Colonies In Agar Cultures Research Articles

Synergistic and inhibitory interactions in the in vitro control of murine megakaryocyte colony formation.

The formation of megakaryocyte colonies in agar cultures of murine bone marrow or spleen cells can be stimulated by the addition of interleukin-3 (IL-3), erythropoietin (EPO), thrombopoietin (TPO), or IL-6. However, greater numbers of colonies developed if combinations of two or more of these stimuli were used, particularly combinations including stem cell factor, with maximal numbers of colonies developing with the combination of stem cell factor plus IL-3 plus EPO. The data indicate that most committed progenitor cells in the megakaryocyte lineage were unusual in that they required stimulation by two or more hematopoietic growth factors. In tests using a range of growth factors, G-CSF was exceptional in that it consistently specifically inhibited megakaryocyte colony formation stimulated by EPO, TPO, or IL-6 but not that stimulated by IL-3. The mechanisms involved in this inhibitory action of G-CSF are unknown, but the inhibitory action could be of relevance for the dose-dependent lowering of platelet levels observed in some subjects injected with G-CSF.

Role of mature leukemic cells in the amplification of leukemic stem cells in a murine model

The recently described PGM-2 leukemia displays a hierarchical structure with bipotential stem cells, B-lymphocyte and macrophage progenitor cells, and post-mitotic end cells. Because the different cell types can easily be identified in vitro by clonal culture assays and simple staining procedures, this leukemia is a useful model for the study of the interactions between different cell compartments in a leukemic clone. Our analysis of the impact of mature leukemic macrophages on the proliferation of stem cells was facilitated by the establishment of long-term cultures producing new stem cells over prolonged periods of time. A prerequisite was the development of an adherent layer of fibroblasts and leukemic macrophages. Enumeration of adherent cells revealed a good correlation between the number of macrophages and the number of stem cells generated, and expansion of the macrophage population by treatment with interleukin 3 (IL-3) resulted in a significant improvement of the culture conditions. Leukemic macrophages were also able to induce the formation of stem-cell colonies in agar culture, suggesting a role for humoral mediators. Antibody neutralization experiments and bioassays identified IL-7 and IL-6 as factors cooperating in the stimulation of stem-cell self-renewal. Feed-back stimulation of leukemic stem cells by mature leukemic cells may also be relevant to human leukemias and have implications for differentiation therapy.

Nectria and Penicillifer

The new species Nectria alata and N. penicilliferi (Ascomycetes, Hypocreales) and their anamorphs, respectively, Penicillifer bipapillatus sp. nov. and P. macrosporus sp. nov., are described. This is the first connection of Penicillifer species to teleomorphs. The generic concept of Penicillifer is restricted to anamorphs of Nectria species having conidiophores that: 1) are unbranched and monophialidic; 2) bear a terminal penicillus of phialides; or 3) are once- or twice-branched with each branch bearing a terminal penicillus of phialides. Conidia are cylindrical, 1-septate, bipapillate, and joined end to end in chains that ultimately break down, leaving the conidia adherent in slime. Colonies in agar culture produce a diffusing brown pigment. A key to accepted species of Penicillifer is presented, as is a key to genera and species of hyphomycetes that have colorless or lightly colored, mononematous conidiophores and slimy chains of colorless conidia. Two distinctive species ofNectria collected in the American tropics were grown in pure culture from isolated ascospores. Both species produced anamorphs identified as Penicillifer van Emden. These Nectria species and their anamorphs are described below. Taxonomy of Penicillifer is clarified, and its relationship to Nectria is discussed. A key to accepted species of Penicillifer is presented, as is a key to the hyphomycetes known to produce colorless conidia in slimy chains.

A simple and rapid in situ staining method for granulocyte‐macrophage colonies in agar culture

This report describes a very simple, quick and effective method for in situ staining of granulocyte-macrophage colonies in agar cultures and for classifying various colony types. The procedure takes only one minute to fix and a few minutes to stain; a few additional minutes are required for preparation of the permanent whole plate. In this process the Riu stain, a modified Romanowsky stain, is used. Besides the ease and rapidity of this procedure, the identification of colony types appears to be enhanced. Thus, the method seems to be very beneficial in routine observations of colony types.

A colorimetric liquid culture assay of a growth factor for primitive murine macrophage progenitor cells

Synergistic factors from media conditioned (CM) by human placentas or the 5637 human bladder carcinoma cell line (SF H-HPCM and SF H-5637 respectively) have the ability to stimulate early progenitor cells in mouse bone marrow to form large colonies in agar cultures after 12–14 days, in the presence of CSF-1. Culture conditions have been examined and a quicker and more convenient liquid culture assay has been developed for this factor, using a tetrazolium salt to quantitate cell proliferation. The use of flat-bottomed vessels, high cell density, supra-optimal doses of CSF-1 or the addition of WEHI-3-CM to these cultures, all resulted in a decrease in the required incubation time. In combination, these modifications reduced the assay time to 4 days.

Persisting colonies in agar cultures containing serum from patients with CML in blastic transformation

Addition of serum from patients with chronic myeloid leukemia (CML) in both chronic phase and blastic transformation, to agar cultures of normal human marrow cells stimulated the growth of persisting colonies (day 35) containing either eosinophils or mast cells. Chronic phase serum stimulated an 800% increase in the total number of these colonies of which only 16% were mast cells. Serial studies using serum from 2 patients demonstrated that the proportion of mast cells increased during the progression to blastic transformation. The emergence of a greater proportion of persisting mast cell colonies and a decrease in absolute number of eosinophil colonies in agar cultures of normal marrow cells containing serum from patients with CML coincides with the emergence of blastic transformation and suggests that a significant change occurs in the absolute and relative concentration of hemopoietic growth factors in these patients.

Stimulation of Persisting Colonies in Agar Cultures by Sera From Patients With CML and AML

Cord plasma contains colony-stimulating activity (CSA) which stimulates the in vitro clonal growth of neutrophils, eosinophils, macrophages, erythrocytes, and persisting mast cells in semisolid cultures. Analysis of day 35 colonies in agar cultures was found to be a suitable means of demonstrating this activity and discriminating between it and granulocyte-macrophage colony-stimulating factor (GM-CSF). Serum (10%) from patients with acute and chronic myeloid leukemia (AML and CML) was added to normal human bone marrow cultures to search for similar activity in these patient's serum. Although the number of colonies on day 12 (predominantly neutrophils and macrophages) was not significantly different from the number of colonies in cultures containing normal serum, the number of colonies increased 500% in cultures containing CML serum on day 35. Serum from patients with AML during regeneration also stimulated an increased number of colonies on day 35. Although both eosinophil and mast cell colonies were still present on day 35, only mast cell colonies persisted for 150 days. On day 35, cultures containing 10% CML serum contained predominantly eosinophil colonies (84%), whereas cultures containing AML serum contained predominantly mast cell colonies (76%). Although serum contains various CSFs, the specific factor which stimulates persisting mast cell colonies may be the human equivalent of murine persisting (P) cell-stimulating factor (Multi-CSF).

Stimulation of persisting colonies in agar cultures by sera from patients with CML and AML

Cord plasma contains colony-stimulating activity (CSA) which stimulates the in vitro clonal growth of neutrophils, eosinophils, macrophages, erythrocytes, and persisting mast cells in semisolid cultures. Analysis of day 35 colonies in agar cultures was found to be a suitable means of demonstrating this activity and discriminating between it and granulocyte-macrophage colony-stimulating factor (GM-CSF). Serum (10%) from patients with acute and chronic myeloid leukemia (AML and CML) was added to normal human bone marrow cultures to search for similar activity in these patient's serum. Although the number of colonies on day 12 (predominantly neutrophils and macrophages) was not significantly different from the number of colonies in cultures containing normal serum, the number of colonies increased 500% in cultures containing CML serum on day 35. Serum from patients with AML during regeneration also stimulated an increased number of colonies on day 35. Although both eosinophil and mast cell colonies were still present on day 35, only mast cell colonies persisted for 150 days. On day 35, cultures containing 10% CML serum contained predominantly eosinophil colonies (84%), whereas cultures containing AML serum contained predominantly mast cell colonies (76%). Although serum contains various CSFs, the specific factor which stimulates persisting mast cell colonies may be the human equivalent of murine persisting (P) cell-stimulating factor (Multi-CSF).

Correlation between production of colony-stimulating activity (CSA) and adipose conversion in a murine marrow-derived preadipocyte line (H-1/A).

The correlation between adipose conversion of cloned H-1 cells (H-1/A) and their production of colony-stimulating activity (CSA) was examined. The production of CSA from H-1/A cells declined after adipose conversion, although H-1/A cells are active producers of CSA during their fibrocytic stage. The addition of 2 X 10(-5) M 5-bromo-2'-deoxyuridine to the cultures almost completely inhibited adipose conversion and there was no reduction of CSA levels after 9 days of culture. On the other hand, the addition of 10(-6) M hydrocortisone sodium succinate to the culture markedly enhanced adipose conversion, and a greater reduction in the CSA level was observed in the supernatants than in the control cultures after 12 days of culture. Indomethacin had no effect on the production of CSA or on adipose conversion. Furthermore, there were no significant differences between the CSA levels of nondialyzed supernatants and dialyzed supernatants from the control cultures during the entire course of the experiment. Supernatants during the adipocyte stage of H-1/A cultures did not inhibit the CSA derived from the fibrocytic stage. There were no differences in colonies in agar cultures stimulated by supernatants derived from cultures that had undergone either of the above treatments. These results suggest that the reduction of CSA is not due to the production of inhibitors, but that the production of CSA declines after adipose conversion of H-1/A cells. Preadipocytes in bone marrow therefore appear to contribute to granulopoiesis during the fibrocytic stage and are hematopoietically inactive when they convert to adipocytes.

Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture

Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal t lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.

Ph1-negative T lymphocytic colonies in agar cultures of peripheral blood in chronic myeloid leukemia.

T lymphocytic colony formation by peripheral lymphocytes separated by discontinuous albumin gradient centrifugation was evaluated in 8 patients with Philadelphia (Ph1)-positive chronic myeloid leukemia (CML). Colonies were obtained using a liquid-on-agar culture system recently introduced (PHA overlayer-leukocyte feeder layer assay) which has been shown to be simple and reliable. The pattern of colony growth in CML and in normal controls was similar, the peak ranging from the 4th to the 6th day. Also the morphological aspects of colonies did not differ in the two groups. Cells recovered from CML lymphocytic colonies were shown to belong to T cell lineage, as they are able to form spontaneous E-rosettes and to respond to mitogenic stimulation in vitro. In contrast, cells recovered from all other cultured fractions failed to display these properties. Cytogenetic analysis showed that T colony cells were Ph1-negative whereas the chromosome anomaly was found in nonlymphoid colonies of the same patients, thus suggesting a nonclonal origin of T lymphocytes in CML.

Tumor-promoting phorbol esters stimulate hematopoietic colony formation in vitro.

Tumor-promoting phorbol esters stimulated mouse bone marrow cells to form myeloid colonies in agar cultures without added colony-stimulating factors. The colony-stimulating ability of various phorbol esters correlated well with their ability to promote skin tumors in vivo. These results suggest that phorbol esters mimic the action of specific colony-stimulating factors that regulate growth.

Oscillations of granulocytic and megakaryocytic progenitor cell populations in cyclic neutropenia in man.

The concentration of granulocytic progenitor cells in the bone marrow of 2 patients with cyclic neutropenia was assessed at intervals during the cycle. Syncroneous oscillations were observed for cells forming neutrophilic and megakaryocytic colonies in diffusion chambers in vivo (CFU-D), suggesting a close relationship between the two cell types. However, an almost reverse relationship was observed between CFU-D and cells forming colonies in agar culture in vitro (CFU-C), which cycled out of phase with CFU-D. This supports the concept that CFU-D and CFU-C denote different cell types.

Direct "wet" staining of tumour or haematopoietic colonies in agar culture.

Direct "wet" staining of tumour or haematopoietic colonies in agar culture.

Diversity of human granulopoietic precursor cells: separation of cells that form colonies in diffusion chambers (CFU-d) from populations of colony-forming cells in vitro (CFU-c) by velocity sedimentation

Normal human bone marrow contains cells that form granulocytic colonies in fibrin clot diffusion chambers implanted intraperitoneally in sublethally irradiated mice (CFU-d). A series of experiments was performed to determine the relationship between CFU-d and the cells that form colonies in agar culture in vitro (CFU-c). Low-density bone marrow cells ( 3 ) were separated by velocity sedimentation and colony-forming cells in each fraction assessed by culture in diffusion chambers and in soft agar. CFU-d sedimented more slowly than the vast majority of CFU-c, with a peak sedimentation velocity of 5.0 ± 0.4 mm/hr. Two different CFU-c populations could be distinguished. One was a rapidly sedimenting cell population (7.3 ± 0.9 mm/hr.) that formed colonies after 1 wk incubation in vitro. On day 14, however, colonies were derived from more slowly sedimenting cells that had a peak sedimentation velocity of 6.0 ± 0.5 mm/hr. Clusters (3-50 cells), scored on day 7 in agar ##

Proliferative State of Normal In Vitro Colony-forming Cells During Development of L5222 Rat Leukemia and Their Reaction to Chemotherapy

In the experimental rat leukemia, L5222, the decrease of normal in vitro colony-forming cells (CFU-C) after chemotherapy with daunomycin is much less than in nonleukemic controls. The leukemia is therefore used here to test the hypothesis that in leukemia the CFU-C are expelled from the active cell cycle to a resting state and are thereby less sensitive to cycle-dependent chemotherapeutic agents. The L5222 leukemia has the advantage that the leukemic blast cells do not form colonies in agar culture so that normal CFU-C can be assessed under leukemic conditions. To compare the proportions of CFU-C in the S-phase in normal and leukemic rats, two S-phase-specific agents, 3H-thymidine and hydroxyurea, were used to kill proliferating bone marrow cells. Following treatment with 3H-thymidine in vitro, about 41% of the CFU-C were killed in normal and about 25% in leukemic bone marrow. Hydroxyurea administered in vivo resulted in the death of about 33% and 26%, respectively. The results indicate that fewer normal CFU-C are in S-phase in the L5222 leukemia, which might help to explain how enough normal stem cells survive chemotherapy to regenerate the bone marrow.

Proliferative state of normal in vitro colony-forming cells during development of L5222 rat leukemia and their reaction to chemotherapy

In the experimental rat leukemia, L5222, the decrease of normal in vitro colony-forming cells (CFU-C) after chemotherapy with daunomycin is much less than in nonleukemic controls. The leukemia is therefore used here to test the hypothesis that in leukemia the CFU-C are expelled from the active cell cycle to a resting state and are thereby less sensitive to cycle-dependent chemotherapeutic agents. The L5222 leukemia has the advantage that the leukemic blast cells do not form colonies in agar culture so that normal CFU-C can be assessed under leukemic conditions. To compare the proportions of CFU-C in the S-phase in normal and leukemic rats, two S-phase-specific agents, 3H-thymidine and hydroxyurea, were used to kill proliferating bone marrow cells. Following treatment with 3H-thymidine in vitro, about 41% of the CFU-C were killed in normal and about 25% in leukemic bone marrow. Hydroxyurea administered in vivo resulted in the death of about 33% and 26%, respectively. The results indicate that fewer normal CFU-C are in S-phase in the L5222 leukemia, which might help to explain how enough normal stem cells survive chemotherapy to regenerate the bone marrow.

Preparation of colony stimulating factors from human placental conditioned medium

Human placental conditioned medium is able to stimulate human bone marrow cells to form neutrophilic granulocyte-monocyte colonies and eosinophil colonies in agar cultures. Methods are described for preparing highly active preparations (capable of supramaximal stimulation of colony formation) by partial purification of the conditioned medium using calcium phosphate absorption and gel filtration chromatography. Partially purified calcium phosphate gel eluates appear to increase in specific activity after concentration by ultrafiltration. This phenomenon was not observed with the active fractions after further purification on Sephadex G-100. Fractionation of the conditioned medium using gel filtration and hydrophobic chromatography (Cibacron Blue-Sepharose) showed that distinct and partially separable factors were responsible for stimulating granulocyte-macrophage and eosinophil colony formation. From the gel filtration data the apparent molecular weight of the eosinophil colony stimulating factor was higher than the molecular weight of the granulocyte-macrophage colony stimulating factor. Cibacron Blue-Sepharose chromatography led to the separation of one molecular species of granulocyte-macrophage colony stimulating factor from eosinophil colony stimulating factor so that a specific stimulus for human neutrophilic granulocyte and/or monocyte-macrophage progenitors is now available.

The physicochemical properties of erythrocyte derived activity which enhances murine bone marrow colony growth in agar culture.

A stable preparation which retains enhancement activity for the growth of murine granulocyte-macrophage colonies in agar culture has been extracted from human and rat erythrocytes by osmotic lysis and high speed centrifugation. Trypsin digestion followed by Sephadex G-25 column chromatography has resulted in the removal of haemoglobin and the isolation of an active fraction with an apparent molecular weight of 900 to 1000 daltons. Enhancement activity was abolished by n-ethylmaleimide (NEM) treatment, suggesting that the enhancement is dependent on the presence of free sulphydryl groups.

Hemopoietic colony forming cells in regenerating mouse liver.

Cells from regenerating mouse liver removed 2-25 days post 68% hepatic resection have been assayed for in vivo colony forming capacity in soft agar (CFU-C), proliferative capacity in liquid culture, and in vivo spleen colony forming capacity (CFU-S). These studies demonstrated low concentrations of CFU-C and CFU-S in normal and sham operated liver, with an appreciable increase of both in regenerating liver, reaching maximum values in tissue removed 5--7 days post hepatic resection. Colony formation in agar by regenerating liver cells occurred in the absence of exogenous colony stimulating factor. Separation of liver cells on the basis of adherent properties prior to culture indicated concentration of CFU-C in the non-adherent fraction, while cells producing colony stimulating factor were concentrated in the adherent fraction. Foci of actively dividing cells of themacrophage and granulocyte series arose in liquid culture from preparations of sham operated and regenerating liver, although total cell formation was greater with regenerating liver. A small proportion of the colonies formed in agar from regenerating liver consisted of cords of epithelioid cells, which resembled hepatocytes and differed from the macrophages or granulocytes found in the majority of colonies, raising the possibility that regenerating hepatocytes form colonies in agar culture.