mechanocytes according to the results of typing them by isoantigens and chromosomal markers]

The bone marrow of radiochimaeras and heterotopic bone marrow transplants were used to study the origin of precursors of the fibroblasts growing in the monolayer cultures of hemopoietic tissue. In the bone marrow explants of the (C57BL/6 X CBA) F1 mice, in which the CBA bone marrow was transplanted following the lethal irradiation, the fibroblasts grown in the colonies were of recipient origin judging by isoantigens in the reaction of indirect immunofluorescence with the anti-C57BL/6-serum. At the same time in the bone marrow explants from heterotopic transplants (CBA leads to CBA X C57BL/6) the fibroblasts grown in colonies were of donor origin. The cultures of hemopoietic cells of the bone marrow of females heterotopically transplanted in the singenic male (guinea pigs Huston) contained only fibroblasts which were of donor origin judging by sex chromosomes in the metaphase plates of dividing cells. Hence, the bone marrow precursors of fibroblasts do not depend histogenetically on hemopoietic cells and are not replaced at the expense of repopulating cells of the second partner.     

Potential use of tuftsin in treatment of candida peritonitis in a murine model

A major complication of continuous ambulatory peritoneal dialysis (CAPD) is peritonitis caused by Candida albicans. Increasing the activity of the peritoneal macrophages, the predominant cell type found in the peritoneal cavity, may be a promising treatment for this infection. Tuftsin was found to increase thioglycollate-elicited mouse peritoneal macrophage activity. 2x10(-7) M tuftsin enhanced two-fold cell association with radiolabelled candida, superoxide aniom production, and killing activity. Thus, a model consisting of mice undergoing peritoneal dialysis was developed in order to study the use of tuftsin as a therapeutic drug against peritoneal candidiasis. Administration of tuftsin (50 micrograms/mouse) before candidiasis induction with a lethal dose of candida (7x10(8) candida per mouse) improved mouse survival up to 70%, compared with 10% in the control group. The potential of tuftsin as a treatment for candidiasis was shown when the infection was induced with a sublethal dose of candida. Daily intraperitoneal injections of tuftsin (50 micrograms) to the sublethally infected mice caused a significant decrease in the number of candida recovered from the peritoneal cavity and from the blood (from 700 +/- 190 to 110 +/- 26 CFU/ml and from 100 +/- 26 CFU/ml to 17 +/- 8 CFU/ml, respectively). In addition, a larger number of peritoneal macrophages with greater phagocytic and killing activity were found in the tuftsin-treated mice. The effect of tuftsin may promote its potential use in the therapy of peritonitis in patients undergoing chronic peritoneal dialysis.     

The effect of inflammation on splenic colony formation after the intraperitoneal administration of bone marrow cells

The number of spleen colonies formed after intraperitoneal injection of bone marrow cells increases approximately 100-fold in mice with inflammation induced by nitrocellulose filters implanted into the intraperitoneal cavity. By transplanting these filters together with cells grown on them into intact animals and replacing them with clean filters we have demonstrated that this effect is associated with inflammation focus in the peritoneal cavity rather than with CFU-S proliferation of the filter surface.     

The effect of allogeneic lymphocytes on colony formation during culturing of hematopoietic precursor cells in the peritoneal cavity

The number of colonies formed in the peritoneal cavity (on the artificial underlayer made of peritoneal cells) and in the spleen of lethally irradiated recipients, (CBA X X C57BL) F1 mice, after the intraperitoneal injection of marrow cells depends on the cell donor's genotype: syngeneic cells and cells from mice of the parent strain CBA form fewer colonies in the peritoneal cavity than in the spleen, while cells from C57BL mice produce the reverse distribution of colonies between the peritoneal cavity and the spleen. Allogenic lymphocytes, when transplanted simultaneously with hematopoietic cells, suppress colony formation in the peritoneal cavity from day 2 of cultivation and eliminate the already developed foci of hematopoiesis by day 5.     

CFU-F circulating in cord blood

CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.     

A study of hemopoiesis on a sublayer of peritoneal cells in the presence of hemopoietic cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

A Study of Hemopoiesis on Sublayer of Peritoneal Cells in the Presence of Hemopoietic Cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

Colony-forming fibroblast precursors of the circulating blood

Colony-forming fibroblast precursors were detected in circulating blood of adult guinea pigs by CFUf in vitro colony assay. SFU amount to 0.9 +/- 0.2 (M +/- m) per 10(5) explanted leucocytes ranging from 0.04 X 10(-5) to 3.0 X 10(-5) for individual donors. The presence of collagen type I and lack of factor VIII antigen and of FC-receptors proved that CFU-derived colonies in the blood cultures were composed of fibroblasts.     

Response of lymphosarcoma LS/BL cells to continuous irradiation

Mouse lymphosarcoma LS/BL cells growing as an ascites tumor in the peritoneal cavity of C57BL mice were continuously irradiated in vivo at a low exposure rate of 1.2 Gy per day (5 rad/hr). The growth of the ascites tumor evaluated by direct counting of the cells in the peritoneal cavity and their capacity to form colonies in livers declined with increasing time of continuous irradiation. The radiosensitivity and repair ability of LS/BL cells were studied by a serial dilution method using host survival time as the end point and by the liver colony assay. The radiosensitivity of continuously irradiated LS/BL-CI cells showed no remarkable change as measured by the Do values, but from the 150th week of irradiation the initial shoulder on the survival curves appeared and its width increased with time of exposure. The extrapolation number (n) increased from 1.0 to 8.4 after 350 weeks of irradiation. The reappearance of the initial shoulder was proved with the split-dose technique.     

Influence of albumin on granulocyte and macrophage colony-forming efficiency

Mouse granulocyte and macrophage precursors were assayed in plasma clot and fibrin clot cultures, and the effect of bovine serum albumin (BSA) on colony formation was investigated. The number of granulocyte colonies (CFU-g) and clusters increased as the albumin concentration was increased and the number of macrophage colonies (CFU-m) and clusters concomitantly decreased. The albumin-mediated suppression of macrophage colony formation was overcome by the addition of more than 10% fetal bovine serum (FBS) to the plasma clot culture. The effect of BSA and fatty-acid-free BSA on colony-forming efficiency was also tested in fibrin clot cultures containing 10% FBS. Both BSA and fatty-acid-free BSA at a final concentration of 0.5-2% enhanced CFU-g colony formation, while both forms of BSA reduced the number of CFU-m colonies. However, neither BSA nor fatty-acid-free BSA had any effect on colony formation in FBS-free fibrin clot cultures, and only BSA enhanced colony formation when transferrin, linoleic acid, alpha-thioglycerol and dextran were added to the culture. The number of CFU-g (15.6 +/- 3.1) was higher in cultures containing BSA, transferrin, etc., than the number (9.8 +/- 2.5) in cultures without BSA and including transferrin, etc., than the number (9.8 +/- 2.5) in cultures without BSA and including transferrin, etc. (p less than 0.01). The number of CFU-m (32.0 +/- 6.8) in cultures containing BSA and the other four factors was lower than the number (72.2 +/- 5.6) in the culture without BSA (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts

Ionizing radiation-induced genomic instability has been demonstrated in a variety of endpoints such as delayed reproductive death, chromosome instability and mutations, which occurs in the progeny of survivors many generations after the initial insult. Dependence of these effects on the linear energy transfer (LET) of the radiation is incompletely characterized; however, our previous work has shown that delayed reductions in clonogenicity can be most pronounced at LET of 108 keV/microm. To gain insight into potential cellular mechanisms involved in LET-dependent delayed loss of clonogenicity, we investigated morphological changes in colonies arising from normal human diploid fibroblasts exposed to gamma-rays or energetic carbon ions (108 keV/microm). Exposure of confluent cultures to carbon ions was 4-fold more effective at inactivating cellular clonogenic potential and produced more abortive colonies containing reduced number of cells per colony than gamma-rays. Second, colonies were assessed for clonal morphotypic heterogeneity. The yield of differentiated cells was elevated in a dose- and LET-dependent fashion in clonogenic colonies, whereas differentiated cells predominated to a comparable extent irrespective of radiation type or dose in abortive colonies. The incidence of giant or multinucleated cells was also increased but much less frequent than that of differentiated cells. Collectively, our results indicate that carbon ions facilitate differentiation more effectively than gamma-rays as a major response in the progeny of irradiated fibroblasts. Accelerated differentiation may account, at least in part, for dose- and LET-dependent delayed loss of clonogenicity in normal human diploid cells, and could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Osteoclast precursors in bone marrow and peritoneal cavity

Osteoclasts differentiate from cells that share some phenotypes with mature macrophages and monocytes, but early precursors for osteoclasts still remain obscure. To characterize osteoclast precursors, using monoclonal anti-c-Fms and anti-c-Kit antibodies, bone marrow cells were separated and the frequency of clonogenic progenitors were measured. Osteoclast precursors in the bone marrow mainly expressed c-Kit and diminished in frequency when they expressed c-Fms. In contrast to bone marrow, the precursors in the peritoneal cavity were enriched with a population of c-Fms⁺. Injection of these antibodies into mice demonstrated that peritoneal osteoclast precursors were sensitive to anti-c-Fms but not to anti-c-Kit antibodies, whereas those in bone marrow only declined in the presence of both antibodies. Meanwhile, c-Fms as opposed to c-Kit played an essential role in the generation of osteoclasts in cultures. We also compared osteoclast precursors with colony forming cells (CFU-M) by a macrophage colony stimulating factor. CFU-M in bone marrow decreased when anti-c-Kit antibody was administered and no CFU-M was detected in peritoneum. In this study, we show differences between proliferative potential osteoclast precursors maintained in bone marrow and peritoneum and between CFU-M and osteoclast precursors. J. Cell. Physiol. 170:241–247, 1997. © 1997 Wiley-Liss, Inc.     

Effect of keratinocyte growth factor on radiation survival and colony size of human epidermal keratinocytes in vitro.

Keratinocyte growth factor (FGF7, also known as KGF) ameliorates the radiation response of mouse oral mucosa and other epithelial tissues. However, the precise mechanisms remain unclear. The aim of the present study was to investigate the effect of FGF7 on the survival and colony size of normal human epidermal keratinocytes in vitro. Primary neonatal keratinocytes (HEKn) were irradiated with doses of 0 and 2 Gy of 200 kV X rays and incubated in the presence or absence of 100 ng/ml FGF7. The plating efficiency (PE) and surviving fraction (SF2) were determined using a clonogenic assay. In cell cultures without FGF7, the mean PE was 4.6 +/- 0.2%. Irradiation with 2 Gy resulted in an SF2 of 51 +/- 2%. In cell cultures with FGF7, the mean PE was identical, and a similar SF2 of 54 +/- 1% was observed (P = 0.4). However, the individual colony size was significantly increased in all cultures incubated with FGF7 compared to those incubated without FGF7. The number of extremely large colonies (> or =2 mm) was clearly higher (P < 0.0001) in cultures with FGF7. This was accompanied by a significant reduction in the diameter of individual cells from 29 microm in controls to 23 microm with FGF7. In conclusion, FGF7 does not affect the survival of keratinocytes after irradiation, but it does stimulate proliferation of surviving cells.     

Cells Forming Spleen Colonies At 7 Or 11 Days After Injection Have Different Proliferation Rates

Abstract In the early periods (7–9 days) after haemopoietic cell injection, colonies produced by CFU-s and by their progeny are identified in the spleen, while at later periods (11 days after injection) only spleen nodules produced by CFU-s persist. the increase in the suicide values of CFU-s after sublethal (2 Gy) irradiation of mice is associated with a higher proliferation rate of precursors of transitory spleen colonies, but not of CFU-s, as measured by different suicide techniques. During the log-phase of cell growth in a lethally irradiated recipient, the injected CFU-s and CFU-tr proliferate at a higher rate. Active proliferation of CFU-s and CFU-tr has been demonstrated in long-term bone-marrow cultures by the hydroxyurea in-vitro suicide assay. CFU-tr may be the cause of artifactual effects during measurement of haemopoietic stem-cell cycling by CFU-s suicide methods.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Proliferative and differentiation potentials of skeletogenic bone marrow colony-forming cells

The clonal nature of bone marrow fibroblast colonies derived from clonogenic bone marrow osteogenic cells (CFUf) was proved by the chromosome analysis. During subsequent passages of multi-colony derived bone marrow fibroblast strains there occurs a pronounced increase in the cell number and in the number of osteogenic units (tested by transplantation in diffusion chambers). Single colony-derived strains are capable of forming bone and cartilage simultaneously. It follows that CFUf or part of them are clonogenic cells with high proliferative potentials and are common precursors for bone and cartilage tissue. Thus, CFUf may be regarded as osteogenic stem cells.     

Heterologous antisera to stromal mechanocytes of the bone marrow]

Heterologous rabbit antifibroblast serum (AFS) to stromal fibroblasts of the guinea-pig bone marrow was obtained. AFS was shown to bind specifically stromal fibroblasts and their precursors (but not macrophages) in the monolayer primary cultures of the bone marrow, thymus and spleen of guinea pigs (in the complement-dependent cytotoxic reaction and in the indirect immunofluorescence test); AFS also bound precursors of blood fibroblasts and peritoneal exudate (in the cytotoxic reaction). Precursors of the thymus fibroblasts were found to be more sensitive to the AFS action than the spleen and bone marrow precursors, this suggesting that the thymic stromal mechanocytes had a greater concentration of common tissue-specific antigens on their surface. Precursors of the stromal fibroblasts in native cell suspensions were found to be essentially more sensitive to the cytotoxic action of AFS than the colony-forming fibroblasts on the passage cultures.     

Characterization of reticulofibroblastoid colonies (CFU-RF) derived from bone marrow and long-term marrow culture monolayers

The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.     

Distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5' -nucleotidase activity

Using a cytochemical assay we were able to show that the peritoneal macrophage population of normal nontreated mice (resident peritoneal macrophages) exhibits a heterogeneity with regard to the expression of the activity of the ecto-enzyme 5′-nucleotidase (5′-N). About 75% of the macrophages express high enzymic activity whereas the remaining 25% express low 5′-N activity. Macrophages accumulating in the peritoneum as a result of an inflammatory response are predominantly of the low activity type. In vitro activation of resident peritoneal macrophages by lymphokines does not result in a decrease in the number of macrophages expressing high enzymic activity though the level of the enzymic activity of these cells is reduced by about 36%. Bone marrow derived mononuclear phagocyte colonies developing in vitro, under liquid culture conditions, from bone marrows of normal mice can be divided into three types with respect to their expression of 5′-N activity: (1) high activity colonies–relatively small colonies in which all the cells express high 5′-N activity (about 20% of the colonies); (2) low activity colonies – relatively large colonies in which all the cells express low 5′-N activity (about 70% of the colonies); and (3) mixed colonies–relatively large colonies in which all the cells express low enzymic activity except for about 8% of cells located at the periphery of the colonies which express high enzymic activity (about 10% of the colonies). During an inflammatory response the frequency of the high activity colonies is significantly reduced. Our results provide evidence for distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5′-N activity and suggest that (1) the resident macrophages derive from a subpopulation of bone marrow precursor cells developing in vitro into high 5′-N activity mononuclear phagocytes, and (2) during an inflammatory response there is a preferential expansion of clones of the low enzymic activity phenotype.