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PubMed1992

Mechanisms responsible for size differences between hemopoietic colonies: an analysis using a CSF-dependent hemopoietic cell line.

Cells of the colony stimulating factor (CSF)-dependent continuous hemopoietic cell line 32Dcl-3 generate strikingly different large, medium and small colonies in semisolid cultures. Reculture of cells from such colonies showed that all colony types contained clonogenic cells able again to form colonies of all three size types. Clone mapping showed that late initiation of colony formation did not account for the final size difference between colonies, a conclusion confirmed by mapping studies on normal CSF-stimulated murine granulocyte-macrophage colonies. Clonogenic cells within 32 D Clone 3 (32Dcl-3) colonies varied in quantitative responsiveness to CSF stimulation. This responsiveness was generally lower than that of the initiating clonogenic cell and was higher with cells from small colonies than with cells from corresponding large colonies. The data eliminated differences in intrinsic proliferative potential, cellular hierarchy, cell cycle times or responsiveness to CSF stimulation as an adequate explanation for colony size differences. Colony size differences may possibly arise from random differences in the sequence of self-generative versus differentiative divisions in clonogenic cells in developing clones.

Animals↗
PubMed2002

Modulation of colony stimulating factor release and apoptosis in human colon cancer cells by anticancer drugs.

Modulation of the immune response against tumour cells is emerging as a valuable approach for cancer treatment. Some experimental studies have shown that secretion of colony stimulating factors by cancer cells reduces their tumorigenicity and increases their immunogenicity probably by promoting the cytolitic and antigen presenting activities of leukocytes. We have observed that human colon cancer cells (HT-29) are able to secrete granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor when stimulated with cytokines (IL-1beta and TNF-alpha). In this study we assessed, for the first time, the effects of several anticancer drugs on colony stimulating factor release or apoptosis in HT-29 cells. Cytokine-induced release of granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor was significantly increased by cisplatin and 6-mercaptopurine. Taxol only increased macrophage-colony stimulating factor release while reduced that of granulocyte-colony stimulating factor. No changes in colony stimulating factor secretion were observed after treatment with methotrexate. Only cisplatin and taxol induced apoptosis in these cells. Secretion of colony stimulating factors by colon cancer cells may contribute to the immune host response against them. Anticancer drugs such as cisplatin and 6-mercaptopurine increase colony stimulating factor secretion by cytokine stimulated cancer cells probably through mechanisms different to those leading to cell apoptosis, an effect that may contribute to their anti-neoplasic action.

Antineoplastic Agents↗
PubMed1989

Colony formation by megakaryocyte progenitors in myelodysplatic syndromes.

In vitro megakaryocytic colony formation by progenitors from the bone marrow was studied in 40 myelodysplastic syndrome patients. Megakaryocytic colonies were decreased in number or absent in 30 patients; only 10 showed normal colony growth. The growth correlated to some extent with the FAB-class. Patients with normal colony formation had either RA or RARS, but also in these two FAB-types half of the patients showed reduced megakaryocytic colony formation. Only 1 out of 15 patients with RAEB or RAEBt had normal megakaryocyte growth. The patients with CMML did not show any megakaryocytic colonies. The growth of erythroid colonies was normal in 3 patients and reduced or absent in the others. All 3 with normal erythroid colony formation also showed normal megakaryocyte growth, and all patients with normal megakaryocyte colony formation also had normal granulocyte-macrophage colony growth. Granulocyte-macrophage colony and cluster formation was normal in 17 patients. Defective formation of megakaryocytic, erythroid, and granulocyte-macrophage colonies was seen in 22 patients, compatible with a defect in a pluripotent stem cell. Megakaryocytic colony formation had no obvious correlation with any specific chromosome abnormality, and the distribution of the growth patterns was almost similar in patients with a normal and those with an abnormal karyotype.

Adult↗
PubMed1982

Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors.

We report identification of a unique class of human hemopoietic colony-forming cells with extensive ability to generate progenitors for secondary colonies. Mononuclear cells isolated from human umbilical cord blood formed colonies consisting of 40-500 blast cells after 25 d of incubation in methylcellulose culture in the presence of erythropoietin and medium conditioned by phytohemagglutinin-stimulated leukocytes. Replating of these blast cell colonies revealed that 100% of the primary colonies had the ability to generate secondary colonies, including multipotential colonies. These colonies could be distinguished from other hemopoietic colonies in situ by the complete absence of signs of terminal differentiation. Replating of granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies, consisting of an average of 2 x 10(4) cells, revealed less capacity for secondary colony formation. This human blast cell colony assay may provide a method for quantitation of more primitive hemopoietic stem cells than progenitors for GEMM colonies (CFU-GEMM) in man.

Cells, Cultured↗
PubMed1989

Feedback suppression of B cell colony formation in healthy individuals.

Proliferation and differentiation of B cells has been extensively studied and the study of feedback suppression of B cell proliferation has been limited to humoral factors. However, very little is known about feedback suppression of B cell proliferation by cellular influences. We have previously reported on the role of T cells and their subsets on B cell proliferation in that we did not observe suppression of B cell colony growth by T cells. We now report on the role of B cells in limiting B cell proliferation. B cell colonies were grown in methyl cellulose for either 3 days or 5-6 days utilizing 2 x 10(5) T cells irradiated with 9,000 rads, and 2 x 10(5) B cells. The B cells were then obtained from these colonies and increasing numbers of cells were added to fresh autologous B cells that were further cultured for 5 days to form new B cell colonies. At the end of this period, B cell colony numbers were determined. Our data show that addition of CD19- and CD20- positive B cells recovered from mature colonies after 5 days to fresh B cells suppressed further B cell colony growth in all cases tested, whereas addition of CD19-positive B cells recovered from immature colonies after 3 days of culture did not suppress further B cell colony growth. Elimination of CD 19- or CD20-positive cells with monoclonal antibody to CD19 and complement or by the technique of panning enhanced colony growth. Supernatants obtained from B cell colonies did not suppress B cell colony formation. Our data suggest that there is feedback suppression of normal progenitor B cell proliferation by constituent B cells and that this effect develops during maturation of colonies during the growth phase.

Antigens, CD19↗
PubMed1979

Difference in the cell proliferation and colony-forming ability of normal human T lymphocytes.

Cell transfer experiments were carried out with phytohaemagglutinin-induced normal human T lymphocyte colonies after 2--10 days of primary colony growth. The cells gave a cloning efficiency of 15% after 2 days of incubation and this decreased to 0.2% with the progressive growth of the colonies. The primary colonies had a proliferative capacity to give about 240 cells after 10 days of incubation, but only contained 0.5 to one cell per colony that could form a new colony. This number of colony-forming cells per colony did not change with an increase in colony size. These results indicate that colonies maintained the same number of colony-forming cells that they started with and that the other cells could proliferate but not form colonies. It is suggested that this ability to distinguish between colony-forming and proliferative T cells may be useful for determining specific deficiencie in either cell type in various diseases.

Cell Division↗
PubMed1988

In myelodysplastic syndromes progression to leukemia is directly related to PHA dependency for colony formation and independent of in vitro maturation capacity.

In an agar-liquid double-layer colony assay in which myeloid leukemia colony-forming cells require the presence of both the lectin PHA and CSF for in vitro proliferation, colony formation of bone marrow cells derived from patients with a myelodysplastic syndrome (MDS) was studied. In five of 14 MDS and all five leukemic transformed MDS cases, colony formation was found to require both PHA and CSF. Three of these five PHA-dependent MDS cases progressed to overt leukemia within 1 year, one progressed from RA to RAEB, and one patient received AML chemotherapy. PHA-dependent colony formation was associated with higher bone marrow blast counts, but not directly to FAB type or cytogenetic abnormalities. In nine other MDS cases only CSF was required for colony formation. In these PHA-independent cases the course of the disease was stable during the observation time (5-17 months). Two types of colonies were observed in this in vitro system: colonies adherent and colonies nonadherent to the agar underlayer. The former consisted of terminally differentiated myeloid cells, and the latter comprised immature cells. This suggests that the percentage of adherent colonies formed in vitro may be used as a measure for the maturation defect in MDS. However, no correlation was found between the percentage of adherent colonies and progression to leukemia of the MDS cases. Our findings suggest that the dependency on PHA for in vitro colony formation of colony-forming cells in MDS is predictive for the progression to leukemia. However, the in vitro differentiation capacity has no apparent prognostic significance.

Adolescent↗
PubMed1986

Activities derived from established human myeloid cell lines reverse the suppression of cell line colony formation by lactoferrin and transferrin.

Myeloid cell lines were evaluated for the release of substances needed for colony formation by their own colony-forming cells (CFC) and by other myeloid cell lines. Dialyzed U937 conditioned medium (CM) had no effect on the cloning efficiency of U937 cells, whether or not U937 CFC had been induced for MHC class-II antigens by preincubation of these cells for 72 h with indomethacin and human gamma interferon (HuIFN gamma). Dialyzed U937 CM, however, restored colony formation of HuIFN gamma-induced U937 cells suppressed by lactoferrin (LF) or transferrin (TF). Dialyzed U937 CM did not restore colony formation of U937 cells suppressed by acidic isoferritins (AIF) or prostaglandin E2 (PGE2). Detection of the growth-restoring effects of U937 CM required that U937 CM be prepared in the presence of indomethacin or that the CM be dialyzed to remove inhibitors of U937 colony formation. Dialyzed U937 CM did not inactivate LF. Dialyzed U937 CM did not stimulate or enhance colony formation of normal human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), or multipotential (CFU-GEMM) progenitor cells, but did contain potent inhibitory activity against these progenitor cells. HL-60, EM2, EM3, and K562 cells were also evaluated. HL-60-, EM3-, and K562-CFC that were not preincubated with HuIFN gamma did not express MHC class-II antigens, and colony formation by these cells was not influenced by LF, TF, or AIF. Noninduced EM2-CFC constitutively expressed MHC class-II antigens, and colony formation by these cells was suppressed by LF, TF, and AIF. After induction of MHC class-II antigens on HL-60- and EM3-CFC by HuIFN gamma, colony formation by these cells was suppressed by LF, TF, and AIF. Colony formation by HuIFN gamma-induced EM2 cells was more responsive to inhibition by LF, TF, and AIF than was colony formation by noninduced EM2 cells. K562 cells were not induced into a responsive state to LF, TF, or AIF by HuIFN gamma. Dialyzed CM from HL-60, EM2, and EM3 cells contained activities that restored colony formation by their own LF-suppressed CFC. The activities present in dialyzed CM from U937, HL-60, EM2, and EM3 cells may be similar since they could each restore LF-suppressed colony formation of U937, HL-60, EM2, or EM3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Line↗
PubMed1987

Blast cell colony assay for umbilical cord blood and adult bone marrow progenitors.

We previously described candidate human blast cell colonies in culture of umbilical cord blood cells. However, their replating efficiencies were low, and we were unable to grow colonies from adult marrow cells. We report here a consistent method of growth and identification of human blast cell colonies that are supported by low serum culture and by delayed addition of medium conditioned by a T lymphoblast cell line, C5MJ. Nonadherent mononuclear cord blood and bone marrow cells were prepared by use of Ficoll-Paque and overnight adherence to plastic. Bone marrow cells were further enriched for progenitors by panning with monoclonal anti-My-10 antibody. Cells were plated in methylcellulose culture containing 2% fetal calf serum and supplemented with bovine serum albumin, lecithin, cholesterol, and transferrin. On day 14 of culture, concentrated C5MJ-conditioned medium was carefully added to each dish. Blast cell colonies consisting of 18 to 100 cells were detected on days 21 to 28. Forty percent to 75% of the blast cell colonies in individual samples yielded secondary colonies upon replating (positive colonies). The replating efficiency of the positive colonies ranged from 3% to 100%. The largest secondary colony contained 7,800 cells. In addition to single-lineage colonies, multilineage colonies revealing two to five lineage combinations were seen. These results suggest that human primitive progenitors are dormant in cell cycle and that they survive in the absence of colony-stimulating factors. Human blast cell colonies may provide a unique population of progenitors for studies of the early process of human hemopoiesis.

Adult↗
PubMed1982

Kinetics of the formation of pure and heterogeneous megakaryocyte colonies by megakaryocyte progenitor cells in vitro.

The formation of pure and heterogeneous megakaryocyte colonies in vitro by megakaryocyte progenitor cells was studied using a whole plate stain for acetylcholinesterase to identify megakaryocyte colonies. In cultures of 75,000 C57BL marrow cells, maximal numbers of 11 +/- 5 (mean +/- standard deviation) pure and 6 +/- 1 heterogeneous colonies were reached after 6-7 days of incubation, after which megakaryocyte colony degeneration was marked. The ratio of pure: heterogeneous colonies remained constant at 2:1 both during the incubation period, and when different concentrations of megakaryocyte colony stimulating factor (MEG-CSF) and different numbers of cells per culture were used. The average size of both pure and heterogeneous colonies increased during the incubation period to maximal colony sizes of 15 +/- 13 cells per pure colony and 264 +/- 300 cells per heterogeneous colony. Sequential analysis of individual colonies in situ failed to demonstrate an increase in the size of individual pure colonies, but the enlargement of individual heterogeneous colonies was observed.

Acetylcholinesterase↗
PubMed1994

Urinary tract infections with low and high colony counts in young women. Spontaneous remission and single-dose vs multiple-day treatment.

BACKGROUND: The aim of this study was to test whether the spontaneous cure rate is higher in dysuric women with low urinary colony counts (10(2) to 10(4) colony-forming units [cfu] per milliliter) than in women with high colony counts (> or = 10(5) cfu/mL) and whether the response of patients with low colony counts to single-dose treatment is better than those with high colony counts. METHODS: Dysuric women underwent a clinical interview, examination, urine culture, and microscopic examination of urine, and were asked to postpone treatment for 2 days. At that time, the urine tests were repeated. Women with bacterial urinary tract infection were randomized to a group given a single dose of 1200 mg of norfloxacin and to a group treated with 400 mg of norfloxacin twice daily for 7 days. Cure rates were tested at 1 and 5 weeks after treatment. RESULTS: Of 146 women with urinary tract complaints, 113 (25 patients with sterile urine cultures, 21 with low colony counts, and 67 with high colony counts) agreed to postpone antibiotic treatment. Two days after the initial urine culture, only one patient with a low initial colony count had a sterile urine culture (5%, 95% confidence interval, 0% to 14%); and 10 patients (48%, 95% confidence interval, 27% to 69%) had a colony count of 10(5) cfu/mL or more. The rate of spontaneous cure in women with high initial colony count was 7%, 95% confidence interval, 1% to 13%. One week after treatment, the cure rates were 48 (84%) of 57 patients given single-dose treatment vs 49 (98%) of 50 patients treated for 7 days. Five weeks after treatment the rates were 63% and 83%, respectively. The efficacy of single-dose treatment in patients with low urinary counts were similar to those with high counts, and less than that achieved by 7 days of treatment. CONCLUSIONS: The spontaneous cure rate for a 2-day period is minimal in patients with both a low and a high colony count, but half of the patients with low urinary counts will have high colony counts after this interval. The response to single-dose treatment is similar in patients with low and high colony counts, and lower than with multiday treatment.

Adolescent↗
PubMed1990

Colony formation of clone-sorted human hematopoietic progenitors.

To characterize human hematopoietic progenitors, we performed methylcellulose cultures of single cells isolated from a population of CD34+ cells by fluorescence-activated cell-sorting (FACS) clone-sorting system. CD34+ cells were detected in bone marrow (BM) and peripheral blood (PB) cells at incidences of 1.0% and 0.01% of total mononuclear cells, respectively. Single cell cultures revealed that approximately 37% of BM CD34+ cells formed colonies in the presence of phytohemagglutinin-leukocyte conditioned medium and erythropoietin. Erythroid bursts-, granulocyte-macrophage (GM) colony-, and pure macrophage (Mac) colony-forming cells were 10% each in CD34+ cells. Approximately 15% of PB CD34+ cells formed colonies in which erythroid bursts were predominant. CD34+ cells were heterogeneous and fractionated by several antibodies in FACS multicolor analysis. In these fractionated cells, CD34+, CD33+ cells formed GM and Mac colonies 7 to 10 times as often as CD34+, CD33- cells. Most of the erythroid bursts and colonies were observed in the fraction of CD34+, CD13- cells or CD34+, CD33- cells. The expression of HLA-DR on CD34+ cells was not related to the incidence, size, or type of colonies. There was no difference in the phenotypical heterogeneity of CD34+ cells between BM and PB. About 10% of CD34+ cells were able to form G colonies in response to granulocyte colony-stimulating factor (G-CSF) and to form Mac colonies in GM-CSF or interleukin-3 (IL-3). Progenitors capable of generating colonies by stimulation of G-CSF were more enriched in CD34+, CD33+ fraction than in CD34+, CD33- fraction. Thus, single cell cultures using the FACS clone-sorting system provide an accurate estimation of hematopoietic progenitors and an assay system for direct action of colony-stimulating factors.

Antigens, CD34↗
PubMed1989

Immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar.

An immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar for up to 50 days was performed. Three morphological variants of developing tumor colonies are reported: 1) large light colonies, 2) small dark colonies, and 3) smooth-edged colonies. The large light colony variant is the most frequently observed in the soft agar assay (approximately 70%), followed by the dark colony variant (approximately 27%), and the smooth-edged colony variant (approximately 3%). Major morphological characteristics are associated with each variant, as shown with light microscopy (LM) and transmission electron microscopy (TEM). Both LM and TEM analyses demonstrated that the large light colony variant was hypomelanotic and contained a microfibrillar extracellular matrix (ECM). The small dark colony variant was found to be hypermelanotic and contained a less demonstrable ECM. The smooth-edged variant has an encapsulated periphery, no demonstrable ECM, and tightly packed cells with desmosome-like junctions. In order to characterize further the ECM in the most commonly observed variant, the large light colony, specific antibodies to fibronectin (FN) and collagen types IV and V (COLs IV and V) were applied and observed with immunofluorescence microscopy and immunoperoxidase. In paraffin sections of melanoma colonies, FN was observed associated with both the cell surface and the ECM. However, no specific staining was seen for COLs IV and V. In addition, ruthenium red was used to preserve and selectively bind to glycosaminoglycans (GAGs) and proteoglycans (PGs). TEM studies reveal GAG-like granules stained with ruthenium red in the fibrillar ECM and a dotted, punctate staining of the cell surface. Understanding the biological and architectural composition of developing melanoma tumor colonies in soft agar could contribute to the development of more efficient chemotherapeutic strategies.

Agar↗
PubMed1988

Analysis of murine megakaryocyte colony size and ploidy: effects of interleukin-3.

Megakaryocytes develop from diploid precursor cells that, after variable numbers of mitoses, cease cell division and then undergo synchronous nuclear endoreduplication (endomitosis). Megakaryocyte colony formation represents an in-vitro model of these processes in which the number and ploidy of colony cells reflect the activity of the mitotic and endomitotic pathways, respectively. We have analyzed the size and ploidization of murine megakaryocyte colonies grown in agar and examined the influence of interleukin-3 (IL-3) on these parameters. Colonies were identified in situ by staining for acetylcholinesterase and the ploidy of colony cells was determined by fluorescence cytophotometry. More than 98% of the megakaryocytes that developed in culture could be analyzed. In cultures of unfractionated marrow cells stimulated by either pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM, a crude source of megakaryocyte colony-stimulating activity) or IL-3, the modal ploidy of day-5 colony megakaryocytes was 16N (range 2N-128N). The distribution of colony size was described by an inverse exponential relationship. Colony size and geometric mean ploidy were inversely correlated under conditions of maximal stimulation with PWM-SCM and at all concentrations of IL-3 tested. Increasing concentrations of IL-3 in cultures of either unfractionated marrow cells or nonadherent T-lymphocyte-depleted (NATD) marrow cells stimulated similar dose-dependent increases in the mean size and numbers of megakaryocyte colonies but did not significantly alter their ploidy distribution. However, the mean ploidy of colony megakaryocytes in IL-3-stimulated cultures of NATD marrow cells was significantly less (P less than 0.001) than the mean ploidy of megakaryocytes in IL-3-stimulated cultures of unfractionated marrow cells. The mean ploidy of megakaryocytes, which developed in PWM-SCM-stimulated cultures, was not affected by initial accessory cell depletion. We conclude that the size and ploidy characteristics of day-5 murine megakaryocyte colonies are structured as continua and that IL-3 stimulates an increase in mean colony size and numbers without affecting ploidization. T-lymphocytes and adherent cells elaborate an activity which promotes endomitosis in vitro; factors in PWM-SCM can substitute for this activity.

Animals↗
PubMed1986

Serum-free culture of primitive murine hemopoietic colony-forming cells.

We report the effect of four sources of hemopoietic growth factors, alone or in combination, on colony growth in serum-free cultures of bone marrow from normal mice or marrow from mice pre-treated with 5-fluorouracil (5-FU-bm). The four supplements were: mouse spleen conditioned medium (SCM, a source of multi-lineage colony-stimulating activity, multi-CSA), human placental conditioned medium (HPCM, a source of synergistic activity), pregnant mouse uterus extract (PMUE, a source of M-CSA) and erythropoietin (Epo). First, in cultures of normal marrow, only PMUE and SCM induced significant colony growth when added alone. The majority of those colonies contained granulocytes and macrophages (myeloid colonies). In Epo-supplemented cultures, only SCM supported the growth of erythroid bursts and mixed erythroid-myeloid colonies. HPCM thus appears to be a poor source of multi-CSA. Second, in cultures of 5-FU-bm, few colonies developed if any of the above supplements were added alone. Only SCM + Epo together stimulated the formation of a low number of very large, mixed erythroid/myeloid/megakaryocyte colonies. HPCM, but not SCM, synergized with PMUE to augment myeloid colony numbers. Hence, SCM appears to be a poor source of synergistic activity (SA). In cultures of 5-FU-bm already supplemented with HPCM + PMUE, the addition of Epo did not change total colony numbers but did induce erythroid differentiation in one third of the colonies present. These data suggest that multi-CSA and SA may be expressed by different factors and that 5-FU pre-treated marrow contains: a population of primitive multipotential progenitors which form large, mixed colonies in the presence of SCM + Epo, and a larger Epo-sensitive population which also requires HPCM + PMUE to form mixed colonies.

Animals↗
PubMed1999

Mode of colony foundation influences the primary sex ratio in ants.

In ants, young queens can found new colonies independently (without the help of workers) or dependently (with the help of workers). It has been suggested that differences in the mode of colony founding strongly influence queen survival and colony development. This is because independent queens are constrained to produce a worker force rapidly, before they deplete their body reserves and to resist the intense intercolony competition during the founding stage. By contrast, queens that found colonies dependently remain with the workers, which probably results in a lower mortality rate and earlier production of reproductive offspring. Consequently, in species that found independently, queens of incipient colonies are expected to produce mostly worker brood by laying a lower fraction of haploid (male) eggs than queens in mature colonies; such a difference would not occur in species founding dependently. We compared the primary sex ratio (proportion of male-determined eggs) laid by queens in incipient and mature colonies of two ant species Lasius nigerLinepithema humile, showing independent and dependent modes of colony founding, respectively. As predicted L. niger queens of incipient colonies laid a lower proportion of haploid eggs than queens from mature colonies. By contrast, queens of L. humile laid a similar proportion of haploid eggs in both incipient and mature colonies. These results provide the first evidence that (1) the primary sex ratio varies according to the mode of colony foundation, and (2) queens can adjust the primary sex ratio according to the life history stage of the colony in ants. Copyright 1999 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed1986

Assessing cultured colonies automatically.

The number of colonies formed by macrophage colony-forming cells and high proliferation potential colony-forming cells was assessed by an image processor. The processor counted and sized colonies accurately, reproducibly, rapidly (2 s/dish) and objectively. The processor also measured the amount of light (in grey levels) the colonies transmitted. The optical density of a colony (the sum of its grey levels) was related to its cellularity. Thus the image processor compared both the number of colonies in samples and their cellularity. Samples of marrow containing high proliferation potential colony-forming cells of different proliferative capacity were prepared by injecting fluorouracil into mice and collecting their marrow 2-10 days later (marrow samples called FU2-FU10). These samples were cultured with one of three sources of synergistic factor titrated over seven dilutions. Colonies contained approx. 5 X 10(4) cells after 11 days culture but the way that FU2-FU10 marrow grew depended on the interval between treating donors with fluorouracil and collecting their marrow. Samples collected 2-4 days after fluorouracil formed more colonies containing more cells with small increases of synergistic factor whereas samples collected after 8-10 days did neither. It was important to culture samples of marrow with the appropriate synergistic factor for the interval after fluorouracil. Factor(s) derived from the 5637 cell line acted optimally on high proliferation potential colony-forming cells in samples collected 2-8 days after fluorouracil, and factor(s) derived from Wehi 3B cells on high proliferation potential colony-forming cells in samples collected 6-10 days after fluorouracil. Factor(s) derived from placental conditioned medium acted well on samples collected between 2 and 10 days. The proliferative capacity of samples of marrow could also be compared by estimating growth curves for high proliferation potential colony-forming cells in samples collected at successive intervals after fluorouracil.

Animals↗
PubMed1985

Development of macrophage and granulocyte colonies from human peripheral blood.

The presence of macrophage and granulocyte progenitor cells in the human peripheral blood enabled the establishment of colonies from this accessible tissue and obviated the need for bone marrow to achieve this task. We have developed a method of obtaining reproducible growth of macrophage/granulocyte colonies from human peripheral blood. Colonies of macrophages and granulocytes were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). At early stages of colony growth both macrophage and granulocyte colonies were detected while following 20-25 days in culture all colonies tested revealed monocyte-macrophage morphology. To obtain higher numbers of colonies, we tested different cell sources, different CM preparations and the effect of steroid hormones on colony development. We found that the mononuclear cells obtained from cord blood (CB) or from some patients with inflammatory bowel disease yielded much higher numbers of colonies than PBM from normal individuals. Colony development from these two sources did not depend on an external source of colony stimulating factor (CSF) but was augmented as a result of CSF supplementation. CM obtained from CB mononuclear cells as well as supernatants from some human monoblastic cell lines were similar in their CSF activity to CM from normal PBM and made possible the development of macrophage/granulocyte colonies. Higher numbers of colonies were induced by including physiological concentrations of estradiol in the culture medium, in the absence of external sources of CSF. The system described above enabled the analysis of cloned macrophages and their circulating progenitor cells as well as the assay of different preparations of CSF.

Adolescent↗