The dependence of the formation of stromal CFU-f colonies on the stimulating action of hematopoietic cells

CFU-f-derived stromal colony formation was accomplished in adherent marrow cell cultures (AMCC) with serum-rich medium. It turned out to require additional stimulation by hemopoietic feeder cells: by irradiated marrow cells and spleen cells if they possess megakaryocytes and platelets or by platelets from the blood. PDGF, EGF and IL-3 did not substitute the colony stimulating activity of feeder cells. Thymus, lymph node cells and blood leucocytes had no colony stimulating activity. At low oxygen concentrations which improve colony formation the stimulating activity of hemopoietic feeder cells was expressed, as well. Thus, CFU-f colony formation depends on stimulation by hemopoietic cells in addition to serum growth factors. In full populations of marrow cells the CFU-f colony formation is stimulated by marrow cells which accompany the CFU-f.     

Production of hemopoietic growth factors and gamma-interferon by large granular lymphocytes from patients with T gamma lymphocytosis

Two patients with T gamma lymphocytosis in whom expanded large granular lymphocyte (LGL) populations were detected in peripheral blood and bone marrow are described. The surface antigen phenotypes of the LGL from these patients were similar with a major portion of cells carrying T3, T8, T11 and Leu7 markers. However, whereas fresh LGL from both patients demonstrated antibody-dependent cell cytotoxicity (ADCC), natural killer (NK) cell function was present in one case but absent in the other. Supernatants from enriched suspensions of the LGL unstimulated by exogenous antigen or mitogens were shown to contain significant amounts of hemopoietic growth factors colony-stimulating activity (CSA) and burst-promoting activity (BPA). In one case gamma-interferon was also detected. This study contributes to the accumulating evidence that LGL are able to generate factors which have the capacity to influence the proliferation of hemopoietic progenitor cells in vitro.     

Heterologous antiserum to rat lymphohemopoietic precursor cells.

Lymphohemopoietic precursor cells in rat bone marrow are members of a subset of lymphocyte-like cells that bears the bone marrow lymphocyte antigen (BMLA) and that lacks antigens present on peripheral B and T cells. This was demonstrated by two experimental approaches. In the first, bone marrow cells with the potential to form hemopoietic colonies in spleen (CFU-S), to repopulate lumphoid tissues and blood, and to rescue lethally irradiated recipients were enriched approximately 10-fold by a fractionation procedure designed to isolate a "null" population of bone marro lymphocytes. In the second approach, the lymphohemopoietic precursor cell activity in bone marrow was completely abrogated by opsonization with rabbit antiserum (ALSBM) raised against this "null" population of bone marrow cells. Precursor cell activity was not affected by treatment with antiserum to T and B cells. Quantitative cross-absorption studies showed that the antigen detected by ALSBM on lymphohemopoietic precursor cells had the same cellular distribution as did the previously described bone marrow lymphocyte antigen. It is likely that this antigen is present both on pluripotent stem cells and on committed progenitors of the myelocytic, erythrocytic and lymphocytic series.     

The release of interleukin-2 (IL-2) and colony stimulating activity (CSA) in aplastic anemia patients: opposite behaviour with improvement of bone marrow function

Peripheral blood cells from patients with aplastic anemia were tested for their ability to release interleukin-2 (IL-2) and colony stimulating activity (CSA) before treatment. IL-2 release--as measured in the mouse thymocyte assay--was abnormally high in 18/34, and abnormally low in 10/34 patients. "Low" release was due to simultaneous release of thymocyte inhibitors. In 18 patients who achieved self-sustaining hemopoiesis after high dose immunosuppressive therapy, excess IL-2 release decreased to low levels (p less than 0.001), and the release of inhibitors disappeared. In contrast, the release of CSA by patient cells--which did not correlate with peripheral blood monocyte counts--either remained high or increased to excessively high values in 24/24 patients tested before and after successful immunosuppressive treatment. Patients with stable hemopoietic grafts after bone marrow transplantation for aplastic anemia, did not release excess CSA. It is concluded that IL-2 and CSA play opposite roles in aplastic anemia. High IL-2 release seems associated with disease activity, whereas high CSA-release appears to reflect a repair mechanism.     

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.     

Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c- MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU- GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.     

Stimulation of genetic resistance to marrow grafts in mice by interferon-alpha/beta

Lethally irradiated mice were infused with syngeneic, H-2 allogeneic, parental strain, or H-2 heterozygous bone marrow cells. They were injected daily with rabbit anti-mouse interferons (IFN)-alpha/beta or gamma or with IFN-alpha/beta. The growth of donor-derived cells was judged 5 days later by measuring splenic incorporation of 5-iodo-2'-deoxyuridine-125I into DNA. Antibodies to IFN-alpha/beta, but not to IFN-gamma, weakened genetic (both hybrid and allogeneic) resistance to marrow cell grafts. IFN-alpha/beta stimulated hybrid and allogeneic resistance, the latter even in genetically "poor responder" mice. Mice pretreated with silica, which weakens genetic resistance, were stimulated by IFN-alpha/beta to resist incompatible marrow cell grafts; however, IFN-alpha/beta failed to reverse the effects of antiasialo GM1 serum on marrow graft rejection. IFN-alpha/beta did not inhibit the growth of syngeneic marrow cells and did not stimulate resistance to H-2 heterozygous bone marrow cells. We propose that genetic resistance occurs in two discrete steps. In the first step, hemopoietic histocompatibility (Hh) antigens are recognized by one host cell type, and this recognition leads to IFN-alpha/beta secretion by a silica-sensitive cell. In the second step, asialo GM1-positive natural killer cells stimulated by IFN-alpha/beta recognize Hh antigens on marrow stem cells and cause rejection. The defects in resistance observed in genetically poor responder mice and in mice treated with silica appear to involve the first step in recognition. The lack of rejection of H-2 heterozygous (Hh-) marrow cells by parental strain mice injected with IFN-alpha/beta indicated that specific Hh recognition is critical in the second step of genetic resistance.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.     

The radioprotective effect of hypothermia on the immune and hematopoietic systems in mammals

The functional activity of the synthetic apparatus (parameter alpha) in blood lymphocytes, bone marrow hemopoietic cells, and thymus cells, as well as the total number of blood and bone marrow cells in rats after y-irradiation at a dose of 8 Gy in the conditions of normothermia and hypothermia (16-18 degrees C) with hypoxia-hypercapnia were investigated after 2 h and on days 1 and 4. The recovery processes in blood in both groups of rats after acute X-irradiation at a dose of 7 Gy for 36 days were analyzed too. Under hypothermia, on days 1-4 after acute gamma-irradiation, a decrease in the synthetic activity in remaining cells and devastation in the hemopoietic system were pronounced to a lesser degree. After X-irradiation, the restoration of synthetic activity in blood lymphocytes was shown to begin earlier and to finish faster in "hypothermic" rats as compared with the animals irradiated in the state of normothermia. The survival of "hypothermic" rats was 100% as compared with 30% in "normothermic" animals. Thus, the data show that hypothermia exerts a radioprotective effect on the cells of the immune and hemopoietic systems, thus enhancing the resistance of the organism to radiation.     

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.     

Formation of capillary networks from bone marrow cultured in collagen gel

The growth of capillaries from mouse bone marrow was studied in collagen gel. When the culture contained sufficient bone marrow cells (more than 1 X 10(6) cells) and cell clusters, short capillaries with lumina appeared about one to two weeks after inoculation, following the proliferation of fibroblastic cells and hemopoietic cells. Four weeks after inoculation, these capillaries formed a network among hemopoietic cells and adipocytes. Electron microscopic observations revealed that these capillaries had thin walls and poorly developed basement membranes, similar to the sinusoids of bone marrow. These capillaries did not appear when the amount of inoculated bone marrow was reduced or dispersed to the point that the marrow cell clusters disappeared. The quantity of the inoculum and the clustering of cells, therefore, seems to play important roles in the appearance of the capillaries.     

Maintenance and proliferation control of primitive hemopoietic progenitors in long-term cultures of human marrow cells

Primitive, high-proliferative potential hemopoietic progenitors can be routinely maintained for many weeks in long-term marrow cultures (LTC) in the absence of added hemopoietic growth factors. Nevertheless, these progenitors are clearly responsive to both positive and negative regulatory control mechanisms that operate within the adherent layer as evidenced by cyclic changes in their proliferative activity each time the medium is replaced. The key event appears to be the addition of a constituent of fresh horse serum that is not found in fetal calf serum. Analogous primitive neoplastic progenitor cell types from CML or PV patients are insensitive to the negative arm of this proliferation control mechanism both in vitro and in vivo. A model to explain the progenitor cell cycle changes normally observed in the LTC system is proposed. This model suggests that perturbations of nonhemopoietic mesenchymal cells determine the net positive or negative influence that these regulatory cells exert on adjacent primitive hemopoietic cells, possibly by a mechanism involving direct cell contact. Recently, we have identified a number of cytokines that can simulate the transient positive effect of fresh horse serum, as well as another cytokine, that is, tumor growth factor-beta (TGF-beta), that can mimic the negative but reversible effect exerted by mesenchymal cells. These studies demonstrating the effects of positive and negative regulatory cytokines on the control of hemopoiesis in the adherent layer of LTC suggest new approaches for analyzing the basis of both normal and abnormal stem cell regulation by marrow stromal elements.     

Growth factors that stimulate human granulocyte—macrophage colony formation produced by the cell line CM-S

CM-S is an autonomous cell line of human hemopoietic precursor cells inducible to monocyte-macrophage differentiation in response to appropriate inducing agents. CM-S cells produce factors that stimulate their own growth and proliferation, and are also capable of stimulating clonal proliferation of human, but not mouse, monocytic and granulocytic bone marrow progenitor cells in viscous medium. Preliminary purification steps have demonstrated at least two species, one of which (MW 30,000–50,000) retains both these activities, while the other (MW ≤ 10,000) apparently retains only the autostimulatory activity. CM-S cells could thus be a useful source for the purification of human colony stimulating factors (CSFs). CM-S cells also respond to factors present in human placenta conditioned medium, known to contain human CSF. This suggests that CM-S cells could provide a homogeneous target cell population for testing CSFs from other human sources.     

Leukaemic peripheral blood plasma and bone marrow plasma: comparison of influence on lymphocyte proliferation

Abstract. Peripheral blood plasma from some children with untreated acute lymphoblastic leukaemia (ALL) exerted an inhibitory effect in vitro on phytohaemagglutinininduced lymphocyte transformation of normal peripheral blood lymphocytes. This occurred at concentrations beyond that required for optimal response as judged by reduction of blast cell formation and tritiated thymidine and tritiated uridine incorporation into DNA and RNA, respectively. In contrast, bone marrow plasma from these patients was non-inhibitory or contained significantly less inhibitory activity. Bone marrow plasma from the majority of healthy controls was superior to their peripheral blood plasma in enhancing phytohaemagglutinin-induced mitogenesis. The difference between an individual's bone marrow- and peripheral blood-derived plasma in enhancing proliferation of patient and healthy control cells was significantly greater amongst the patients than the healthy control group; this was attributed mainly to the increased inhibitory activity of ALL peripheral blood plasma compared with normal plasma. Medium conditioned by phytohaemagglutinin-stimulated normal peripheral blood lymphocytes was effective in neutralizing the inhibitory activity of ALL peripheral blood plasma. Taken together, these in vitro results are at least suggestive that in vivo , in healthy subjects, the rapidly proliferating cells in the bone marrow and the 'resting' blood cells in the circulation may be under the influence of a fine balance of different types and/or levels of humoral growth stimulatory and inhibitory factors and that in ALL an unstable balance of these factors exists. The decreased proliferation of circulating blast cells compared with bone marrow blasts in ALL may be attributed, at least in part, to exposure to the different levels of inhibitor(s) in the circulation and bone marrow as demonstrated in vitro by our results.     

Photoinactivation of lymphohemopoietic cells: studies in transfusion medicine and bone marrow transplantation.

Ultraviolet (UV) irradiation affects eukaryotic cells in numerous ways. Exposure of blood transfusion products to UVC (200-280 nm) or UVB (280-320 nm) reduces or abrogates their immunogenicity and thereby prevents allosensitization and transfusion refractoriness in several models. Although the exact mechanism is not known, in vitro studies suggest that UV exposure results in a loss of class II histocompatibility antigens from the cell surface, alterations of calcium homeostasis, and a lack of interaction between antigen presenting and responding cells. In the UVB and UVA (320-400 nm) range, lymphocytes appear to be more sensitive than hemopoietic cells. In murine transplant models, UVB irradiation of spleen and marrow cells can be used to prevent the development of graft-versus-host disease while allowing for complete hemopoietic reconstitution. Furthermore, in clinical marrow transplantation, pilot studies of UVA in conjunction with psoralen administration have yielded encouraging results in patients with steroid refractory graft-versus-host disease of the skin. Thus, UV irradiation provides an interesting tool to study cell/cell and donor/host interactions and may have some applications in transfusion medicine and bone marrow transplantation.     

Collection and freezing of blood stem cells in acute nonlymphoblastic leukemia

Blood-derived hemopoietic stem cells were collected using a continuous (10 patients) or a semi-continuous flow separator (2 patients) in some patients with acute non-lymphocytic leukemia. For ten out of those patients, five to seven leukaphereses were performed during a short period (median = 11 days) of marrow recovery following severe aplasia induced by an intensive chemotherapy. The mean number of CFU-GM cells collected per leukapheresis and per patient respectively was 6.7 X 10(4)/kg and 38.5 X 10(4)/kg. This latter number was similar to that obtained during a marrow harvest performed for a bone marrow transplantation, suggesting that high numbers of hemopoietic stem cells can be collected from the peripheral blood in leukemic patients and used for autologous transplantation.     

Developments in modern hematology.

In the past 40 years our concepts about hemopoiesis have been changed dramatically. The results of bone marrow transplantation into lethally irradiated mice since the mid-fifties suggested the existence of a hemopoietic stem cell, which was initially identified as a spleen colony forming cell (CFU-S). Later experiments showed that the stem cell compartment is rather heterogeneous and that the most primitive stem cell, unlike the CFU-S, has the ability for long-term engraftment of an irradiated recipient. Daughter cells of such primitive quiescent stem cells lose their capacity for self-generation gradually with each mitosis and become more and more committed to a specific differentiation lineage. In vitro culture techniques in a serum-free semi-solid medium enabled the establishment and analysis of specific hemopoietic growth factors. Such factors, which are essential for the maintenance, proliferation and differentiation of progenitor cells and the functional activity of mature cells can now be produced with recombinant DNA techniques in pure form and large quantities. Hemopoiesis requires an appropriate microenvironment, consisting of various stromal cell types and an extracellular matrix. Intercellular contacts, adhesion of cells and growth factors to the matrix molecules seem essential in the regulating action of this hemopoietic microenvironment. In long-term bone marrow cultures the development of a stromal hemopoietic microenvironment can facilitate long-term maintenance of stem cells and hemopoietic differentiation. For bone marrow transplantation and infusion of hemopoietic growth factors many clinical indications are well established and our possibilities to interfere in the regulation of hemopoiesis are still growing.     

Collection of hematopoietic stem cells from canine peripheral blood and their ability to regenerate hematopoiesis]

A procedure is presented for the collection of a large number of hemopoietic stem cells from the peripheral blood of dogs by means of a single leukapheresis using the NCI-IBM Blood Cell Separator. In the course of a leukapheresis of about 285 min duration a mean of 23 x 10-9 leukocytes is collected from the blood. The hemopoietic stem cells among such separated leukocytes initiate repopulation of bone marrow within 10 days after whole body X-irradiation with 1200 R. The cell numbers in a defined histological section of femoral bone marrow are evaluated 9 to 10 days after irradiation and subsequent autologous transfusion of 6.72 x 10-9 separated mononuclear leukocytes. The results indicate that the bone marrow cell numbers of transfused dogs are significantly greater than in dogs given only 1200 R and reach a level of approximately 49% of the normal value. Possible ways of increasing the yield of hemopoietic stem cells from the peripheral blood will be considered.     

Evaluation of the marrow culture on the macrophage layer covering intraperitoneally implanted membrane as an assay for hemopoietic progenitor cells

When cellulose acetate membranes are implanted into abdominal cavity of mice they turn into a foreign body overgrown with macrophages. Such macrophage layer has been shown by other authors to be able to support the growth of hemopoietic colonies formed by intraperitoneally injected hemopoietic cells. This study confirms and extends this observation by showing that both granulopoietic and erythropoietic colonies may be observed. The number of colonies grown is in linear correlation with that of injected hemopoietic cells. The frequency of erythropoietic colonies was greatly enhanced by blood letting of the host mice. Colony forming cells were most numerous in the bone marrow then in the spleen and peripheral blood and hardly in the thymus. Prior irradiation of the host mice was essential for obtaining colony growth and the optimal dose was determined to be 6.0 Gy. This technique opens the way to studies into hemopoietic progenitor cells for laboratories having no sophisticated tissue culture equipment and where necessary reagents are easily available.     

The neuronal EGF-related genes NELL1 and NELL2 are expressed in hemopoietic cells and developmentally regulated in the B lineage

NELL1 and NELL2 (neural epidermal growth factor-like 1 and 2) are recently described members of the epidermal growth factor gene family that have previously been shown to be expressed almost exclusively in brain tissue. Here we demonstrate regulated expression of NELL1 and NELL2 in human hematopoietic cells. Mature NELL1 mRNA is not detected in any normal hemopoietic cell type, although the gene is transcribed during a narrow window of pre-B cell development, and cell lines at the same developmental stage express the NELL1 mRNA. The related NELL2 gene is expressed by all nucleated peripheral blood cells examined (B, T, monocyte, and natural killer cells), but not in any of the bone marrow B lineage cells at earlier stages of development. However, leukemic cell lines corresponding to the same early differentiation stages express abundant NELL2 mRNA. These results suggest normal and possible oncogenic roles for the NELL proteins in hemopoietic cells.