Showing papers on "Haematopoiesis published in 1982"

Transient nature of early haematopoietic spleen colonies

Abstract: Certain cells (CFU-S) in the haematopoietic tissues of mice are able to form macroscopic nodules in the spleen 7–14 days after injection in heavily irradiated recipient mice1. At 7–9 days, most of the spleen colonies contain recognizable cells of one predominant haematopoietic lineage2, and do not contain cells capable of spleen colony formation on injection in secondary hosts3. However, by 14 days most of the spleen colonies do contain cells of more than one line of haematopoietic differentiation2, as well as precursor cells capable of again generating similar multilineal spleen colonies on retransplantation3. These observations have led to the widely accepted conclusion that most spleen colonies are derived from pluripotential ‘stem’ cells3, and to the suggestion that the apparent transformation in character of spleen colonies from unilineal at early times to mixed later on reflects the local operation of ‘instructive’ differentiative signals in the splenic environment2. The reasoning behind these suggestions implicitly assumed that late colonies simply represented a further stage in the development of the same colonies observable at earlier times. Here, we present direct evidence against this assumption. Our data indicate that most spleen colonies identified as surface nodules at days 7–8 are neither multipotential nor self-maintaining, but rather are destined to disappear from the spleen within 72 h. The observations set limits on the validity of the spleen colony method as an assay for self-maintaining pluripotential progenitor cells, and give cause for reassessment of the data on which widely accepted views concerning the regulation of such cells are based.

Transcripts from the cellular homologs of retroviral oncogenes: distribution among chicken tissues.

Abstract: The oncogenes (v-onc genes) of rapidly transforming retroviruses have homologs (c-onc genes) in the genomes of normal cells. In this study, we characterized and quantitated transcription from four c-onc genes, c-myb, c-myc, c-erb, and c-src, in a variety of chicken cells and tissues. Electrophoretic analysis of polyadenylated RNA, followed by transfer to nitrocellulose and hybridization to cloned onc probes showed that c-myb, c-myc, and c-src each give rise to a single mature transcript, whereas c-erb gives rise to multiple transcripts (B. Vennstrom and J. M. Bishop, Cell, in press) which vary in abundance among different cells and tissues. Transcription from c-myb, c-myc, c-erb, and c-src was quantitated by a "dot-blot" hybridization assay. We found that c-myc, c-erb, and c-src transcription could be detected in nearly all cells and tissues examined, whereas c-myb transcription was detected only in some hemopoietic cells; these cells, however, belong to several different lineages. Thus, in no case was expression of a c-onc gene restricted to a single cell lineage. There appeared to be a correlation between levels of c-myb expression and hemopoietic activity of the tissues and cells examined, which suggests that c-myb may be expressed primarily in immature hemopoietic cells. An examination of c-onc RNA levels in target cells and tissues for viruses carrying the corresponding v-onc genes revealed no obvious correlation, direct or inverse, between susceptibility to transformation by a given v-onc gene and expression of the homologous c-onc gene.

Differential expression of the amv gene in human hematopoietic cells.

Abstract: Total cellular RNAs from a variety of fresh and culture-derived human hematopoietic neoplastic cell types at various stages of differentiation and human sarcoma, carcinoma, melanoma, and glioblastoma cell lines were enriched for poly(A)- containing sequences, fractionated by gel electrophoresis, and blot hybridized to a cloned DNA probe containing the transforming sequences (v-amv) of avian myeloblastosis virus (AMV), a virus known to cause myeloid leukemias in chickens. Expression of RNA sequences homologous to AMV was detected in all immature myeloid and lymphoid T cells in addition to the single erythroid cell line examined, but not in mature T cells or in B cells, including lymphoblast cell lines derived from patients with Burkitt lymphoma. In addition, induction of the cell line HL60, a promyelocytic leukemia line, to differentiate with dimethyl sulfoxide or retinoic acid resulted in a reduction of the level of expression of the human cellular gene c-amv homologous to v-amv. There was no detectable c-amv mRNA in any of the solid tumor cell lines examined. Thus, expression of the human c-amv gene could be correlated with the stage of differentiation of different hematopoietic cell types determined by morphologic and marker studies. Expression of c-amv could not be correlated with the extent of methylation in HL60 and in HL60 induced to differentiate with dimethyl sulfoxide.

Biochemical characterization and cellular distribution of a polymorphic, murine cell-surface glycoprotein expressed on lymphoid tissues

Abstract: A murine leukocyte surface glycoprotein (Mr = 95 000) has been defined by means of xenogeneic monoclonal antibodies. In normal hematopoietic tissues, the glycoprotein is found in highest amounts in the bone marrow. Flow cytometric analysis shows that essentially all bone-marrow cells express the glycoprotein and that it is a major component of a subpopulation of cells containing predominantly granulocytic precursors. In contrast, only about 5 percent of thymocytes express sufficient glycoprotein to be detected by flow cytometric analysis, although under stringent conditions up to 20 percent of thymocytes are susceptible to complement-mediated cytotoxicity using a monoclonal antibody against the glycoprotein. Functional assays showed that both prothymocytes and colony forming unit-spleen express the glycoprotein which is broadly distributed on murine hematopoietic tumor cell lines. However, although some Thy-I+ (T) cell lymphomas express large amounts of the glycoprotein, others do not express detectable quantities of the molecule. The glycoprotein is not restricted to hematopoietic cells and can be detected on lung, kidney, brain, and liver as well as cultured fibroblasts. Monoclonal antibodies against the glycoprotein cross-react with an antigen present on human cells. As described in the accompanying paper, the glycoprotein exists in two antithetical allelic forms and we show that it is identical to a polymorphic surface molecule independently characterized by Colombatti and co-workers.

Hematological characterization of congenital osteopetrosis in op/op mouse. Possible mechanism for abnormal macrophage differentiation.

Abstract: Compared with normal littermates, the op/op mice had very few macrophages in the peritoneal cavity and severely reduced numbers of monocytes in the peripheral blood. Moreover, osteopetrotic animals demonstrated an altered distribution of hemopoietic tissue with a 10-fold decrease in the number of marrow cells. Liver hemopoiesis persisted in 4-wk-old mice as evidenced by the presence of hemopoietic stem cells (HSC). Moreover, the concentration of HSC was decreased in marrow and increased in the spleen of op/op mice. In spite of the paucity of cells of monocyte-macrophage lineage in vivo, progenitor cells from hemopoietic tissues of op/op mice formed increased numbers of monocyte-macrophage colonies in vitro in the presence of exogenous colony-stimulating activity (CSA). The source of this critical CSA was a medium conditioned by stromal fibroblastoid colonies formed in vitro by normal marrow cells. Therefore, these data suggest that op/op mice possess normal monocyte-macrophage-osteoclast progenitor cells but these cells are unable to fully differentiate in the op/op mouse microenvironment. In support of this, in cultures of stromal fibroblastoid colonies from op/op marrow or spleen, the concomitant growth of macrophages, normally very dense, was drastically reduced. Moreover, transplantation of op/op spleen cells into lethally irradiated littermate recipients resulted in their hemopoietic reconstitution without signs of macrophage defect. Thus, the op/op splenic cells do not transfer the disease and are capable of normal differentiation in normal in vivo environment. These observations support the hypothesis that the defect in op/op mice is a result of the failure of hemopoietic stromal fibroblastoid cells to release sufficient amounts of CSA necessary for normal differentiation of cells of the monocyte-macrophage lineage.

Donor origin of the in vitro haematopoietic microenvironment after marrow transplantation in man

Abstract: The method for long-term culture of marrow cells in vitro as described by Dexter1 has recently been successfully applied to human marrow2,3 and is dependent on the development of an adherent stromal cell layer consisting of cells described as “endothelial-like cells, fat cells, and macrophages” 4. The present study was designed to determine the origin and composition of the stromal cells forming the in vitro ‘microenvironment’ and maintaining haematopoeisis in long-term cultures grown from marrows of 14 patients who received marrow transplants from HLA identical siblings of the opposite sex. The presence of a Y chromosome was used as a marker to establish the donor or recipient origin of the cells. We found that the stromal cells became progressively donor in origin with time after transplantation and some reacted with antibody directed against factor VIII-associated antigen. In addition, donor-derived in vitro stromal cells synthesized both interstitial and basal lamina collagen types, indicating that the in vitro microenvironment is transplantable and composed in part of endothelial-like cells.

Inhibition of in vitro granulopoiesis by autologous allogeneic human NK cells.

Abstract: This study demonstrates the ability of human NK cells to inhibit in vitro granulopoiesis of autologous and allogeneic BM cells. NK lytic activity and GM-CFC inhibition was present among nonstimulated lymphocytes from healthy donors and could be increased by treatment of PBL with IFN. Both the cytotoxic NK cells and the GM-CFC inhibitory cells could be enriched for among nonadherent, low-density cells. High-density cells were not cytotoxic, only inhibitory to a small extent, and could become neither cytotoxic nor more inhibitory after IFN treatment. In contrast, low-density cells showed an increased cytotoxic and GM-CFC inhibitory capacity after IFN treatment. The NK mediated GM-CFC inhibition was dependent on cell contact with BM cells, increased with longer preincubation times, and was most efficient against 7-day GM-CFC as compared with 14 day GM-CFC progenitors. In conclusion, these data provide new information about the human NK cell as a potent inhibitor of in vitro granulopoiesis and also as a possible regulator of hematopoiesis in vivo.

A stochastic model of self-renewal and commitment to differentiation of the primitive hemopoietic stem cells in culture.

Abstract: We recently identified a murine hemopoietic stem cell colony which consists of undifferentiated (blast) cells and appears to be more primitive than CFU-GEMM in the stem cell hierarchy. The progenitors for the colony which we termed "stem cell colony" possess an extensive self-renewal capacity and the ability to generate many secondary multipotential hemopoietic colonies in culture. We replated a total of 68 stem cell colonies from cultures of murine spleen cells and analyzed the number of stem cell--and granulocyte(neutrophil)-erythrocyte-macrophage-megakaryocyte (GEMM) colony-forming cells in individual stem cell colonies. Of the 68 stem cell colonies, 35 contained progenitors (abbreviated as "S"-cells) for stem cell colonies. The distributions of S-cells and CFU-GEMM in individual stem cell colonies were extremely heterogeneous. Neither the frequency distributions of S-cells nor CFU-GEMM in stem cell colonies could be fitted well by Poisson distribution. Rather, the frequency distribution of the s-cells could be approximated by a geometric distribution and that of CFU-GEMM by an exponential distribution, both of which are variates of the gamma distribution. Our observations are in agreement with those on the distributions of CFU-S in individual spleen colonies and provided support for a stochastic model for stem cell self-renewal and commitment in culture. Application of the theory of the branching process to the distribution of S-cells revealed a distributional parameter "p" of 0.589 which is also in agreement with the earlier report on the p value for reproduction of CFU-S.

Molecules stimulating early red cell, granulocyte, macrophage, and megakaryocyte precursors in culture: similarity in size, hydrophobicity, and charge.

Abstract: Molecules in conditioned medium from stimulated lymphocyte populations or from certain cell lines are known to stimulate cells committed to various hemopoietic lineages as well as pluripotential cells to form colonies in culture. In this study, the relationship between molecules active on pluripotential cells and early cells committed to granulocyte, macrophage, megakaryocyte, or red cell production was explored using techniques of chemical separation. After separation on the basis of charge, or after sequential purification using methods of high resolving power based on hydrophobicity and size, these activities remained associated with one another. The observations provide support for a model which proposes that pluripotential hemopoietic precursor cells as well as their early committed progeny may all be responsive to a single lineage-indifferent factor. Responsiveness to "lineage-specific" factors such as erythropoietin is proposed to be a feature only of later cells after they have made the appropriate receptors as part of their differentiation program.

A new preadipose cell line derived from newborn mouse calvaria can promote the proliferation of pluripotent hemopoietic stem cells in vitro

Abstract: A clonal preadipose cell line MC3T3-G2/PA6, established from newborn mouse calvaria, responds to glucocorticoids and converts to adipose cells in a fashion similar to bone marrow preadipocytes. We investigated the effect of the cells on in vitro hemopoiesis of mouse bone marrow cells by cocultivation. When bone marrow cells were inoculated into confluent cultures of MC3T3-G2/PA6 cells (10(4)-10(6) cells/25-cm2 flask), the number of hemopoietic stem cells (CFU-S) significantly increased during 7-day cultivation in proportion to inoculum size. Under these conditions, active replication of CFU-S was maintained for several weeks until MC3T3-G2/PA6 cell layers detached from the substratum. This capacity of the MC3T3-G2/PA6 line was unique because other established cell lines, including the MTF preadipose line, failed to support CFU-S growth. When bone marrow cells were not allowed to contact the MC3T3-G2/PA6 cell layer, only a small number of CFU-S survived for 7 days. Moreover, MC3T3-G2/PA6 cell-conditioned medium did not show any growth-promoting activity for CFU-S. These results indicate that the MC3T3-G2/PA6 cell line has the ability to promote the proliferation of CFU-S through a short range cell-to-cell interaction by providing an in vitro microenvironment probably similar to that for in vivo hemopoiesis.

Serial depletion and regeneration of the murine hematopoietic system. Implications for hematopoietic organization and the study of cellular aging

Abstract: The mouse hematopoietic system was subjected to repeated depletion and regeneration either by serial transfer of bone marrow cells through lethally irradiated recipients or by repeated treatment with the cycle-active drug hydroxyurea (HU). The capacity of surviving stem cells to proliferate and self-renew was assayed at intervals by two methods: (a) the spleen colony method; and (b) competitive repopulation of irradiated recipients using chromosome markers, with normal bone marrow cells as an internal control. The progressive decline in stem cell function that occurred during serial transfer of bone marrow and that had already begun after a single transfer was not seen during HU treatment; up to 25 pairs of HU injections given over more than 1 yr had no discernible effect on the number of stem cells present 3 wk after the final injection or on their capacity to self-renew. Within 2 d after exposure to HU, the average self-renewal capacity of surviving stem cells was enhanced. This implies that the drug selectively eliminates poorly self-renewing stem cells and hence that these enter cycle more readily than stem cells with a high self-replicative potential. However, the fact of being in cycle at the time of injection did not of itself affect self-renewal. The results show that serial transfer of bone marrow is not a valid method for studying clonal aging phenomena because it does not fulfill the assumptions on which such studies are based. No evidence was obtained for any intrinsic limitation in the capacity of bone marrow populations for repeated regeneration after HU-induced depletion. However, this does not necessarily imply that individual hematopoietic clones are capable of indefinite expansion because hematopoiesis may (as suggested by the relative resistance of highly self-replicative stem cells to mitogenic signals) proceed on the basis of clonal succession.

Retinoic acid enhances growth of human early erythroid progenitor cells in vitro.

Abstract: We studied the effect of retinoic acid on the clonal proliferation of normal human early erythroid progenitor cells in vitro. Normal peripheral blood cells were cultured in methylcellulose with erythropoietin and the number of burst-forming units-erythroid (BFU-E) colonies were scored on day 12 of culture. All-trans retinoic acid increased the number of colonies in a dose-response fashion. Maximal stimulation occurred at 30 nM retinoic acid, which increased the number of BFU-E by a mean of 225 +/- 25% (+/- SE) over plates containing erythropoietin alone. Colony formation increased even in the presence of maximally stimulating concentrations of erythropoietin. The 13-cis retinoic acid stimulated BFU-E proliferation in a parallel manner as the trans analogue, while retinol (vitamin A) did not affect clonal growth. This data supports further the thesis that retinoic acid, in addition to its known effect on epithelial cells, may be involved in the growth of normal hematopoietic cells.

Selective killing of leukaemia cells by antibody–toxin conjugates: implications for autologous bone marrow transplantation

Abstract: The recent development of tumour-reactive monoclonal antibodies has stimulated interest in their use in targeting toxic agents to tumour cells. One such approach involves the covalent coupling of tumour-reactive antibodies to the toxic peptide of the plant toxin, ricin. Such conjugates are highly effective in killing various tumour cells in vitro1–6. Here we have used the murine B-cell tumour, BCL1, in an adoptive transfer system capable of detecting 1–10 tumour cells, to demonstrate the effectiveness and specificity of an anti-immunoglobulin(Ig)–ricin A chain conjugate in deleting tumour cells from infiltrated murine bone marrow. We report that the transferred cells were able to repopulate the haematopoietic system of lethally irradiated mice and that in most mice the tumour did not recur. We discuss the implications of this approach for autologous bone marrow transplantation following supralethal tumour therapy.

Pluripotential hemopoietic stem cells in adult mouse brain.

Abstract: Single cell suspensions of adult mouse brain were shown to contain large numbers of pluripotential hemopoietic stem cells as detected by the ability to form hemopoietic colonies in the spleens of irradiated hosts. These colony forming unit, spleen (CFU-s) cells derived from brain gave rise to colonies identical in morphology and histology to those of bone marrow-derived CFU-s. The average number of CFU-s obtained per 10(5) dissociated adult brain cells was 14, whereas other adult tissues such as lung, kidney, heart, and thymus contained insignificant CFU-s levels when tested. As the level of CFU-s in adult blood is less than 1 per 10(6) nucleated cells, blood contamination does not contribute to the high levels found in adult brain. Individual spleen colonies isolated from irradiated CBA (H-2k) recipients injected with (BALB/c x CBA)F1 (H-2d x H-2k) brain cells were shown by immunofluorescence to contain cells bearing surface H-2d molecules, thus indicating that the colonies arose from the brain cell inoculum and were not endogenously derived. The surface phenotype of brain- and bone marrow-derived CFU-s was found to differ in that brain CFU-s could be inhibited by prior incubation with a monoclonal antibrain antibody B2A2, whereas bone marrow CFU-s were not. Further differences were found between brain and bone marrow CFU-s in the congenitally anemic Wf/Wf mice. These mice were shown to have a very few CFU-s in the adult bone marrow, whereas the brain contained normal adult levels. The large number of hemopoietic stem cells in the brain may indicate an essential requirement for the continual generation of cells such as microglia or phagocytic cells, without the disruption of the blood-brain barrier.

Participation of myeloid leukaemic cells injected into embryos in haematopoietic differentiation in adult mice.

Abstract: It is of interest to determine to what extent malignant cells are still subject to control mechanisms that govern the growth and differentiation of their normal counterparts1–4. One approach has been the introduction of malignant cells into a normal embryo. Such studies have shown that mouse teratocarcinoma cells injected into blastocysts can participate in normal development to produce adult chimaeric animals with markers derived from teratocarcinoma cells1–3,5–7. Other types of malignant cells may also be able to participate in normal morphogenesis, provided that they have the ability to differentiate and are introduced into the embryo at an appropriate stage of development. Here we have examined myeloid leukaemic cells and our results indicate that injection of these cells into embryos in utero at 10 days of gestation can produce apparently healthy adult mice whose granulocytes contain a marker derived from the leukaemic cells.

Clonal preadipocyte cell lines with different phenotypes derived from murine marrow stroma: factors influencing growth and adipogenesis in vitro.

Abstract: We have isolated continuously growing cell lines derived from mouse bone marrow stroma. These cell lines were independently obtained, and though they showed morphologies ranging from the epithelioid to the fibroblastoid patterns, they all differentiated into adipocytes. Subclones obtained from two cell lines had a very high frequency (90-100%) of differentiation into adipocytes after two or three weeks of arrested growth. Though extensive accumulation of lipid often mechanically impaired mitosis, the cells committed to adipocytes did not suffer an irreversible loss of proliferative capacity. Adipogenesis was obtained in conditions similar to those required for fat cell formation in long-term bone marrow culture. The cell lines were found to be insensitive to insulin as a signal of adipocyte differentiation. The ultrastructural characteristics of the preadipocytes and fat cells are also similar to those of the fat cells developing in long-term bone marrow culture. As such, these cell lines should prove useful for analysing cell/cell interactions in haemopoiesis.

Bone marrow transplantation across major histocompatibility barriers in mice: II. T cell requirement for engraftment in total lymphoid irradiation-conditioned recipients

Abstract: Studies were undertaken to examine the role of T lymphocytes in engraftment of bone marrow (BM) in animals conditioned with total lymphoid irradiation (TLI) prior to transplantation across major histocompatibility barriers. Donor BM (added as a source of lymphohematopoietic stem cells) and spleen cells (added as a source of graft-versus-host disease (GVHD)-causing cells) were pretreated in vitro with monoclonal anti-Thy-1.2 plus complement (C). T cell-depleted grafts were then give to allogeneic mice conditioned with 900 rad of single dose TLI plus cyclophosphamide (CY). These mice did not engraft. Even in the absence of added spleen cells, elimination of the small T cell population from donor BM grafts prevented engraftment compared with animals that received the same conditioning regimen and untreated donor cells. These control animals demonstrated uniform evidence of engraftment about 1 month after transplantation. Similar findings were reported when recipients were conditioned with fractionated 17 x 200-rad TLI. In TLI plus CY-conditional recipients, we have also observed that increasing the donation of treated bone marrow cells still did not result in significant engraftment. Furthermore, graft failure in mice receiving normal dosages of anti-Thy-1.2 plus C-treated donor cells was not a strain-restricted phenomenon. Moreover, removal of bone marrow T cells with monoclonal anti-Lyt-1 plus complement also resulted in graft failure in TLI-conditioned recipients. In contrast to TLI conditioning, when Thy-1.2 plus C-treated donor cells were given to recipients conditioned with total body irradiation (TBI), a high percentage of engraftment was demonstrated by an H-2 microcytotoxicity assay. Plausible mechanisms for there findings are discussed.

Isolation and synthesis of a hemoregulatory peptide.

Abstract: A peptide was isolated in pure form from human leukocytes which strongly inhibits the proliferation of immature myeloid cells in vitro (committed stem cells). Structural investigations yielded pGlu-Asp or Glu-Asp or Glu-Cys-Lys-OH as the probable sequence of this peptide. The Glu2, Asp3-analog, prepared synthetically, displayed similar activities and when applied in vivo showed effects on the hemopoietic system ranging from an inhibition of pluripotent and committed stem cells to variations in the bone marrow proliferation and alterations in peripheral blood counts.

Effect of colony stimulating factor on murine macrophages. Induction of antitumor activity.

Abstract: Colony stimulating factor (CSF) was assessed for its capacity to stimulate antitumor activity in macrophages. Murine peritoneal macrophages incubated with CSF for 48 h inhibited [3H[thymidine (TdR) incorporation by P815 tumor cells to approximately 20% of control values. Inhibition of CSF-stimulated macrophages was significantly greater than inhibition by unstimulated macrophages (P less than 0.001). CSF had little direct effect on the proliferation of either tumor cells or macrophages alone, indicating that the antitumor activity of CSF was mediated by macrophages. it is unlikely that impurities in the CSF preparations were responsible for the effect since CSF that had been purified to homogeneity was as active as crude preparations. Furthermore the activity of CSF on macrophages was blocked by addition of purified anti-CSF antibodies. In addition to being tumoristatic, CSF-stimulated macrophages were tumoricidal as determined by a tumor colony growth assay. Tumor cells that had been incubated with CSF-stimulated macrophages showed a significant reduction in tumor colony-forming units (P less than 0.01). Thus, in addition to its effect on hemopoietic stem cells, CSF induces certain effector functions in mature macrophages that may enhance endogenous antitumor host defenses.