The production of committed hemopoietic colony-forming cells from multipotential precursor cells in vitro

Murine fetal liver cells have been fractionated by fluorescence- activated cell sorting into two fractions termed. For convenience, the pre-CFC and CFC (colony-forming cell) fractions, which differ in their relative binding of the lectin, pokeweed mitogen. The CFC fraction contained a high frequency of CFC (26%) and in liquid cultures stimulated with colony-stimulating factors generated large numbers of differentiated progeny. Consequently, residual CFC could not be detected in such cultures after day 4. In contrast, the pre-CFC fraction contained relatively few CFC (1%) but contained the majority of CFU-S and was able to generate large numbers of new CFC after 5-7 days in liquid culture. This production of CFC from precursor cells was absolutely dependent on factors present in pokeweed-mitogen-stimulated spleen cell conditioned medium (PWM-SCM) or postendotoxin serum (ES) but was not stimulated by known granulocyte or macrophage colony- stimulating factors. CFC production occurred from multipotential precursor cells and all types of CFC were detected (excluding lymphocytes). Clonal analysis of pre-CFC showed that most of the generated CFC arose from relatively few multipotential precursors some of which could generate up to 500 CFC. The data suggested a differentiation sequence of the multipotential precursor cells in which differentiation potentials are successively restricted in the order macrophage, eosinophil, granulocyte-macrophage leads to granulocyte, megakaryocyte, erythroid. The frequency of CFC-generating cells in the pre-CFC fraction was significantly less (ten-fold) than that of CFU-S, suggesting that pre-CFC may be a more ancestral subset of CFU-S. The cell fractions and assay systems described should be of use in defining and purifying factors regulating early phases of hemopoietic differentiation and in defining the differentiation steps involved in the restriction of potentialities of multipotential cells.     

Identification of osteoclast precursors in multilineage hemopoietic colonies

The osteoclast is known to be derived from the hemopoietic stem cell, but its lineage and the mechanisms by which its differentiation is regulated are largely unknown. There is evidence that osteoclastic differentiation is induced through a contact-dependent interaction between bone marrow stromal cells and hemopoietic precursors. To analyze osteoclastic lineage, colonies were generated in semi-solid medium from mouse spleen cells in the presence of erythropoietin with either Wehi 3B-conditioned medium or interleukin 3 (IL3). After 7 days, individual colonies were picked. Half of each colony was phenotyped by the morphology of cells in cytospin preparations; the second half of each was incubated for 7 days with a bone marrow-derived cell line (ts8) that induces osteoclastic differentiation from hemopoietic cells, on bone slices in the presence of 1,25-dihydroxyvitamin D3. After incubation, bone resorption was assessed by scanning electron microscopy. No resorption was induced in cells derived from single-lineage colonies, but resorptive cells differentiated in 17% of granulocyte-macrophage (GM) colonies and 38% of multilineage colonies. Since only a minority of GM colonies contained osteoclastic precursors, this suggests that the GM colonies that contained osteoclasts were not typical GM colonies but may have been a form of multilineage colony analagous to other multilineage colonies that contain granulocytes, macrophages, and a third cell type. No resorptive cells were formed when IL3-derived colonies were incubated on bone slices without ts8 cells. The results suggest that osteoclasts are derived from a multilineage precursor, upon which IL3 acts to generate cells capable of osteoclastic differentiation, which form resorptive cells upon incubation with bone marrow stromal cells in the presence of 1,25-dihydroxyvitamin D3.     

Identification and characterization of human hemopoietic mast cell colonies

Persisting mast cell colonies from human bone marrow and cord blood cells grown in semisolid agar cultures for over 56 days have been positively identified and characterized using morphology and cytochemistry. Mast cells demonstrated the following features: Cytoplasmic granules frequently contained the specific and characteristic papyrus rolls (transmission electron microscopy); mature cells were positive to the mouse monoclonal antibody YB5.B8 specific for human mast cells (raised against acute myeloid leukemia cells) and RPA-M1 specific for human monocytes but negative to the human basophil monoclonal antibody Bsp-1; morphologically the cells were large (diameter 20-25 micron), deeply basophilic, and contained granules that measured up to 2 micron in diameter (May-Grünwald-Giemsa stain); the presence of heparin by the thrombin clotting time and positive staining with toluidine blue and alcian blue; the presence of histamine by a positive fluorescent o-phthalaldehyde stain; the presence of IgE receptor sites with human IgE and a rabbit anti-human IgE second antibody; and a unique zone of lysis around mast cell colonies occurred when cultured on peripheral blood feeder layers in agar plates that was not present around monocytic, neutrophilic, or eosinophilic colonies under the same culture conditions. Our results identify the cells in persisting colonies as mast cells and describe some specific characteristics that distinguish these cells from basophils.     

Identification of T lymphocytes in human mixed hemopoietic colonies

The addition of a T-cell growth-promoting medium (PHA-TCM) to culture conditions that support growth of multi-lineage hemopoietic colonies enhances the proliferation of cells with lymphoid morphology within these colonies. These cells were identified as T lymphocytes by their ability to form rosettes with SRBC and their reaction with monoclonal antibodies (OKT3, OKT4) directed against T-cell-specific surface components. They continue to proliferate extensively under the influence of PHA-TCM after transfer of mixed colonies into liquid suspension culture. Supportive evidence for a common progenitor of myeloid and lymphoid cells within single mixed colonies is provided by Y-chromatin body analysis of E-rosette positive and negative cells in colonies grown in cocultures of male and female bone marrow cells.     

Murine hemopoietic colonies in culture containing normoblasts,macrophages, and megakaryocytes

Murine marrow cells, when incubated in methylcellulose culture in the presence of erythropoietin and conditioned medium for two weeks, produced large macroscopic bursts containing normoblasts, macrophages, and often megakaryocytes. The clonal nature of these mixed colonies was supported by linearity studies and analyses of the percentages of constituent cells in different plating conditions. Time course observations and studies of the effects of L-cell-conditioned medium revealed that colony-forming cells for the mixed colonies (CFU-mix) are at earlier stages of hemopoietic development than burst-forming units (BFU-E). The mean of the modal sedimentation velocities of CFU-mix was 3.4 mm/hr and was in close agreement with that reported for the spleen colony-forming units. Almost none of the CFU-mix was in a DNA synthetic phase as measured by short-term exposure to tritiated thymidine. These results strongly indicate that CFU-mix represent a population of pluripotent hemopoietic stem cells in murine marrow.     

Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures.

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.     

Receptor specificity in the self-renewal and differentiation of primary multipotential hemopoietic cells.

To determine whether cytokine-induced signals generate unique responses in multipotential hemopoietic progenitor cells, the signaling domains of 3 different growth factor receptors (Mpl, granulocyte-colony-stimulating factor [G-CSF] receptor, and Flt-3) were inserted into mouse primary bone marrow cells. To circumvent the activation of endogenous receptors, each signaling domain was incorporated into an FK506 binding protein (FKBP) fusion to allow for its specific activation using synthetic FKBP ligands. Each signaling domain supported the growth of Ba/F3 cells; however, only Mpl supported the sustained growth of transduced marrow cells, with a dramatic expansion of multipotential progenitors and megakaryocytes. These findings demonstrate that the self-renewal and differentiation of multipotential progenitor cells can be influenced through distinct, receptor-initiated signaling pathways.     

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.     

Effect of bleeding on in vivo in vitro colony-forming hemopoietic cells.

The effect of bleeding on spleen colony-forming units (CFU-S) and on in vitro colony-forming cells with colony-stimulating factor (CFU-C) and erythropoietin (CFU-E) has been evaluated. The in vivo and in vitro colony-forming cells of the bone marrow show a decrease which for the CFU-E, CFU-C follows a short-lived increase. In the spleen, all progenitor cells assayed have shown a significant and sustained increase.     

Effect of benzene on fibroblastoid colony-forming units in mice.

After exposure of C57BL6 x DBA/2 mice to benzene in air their number of bone marrow fibroblastoid precursor cells, CFU-F, was determined. The CFU-F exhibited an increasing plating efficiency, giving rise to a larger number of colonies and to colonies of greater size. This effect was dose dependent. When the mice were exposed for 16 weeks and were then allowed to rest, their CFU-F plating efficiency returned to normal within 6 weeks, but then increased again. Hematopoietic stem cells, such as CFU-S and CFU-C exhibited a dose-dependent depression. The in vitro exposure of bone marrow cells to benzene metabolites resulted in a dose-dependent depression of CFU-F numbers.     

Effects of anti-myeloid antibodies on the generation of hematopoietic colony-forming units in long-term bone marrow culture

We have prepared and characterized several monoclonal antibodies (MoAbs), PM-18 (CD 15),AML-2-23 (CD 14), and AML-1-99 (no cluster designation) reactive with antigens expressed on myeloid cells. Previous studies using complement-dependent lysis have determined the reactivity of these MoAbs with hematopoietic cells in vitro. PM-81 and AML-2-23 react with variable percentages of CFU-GM but not BFU-E or CFU-Mix. AML-1-99 reacts with greater than 90% of CFU-GM. CFU-E, and CFU-Mix. In order to determine the reactivity of these MoAbs with the bone marrow-derived precursors of in vitro colony-forming cells we have performed complement-dependent lysis and fluorescence activated cell sorting of bone marrow cells followed by long-term culture of surviving or sorted cells. Bone marrow cells from four normal subjects were subjected to various combinations of MoAbs and complement and assayed for residual colony-forming cells. Total surviving cells were then placed in flasks which contained a monolayer of irradiated bone marrow-derived adherent cells previously obtained from allogeneic donors. The cultures supported production of non-adherent colony-forming cells for up to 6 weeks as determined by serial in vitro colony-forming assays in methylcellulose. Cultures treated with one, two or three MoAbs and complement demonstrated variable reductions in colony-forming cells at the initiation of the experiments. However, cumulative production of colony-forming cells in anti-MoAb-treated cultures was usually at least as great as in control cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single-cell origin of mouse hemopoietic colonies expressing multiple lineages in variable combinations.

By using a micromanipulator, single cells from blast cell colonies were individually transferred to 35-mm culture dishes for secondary colony formation. When individual colonies appeared to be mature, they were examined for cellular composition by May-Grunwald-Giemsa staining and were replated for determination of unexpressed hemopoietic potentials. We describe here a total of 50 mixed hemopoietic colonies. Seven types of colonies consisting of cells in two different lineages were seen--i.e., neutrophil-macrophage, neutrophil-eosinophil, macrophage-eosinophil, macrophage-mast cell, macrophage-megakaryocyte, macrophage-erythrocyte, and erythrocyte-megakaryocyte. Six types of colonies revealed three cell lineages--i.e., neutrophil-macrophage-eosinophil, neutrophil-macrophage-mast cell, neutrophil-macrophage-erythrocyte, macrophage-mast cell-erythrocyte, neutrophil-macrophage-megakaryocyte, and neutrophil-erythrocyte-megakaryocyte lineages. In addition, multilineage colonies expressing terminal differentiation in varying combinations of more than three lineages were present. Replating studies confirmed that the progenitors for many of these colonies are terminally committed to differentiation only in the lineages disclosed by staining. This study, thus, provides a proof for the single-cell origin of mouse hemopoietic colonies expressing various combinations of cell lineages. It also supports the hypothesis that the differentiation of multipotential hemopoietic progenitors is through progressive and stochastic restriction in cell lineages.     

Quantitation of insulin-like growth factor-I binding to highly purified human erythroid colony-forming units.

A method has been developed by which erythroid colony-forming units (CFU-E) may be obtained from human blood in sufficient number and purity for quantitative studies of growth factor binding. Studies in serum-free medium have shown that CFU-E require the addition of only two growth factors, erythropoietin (EP) and insulin-like growth factor-I (IGF-I), for growth and differentiation. The IGF-I may be replaced by higher (100-fold) concentrations of insulin. Incubation of CFU-E with 125I recombinant human IGF-I (rhIGF-I) at 4 degrees C has demonstrated specific binding that is directly proportional to the cell concentration. Competition with unlabeled rhIGF-I markedly decreased binding, whereas other growth factors such as granulocyte-monocyte colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and epidermal growth factor (EGF) had no significant effect on the binding of [125I]rhIGF-I. The binding was saturable at an [125I]rhIGF-I concentration of 10 ng/ml (1.2 nM). Scatchard analysis revealed two classes of IGF-I receptors present on the CFU-E cell surface: a low-affinity class of 549 receptors with Kd = 0.44 nM and a high-affinity class of 341 receptors with Kd = 0.04 nM.     

Lithium carbonate increases marrow granulocyte-committed colony-forming units and peripheral blood granulocytes in a canine model.

Psychiatric patients given lithium carbonate (LC) commonly develop granulocytosis of the peripheral blood. This is an important observation with several potential applications in clinical hematology and oncology. In order to study further this phenomenon, we have developed an animal model of LC-induced granulocytosis. Four mongrel dogs weighing 23--28 kg were given commercially available LC in 300 mg capsules by mouth twice daily. Peripheral blood was examined twice weekly and marrow granylocyte-committed colony forming units (CFU-c) were studied weekly. Significant increments of peripheral granulocytes (P = 0.003) and of marrow CFU-c (P =0.001) were noted. An increased number of marrow CFU-c has not been previously reported but is consistent with other knowledge about this phenomenon. We conclude that this convenient animal model may be of value in further studies of LC-induced granulocytosis and its possible applications.     

Mesenteric hemopoietic colonies: occurrence in BALB/c mice after transplantation of syngeneic normal or leukemic hemopoietic cells.

Intraperitoneal (ip) inoculation of BALB/c mice with syngeneic hemopoietic cells results in the formation of 'Mesenteric Hemopoietic Colonies' (MHC). In lethally irradiated mice actively growing erythroid, myeloid and megakaryocytic, or mixed colonies form and soon become confluent. It is therefore concluded that in mice the mesentery is a suitable site for growth of hemopoietic cells. The mesentery might play an important role in the recovery of the hemopoietic system in lethally irradiated mice, being the primary site of proliferation of stem cells and/or CFU before their migration to bone marrow and spleen. Bone marrow and spleen cells from animals infected with Rauscher Leukemia Virus (R-MuLV) also produce MHC and spleen colonies after ip injection into lethally irradiated mice. In addition to the undifferentiated cells in the MHC, cells with limited differentiation and/or retarded maturation were identified. The cytologic pattern of the majority of cells in MHC was of mixed type.     

Kinetics of hemopoietic stem cells in a hypoxic culture

The influence of low oxygen tension on the clonal growth of hemopoietic stem cells in vitro was examined. The numbers of colonies of neutrophil, macrophage, and eosinophil progenitors (CFU-C), derived from human bone marrow, increased at a rate 1.7 times higher in low oxygen tension (7% O2) than in a gas phase that contained air (19% O2). The erythroid (BFU-E) and multipotential (CFU-mix) progenitors increased about 2.4 times in 7% O2, and the increase in the composed cell type of mixed colonies showed no rate difference in either gas phase. Under atmospheric conditions, a mouse mast cell progenitor (CFU-mast) formed colonies, with the addition of 2-mercaptoethanol (2-ME). Under low oxygen tension, the CFU-mast formed colonies without 2-ME, but a further enhancement was observed with the addition of 2-ME. Blood gas analysis of human bone marrow showed a pO2 of 51.8 +/- 14.5 mmHg, which was closed to O2 tension in a gas phase culture media containing 7% O2. This data shows that the physiological O2 tension enhances hemopoietic stem cell proliferation in vitro, and that a part of the enhancing effect by 2-ME is due to a prevention of O2 toxicity at 19% O2.     

Onset of erythropoietin response in murine erythroid colony-forming units: assignment to early S-phase in a specific cell generation.

Murine erythroid colony-forming units (CFU-E) representing successive cell generations in a six-generation long in vitro maturation sequence were tested for their response to erythropoietin (Epo) by measurement of Epo-exposure times necessary to stimulate heme biosynthesis. Generation I CFU-E, which produce mainly 32-cell erythroid colonies, were isolated in 82% average purity from spleens of thiamphenicol-treated anemic animals via differential centrifugation. Generation II CFU-E, which produce mainly 16-cell colonies, were similarly isolated in 51% average purity. Although both types of CFU-E had equivalent dose sensitivity to and affinity for Epo, generation II CFU-E responded to shorter pulses of Epo than did generation I. Correlations between DNA cell-cycle profiles and 59Fe-heme biosynthesis resulting from pulsed exposures established that appreciable Epo response only begins when CFU-E attain early S-phase of generation II. Because CFU-E did not require Epo or other serum factors to pass from generation I to II and because the onset of Epo responsiveness coincided with the beginning of DNA replication in generation II, we suppose that differentiation has reprogrammed one or more of the events associated with generation II S-phase in CFU-E and that these alterations allow Epo to act. Further comparisons between CFU-E from generation I and II may allow us to identify the alterations in question and the nature of their interaction with Epo.     

Mouse yolk sac and intraembryonic tissues produce factors able to elicit differentiation of erythroid burst-forming units and colony-forming units, respectively.

This work was aimed at elucidating the environmental conditions that account for the production of embryonic erythrocytes in the mouse yolk sac (YS), while the adult-type hemoglobin and erythrocytes are generated in the fetal liver. Differentiation of YS hemopoietic stem cells (YS-HSC) of 9.5-day mouse embryos (prior to the colonization of the liver rudiment by HSC) was investigated in vitro. The influence of well-characterized erythroid growth factors, burst-promoting activity (BPA) and erythropoietin (EPO), which trigger the differentiation of early erythroid burst-forming units (BFU-E) and late erythroid colony-forming units (CFU-E), respectively, was tested on the YS-HSC in two different systems of culture: (i) organ culture and (ii) clonal culture in methylcellulose. When studied in organ culture, where the YS microenvironment was maintained, YS-HSC required only additional EPO to attain complete maturation into adult erythrocytes within 7 days. In contrast, YS hemopoietic single cells grown in methylcellulose, and thus released from the influence of the YS, required the presence of both BPA and EPO to generate BFU-E-derived colonies synthesizing high concentrations of hemoglobin. It is concluded that 9.5-day YS from mouse embryos is by itself able to promote the first differentiation steps of the adult lineage YS-HSC due to an intrinsic production of a BPA-like activity. In contrast, these experiments demonstrate that EPO or an EPO-like activity is not produced by YS tissues. As demonstrated earlier, if embryonic tissue is added to YS organ culture, although separated from it by a filter preventing cell contact, YS-HSC differentiate into adult erythrocytes producing adult-type hemoglobins. This shows that, in contrast to YS tissues, the early embryo produces EPO or a factor that can substitute for EPO.     

Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor.

Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.