Colony-forming and colony-stimulating cells in normal human peripheral blood

Peripheral white blood cells (WBC) from normal persons form colonies of granulocytic cells in vitro in soft agar. Stimulus was provided by a feeder layer of peripheral WBC. By centrifugation through an Isopaque-Ficoll gradient, the cells were separated into a mononuclear and a granulocytic fraction with a purity of 96–98% in each fraction. Both the colony-forming cells and the cells inducing colony formation were found in the mononuclear cell fraction. Further fractionation of these mononuclear cells on adherence glass bead columns showed that the colony-forming and colony-inducing cells do not belong to the small lymphocyte population, but were found in the glass adherent fraction containing large monocyte-like and atypical mononuclear cells.     

COLONY-FORMING ABILITY OF BONE MARROW CELLS OF W ANAEMIC MICE ON MACROPHAGE LAYER FORMED IN PERITONEAL CAVITY OF MICE

The colony-forming ability of haematopoietic cells of W anaemic mice was examined on the macrophage layer formed in the peritoneal cavity of mice. Bone marrow cells of W anaemic mice formed a considerable number of colonies on the macrophage layer, notwithstanding they did not form any colonies in the spleen of the same recipients. As the colony-forming ability of the bone marrow cells was not reduced by the incubation with ³H-thymidine, most of the cells which formed colonies on the macrophage layer seemed to stay in G₀ state. The interrelationship between the spleen colony-forming cells, the macrophage-layer colony-forming cells, and in vitro colony-forming cells was discussed.     

Formation of Mononuclear Phagocyte (Macrophage) Colonies by Mouse Spleen Cells in Liquid Culture

When spleen cells of the adult mouse were tested for the formation of mononuclear phagocyte (macrophage) colonies by the liquid culture technique with an incubation period of 7–8 days, about 100 macrophage colonies were produced from 1 × 10⁶ cells. The number of macrophage colonies appearing after 2 days of incubation was small, but thereafter increased progressively up to at least 8 days. In the later stages of incubation (after day 6) large colonies consisting of more than 100 cells appeared. Macrophage colonies in the early stages consisted almost solely of macrophages. On day 6 significant numbers of small round mononuclear cells with no detectable phagocytic activity were seen in the center of large colonies, and by day 8 marked crowding of these cells had occurred. The peripheral region of the large colonies consisted mainly of macrophages and the intermediate region of middle-sized round or slightly stretched cells with weak phagocytic activity. Approximately two-thirds of the colony-forming cells still remained after glass-adherent cells were removed from the spleen cells by passing over a glass-bead column. In cultures of glass-nonadherent cells macrophage colonies were not generated in the early stage. The number of colony-forming cells did not change significantly even after actively phagocytic cells were rigorously removed from the spleen cells. In addition, no macrophage colonies were generated in cultures of spleen cells treated with mitomycin C.     

Infection of granulocyte/monocyte progenitor cells with HIV1

In order to study whether cytopathic HIV1 infection of haemopoietic progenitor cells is involved in the derangement of haemopoiesis in patients with HIV1 infection, we infected enriched progenitor cells with HIV1, by addition of viral inoculate supernatants from HIV1-infected peripheral blood mononuclear cells or by coculture with HIV1-infected monocytes/macrophages. Progenitor cells were seeded into colony assays and single colonies were chosen for HIV1 mRNA determination by in situ hybridization. Growth of progenitors was not affected by infection. However, up to 42% of colonies of pluripotent progenitor cells (colony-forming unit/granulocyte-erythrocyte-monocyte; CFU-GEM) and committed progenitor cells CFU/granulocyte-monocyte (CFM-GM) contained HIV1 mRNA-expressing cells.In addition, we studied HIV1 infection of progenitor cells from the bone marrow of 6 patients with AIDS or AIDS-related complex. Two patients were negative, two had a few colonies expressing HIV1 mRNA in a minority of cells, and in the remaining two, up to 11% of CFU-GM contained HIV1-expressing cells.Thus, infection of progenitor cells with HIV1 was achieved experimentally in vitro and occurs in vivo. However, growth of progenitors after in vitro infection continues and therefore HIV1 infection does not seem to contribute directly to the reduced incidence of haemopoietic progenitor cells in vivo.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Effects of Yersinia enterocolitica on bone marrow cells proliferation in mice

Intraperitoneal (i.p.) infection of mice with virulent Yersinia enterocolitica, that possess the virulence plasmid encoding calcium requirement, caused a significant reduction in the number of nucleated cells per femur, but increased significantly the ratio of both mitosis and in vitro colony-forming units (CFU) to marrow cells. A plasmid-less, isogenic avirulent derivative did not cause such differential effects on marrow cellularity and mitosis ratio. Thus, increase of granulocyte and mononuclear phagocyte progenitor cells by Y. enterocolitica was associated with virulence plasmid presence.     

Comparative Investigation of Mesenchymal Stem Cells Isolated from the Bone Marrow and Fetal Liver of Mouse and Rat

We studied the properties of cells forming fibroblast colonies from the bone marrow and fetal liver of mouse and rat. Bone marrow and fetal liver cells formed colonies in vitro including fibroblasts as well as a considerable proportion of macrophages. The colonies formed from bone marrow and hepatic cells of rat differed from the murine ones by a higher proportion of fibroblasts. Most colonies derived from the bone marrow of both mouse and rat included a fraction of cells expressing alkaline phosphatase, and hence, capable of osteogenic differentiation; the colonies derived from the fetal liver included low proportions of such cells. The cell layers derived from the colony-forming fibroblasts of both bone marrow and fetal liver of mouse maintained hematopoiesis in the peritoneal cavity of irradiated mice, which indicated that these progenitor cells can form hematopoietic microenvironment.     

Interactions between purified GM-CSF, purified erythropoietin and spleen conditioned medium on hemopoietic colony formation in vitro.

Preincubation of C57BL adult marrow cells or CBA fetal liver cells with a 250-fold excess concentration of purified GM-CSF failed to reduce the frequency of cells forming eosinophil, megakaryocyte or erythroid colonies in subsequent agar cultures. When excess concentrations of purified GM-CSF were added to agar cultures stimulated by pokeweed mitogen-stimulated spleen conditioned medium (SCM), no reduction was observed in the frequency of eosinophil, megakaryocyte or erythroid colonies. Addition of 4 units of purified erythropoietin (EPO) to cultures of fetal liver or adult marrow cells stimulated by SCM increased the number of erythroid colonies but did not reduce the number of non-erythroid colonies or the non-erythroid content of mixed erythroid colonies. Although neither GM-CSF nor EPO alone was able to stimulate erythroid colony formation in agar cultures of fetal liver cells, small numbers of large erythroid colonies were stimulated to develop in cultures containing both purified regulators. Purified GM-CSF was also able to support the survival in vitro of a small proportion of erythroid colony-forming cells in fetal liver populations cultured initially in the absence of SCM and the survival of some eosinophil and megakaryocyte colony-forming cells in similar cultures of adult marrow cells. The results do not support the hypothesis that GM-CSF and EPO compete for a common pool of uncommitted progenitor cells. On the contrary, the data indicate that GM-CSF und EPO are able to collaborate in stimulating the proliferation of some erythropoietic cells. Furthermore, purified GM-CSF appears to be able to support temporarily the survival and/or initial proliferation of at least some cells forming erythroid, eosinophil and megakaryocyte colonies, even though GM-CSF is unable to stimulate the formation of colonies of these types.     

Mononuclear phagocyte colony formation of human spleen cells in liquid culture

We found that mononuclear phagocytes formed a distinct number of clusters and colonies on the bottom of a culture dish 7 days later but granulocytes did not, when a large number of human spleen cells were cultured in liquid medium. In all gastric cancer bearers and patients with portal hypertension operated on, however, colony formation was restricted to spleen cells from patients with advanced gastric cancer and from a group of patients with portal hypertension. These spleen cells formed mononuclear phagocyte colonies without the help of exogeneous colony stimulating factor (CSF). We further demonstrated that the colony-forming cells were glass non-adherent and nylon wool adherent, and that spontaneous colony formation required cooperation between the colony-forming cells and colony-stimulating cells adherent to a plastic surface.     

The clonogenic potential of hematopoietic stem cells and mesenchymal stromal cells in various hematologic diseases: a pilot study

Background aimsMesenchymal stromal cells (MSC) are the most popular cells used in regenerative medicine and biotechnology. The clonogenic potential of these cells is defined by colony-forming unit-fibroblasts (CFU-F). It is well known that there is an interaction between hematopoietic cells and stromal cells in disease formation pathogenesis. Therefore we hypothesized that there should be a quantitative and qualitative relationship between MSC colonies (CFU-F) and hematopoietic stem cell colonies (colony-forming unit-granulocyte-macrophages; CFU-GM) among patients with and without hematologic diseases.MethodsForty-two patients were included in this study. Patients were divided into three groups: group A, patients with hematologic malignancies (n = 20); group B, patients with bone marrow (BM) failure (n = 11); group C, patients without hematologic diseases (n = 11). BM aspirates were plated in different densities for CFU-F culture. The plating density was the same for CFU-GM culture.ResultsCFU-GM colonies grew in 90% of group A cells and all of group B and C cells (P = 0.0001). CFU-F colonies became visible on the ninth day of plating in group A and on the eight day in groups B and C. There was no statistically significant difference between the groups for the duration of CFU-F colony formation (P = 0.12). There were differences in the morphology of the colonies among the groups.ConclusionsThis is the first study that has compared the clonogenic potential of stromal cells and hematopoietic stem cells in the same subjects with and without hematologic diseases. No correlation was shown between the clonogenic potential of stromal cells and hematopoietic cells.     

Nonadhesive populations in cultures of mesenchymal stromal cells from hematopoietic organs in mouse and rat

The study of adhesive properties of multipotent mesenchymal stromal cells evaluated from fibroblast colony-forming units in the bone marrow of adult mice and rats in populations of cells attached and unattached to plastic substrate after 2 h to 7 days in culture demonstrated both similarities and differences. The increase in the fibroblast colony-forming units in the adhesive population peaked on day 7 of in vitro culture in both cases; however, nearly no fibroblast colony-forming units were observed in the nonadhesive population from the mouse bone marrow in this period. Conversely, the number of colonies from the rat bone marrow nonadhesive population on day 7 of culture considerably increased, and this nonadhesive population in long-term culture became the source for subsequent nonadhesive subpopulations containing fibroblast colony-forming units. After 7 days of in vitro culture, the suspension of cells isolated from the liver of 17-day-old rat fetuses also contained a fraction of unattached fibroblast colony-forming units. In the nonadhesive subpopulations from the bone marrow and fetal liver, fibroblast colony-forming units were observed up to day 48 and 30, respectively. Stromal cell precursors of nonadhesive subpopulations from the rat bone marrow featured a period of colony formation reduced to 7 days (i.e., they were formed 1.5-2 times faster compared to the primary culture). The total number of fibroblast colony-forming units from all nonadhesive subpopulations was roughly 6 and 7.4 times that of the adhesive population of the primary culture from the bone marrow and fetal liver, respectively. Considering that the mammalian bone marrow remains the preferred source of mesenchymal stromal cells, using nonadhesive subpopulations in the presented culture system can considerably increase the yield of stromal precursor cells     

Human megakaryocytic progenitors (CFU-M) assayed in methylcellulose: Physical characteristics and requirements for growth

The basic culture requirements and several physical characteristics were defined for megakaryocytic colony-forming cells (CFU-M) from normal human marrow growing in methylcellulose. Ficoll-hypaque separated mononuclear cells from human, marrow gave rise to megakaryocytic colonies in the presence of normal human plasma and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Their identity as megakaryocytic colonies was confirmed by immunofluorescence staining with a monoclonal antibody to human factor VIII antigen and by electron microscopy of individually harvested colonies. Demonstration of the single-cell origin of the colonies was provided by analysis of the glucose-6-phosphate dehydrogenase (G-6-PD) enzyme type of individually harvested colonies grown from a G-6-PD heterozygote. The colonies grew best in heparinized or citrated plasma as opposed to serum. Detailed studies suggested that platelet-release products were responsible for this difference. Tritiated thymidine suicide studies showed that the percentage of CFU-M in DNA synthesis was 23 ± 8% (n = 10). The modal velocity sedimentation rate of CFU-M was 4.9 ± 0.6 mm/hr (n = 4) while that of concurrently studied granulocyte/macrophage colony-forming cells (CFU-GM) was 5.7 ± 0.5 mm/hr. Examination of the PHA-LCM dose-response characteristics suggested the presence in the conditioned medium of an inhibitor to megakaryocyte colony growth which was partially removed by chromatography of the medium on Sephadex G-100. The resulting conditioned medium increased the cloning efficiency for CFU-M compared with that with crude PHA-LCM (15.3 ± 7.0 and 8.2 ± 5.3/10⁵ marrow cells, respectively).     

Crucial Role of Extracellular Polysaccharides in Desiccation and Freezing Tolerance in the Terrestrial Cyanobacterium Nostoc commune

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and has a cosmopolitan distribution. In this study, the role of extracellular polysaccharides (EPS) in the desiccation tolerance of photosynthesis in N. commune was examined. Although photosynthetic O₂ evolution was not detected in desiccated colonies, the ability of the cells to evolve O₂ rapidly recovered after rehydration. The air-dried colonies contained approximately 10% (wt/wt) water, and field-isolated, natural colonies with EPS were highly water absorbent and were rapidly hydrated by atmospheric moisture. The cells embedded in EPS in Nostoc colonies were highly desiccation tolerant, and O₂ evolution was not damaged by air drying. Although N. commune was determined to be a mesophilic cyanobacterium, the cells with EPS were heat tolerant in a desiccated state. EPS could be removed from cells by homogenizing colonies with a blender and filtering with coarse filter paper. This treatment to remove EPS did not damage Nostoc cells or their ability to evolve O₂, but O₂ evolution was significantly damaged by desiccation treatment of the EPS-depleted cells. Similar to the EPS-depleted cells, the laboratory culture strain KU002 had only small amount of EPS and was highly sensitive to desiccation. In the EPS-depleted cells, O₂ evolution was also sensitive to freeze-thaw treatment. These results strongly suggest that EPS of N. commune is crucial for the stress tolerance of photosynthesis during desiccation and during freezing and thawing.     

Correspondence between the development of hemopoietic tissue and the time of colony formation by colony-forming cells

The development of a haemopoietic tissue and the time when colony-forming cells in it formed detectable colonies were studied with in vivo spleen colony-forming units (CFUs) and in vitro high-proliferation-potential colony-forming cells (HPP CFC). Cells that form colonies first are developmentally more mature than those doing so later. Marrow containing mature spleen colony-forming cells formed fewer cells in the femora of recipients than that which contained early colony-forming cells. The growth curve of developmentally early high-proliferation potential-colony-forming cells was steeper than that of later cells. The time period before colony-formation occurs is a property of the colony-forming cell and is not due to regulatory mechanisms in the animal or to regulatory cells in the haemopoietic stroma.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

Partitioning of CO2 Fixation in the Colonial Cyanobacterium Microcystis aeruginosa: Mechanism Promoting Formation of Surface Scums

Constraints on inorganic carbon (C_i) availability stimulated buoyancy in natural, photosynthetically active populations of the colonial blue-green alga (cyanobacterium) Microcystis aeruginosa. In nonmixed eutrophic river water and cultures, O₂ evolution determinations indicated C_i limitation of photosynthesis, which was overcome either by CO₂ additions to the aqueous phase or by exposure of buoyant colonies to atmospheric CO₂. Microautoradiographs of M. aeruginosa colonies revealed partitioning of ¹⁴CO₂ fixation and photosynthate accumulation between peripheral and internal cells, particularly in large colonies. When illuminated colonies were suspended in the aqueous phase, peripheral cells accounted for at least 90% of the ¹⁴CO₂ assimilation, whereas internal cells remained unlabeled. However, when ¹⁴CO₂ was allowed to diffuse into colonies 15 min before illumination, a more uniform distribution of labeling was observed. Resultant differences in labeling patterns were most likely due to peripheral cells more exclusively utilizing CO₂ when ambient C_i concentrations were low. Among colonies located at the air-water interface, internal cells showed an increased share of photosynthate production when atmospheric ¹⁴CO₂ was supplied. This indicated that C_i transport was restricted in large colonies below the water surface, forcing internal cells to maintain a high degree of buoyancy, thus promoting the formation of surface scums. At the surface, C_i restrictions were alleviated. Accordingly, scum formation appears to have an ecological function, allowing cyanobacteria access to atmospheric CO₂ when the C_i concentration is growth limiting in the water column.     

Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium

Erythroid colony formation in agar cultures of CBA cells was stimulated by the addition of pokeweed mitogen-stimulated C57BL spleen conditioned medium. Both 48-hour colonies ("48-hour benzidine-positive aggregates") and day 7 large burst or unicentric erythroid colonies ("erythroid colonies") developed, together with many neutrophil and/or macrophage colonies. In CBA mice, the cells forming erythroid colonies occurred with maximum frequency (650/10(5) cells) in 10- to 11-day-old yolk sac and fetal liver but were present also in fetal blood, spleen and bone marrow. The frequency of these cells fell sharply with increasing age and only occasional cells (2/10(5) cells) were present in adult marrow. A marked strain variation was noted, CBA mice having the highest levels of erythroid colony-forming cells. The erythroid colony-forming cells in 12-day CBA fetal liver were radiosensitive (DO 110-125 rads), mainly in cycle and were non-adherent, light density, cells sedimenting with a peak velocity of 6-9 mm/hr. These properties are similar to those of other hemopoietic progenitor cells in fetal tissues. The relationship of these apparently erythropoietin-independent erythroid colony-forming cells to those forming similar colonies after stimulation by erythropoietin remains to be determined.     

Comparison of Fibronectin and Collagen in Supporting the Isolation and Expansion of Endothelial Progenitor Cells from Human Adult Peripheral Blood

Background

Endothelial colony-forming cells (ECFCs), are circulating endothelial progenitor cells increasingly studied in various diseases because of their potential for clinical translation. Experimental procedures for their ex vivo culture still lack standardization. In particular two different extracellular matrix proteins, either fibronectin or collagen, are commonly used by different Authors for coating plastic plates, both allowing to obtain cells that have all the features of ECFCs. However, possible differences in the impact of each substrate on ECFCs have not been analysed, so far. Therefore, in this study we investigated whether fibronectin and collagen may differentially affect ECFC cultures.

Methodology/Principal Findings

ECFCs were isolated and cultured from peripheral blood mononuclear cells of healthy donors. The impact of fibronectin compared with collagen as the only variable of the experimental procedure was analysed separately in the phase of isolation of ECFC colonies and in the following phase of cell expansion. In the isolation phase, although similar frequencies of colonies were obtained on the two substrates, ECFC colonies appeared some days earlier when mononuclear cells were seeded on fibronectin rather than collagen. In the expansion phase, ECFCs cultured on collagen showed a longer lifespan and higher cell yields compared with ECFCs cultured on fibronectin, possibly related to the higher levels of IL-6 and IL-8 measured in their supernatants. ECFCs cultured on both substrates showed similar immunophenotype and ability for in vitro tube formation.

Conclusions/Significance

Overall, the results of this study indicate that, although both fibronectin and collagen efficiently sustain ECFC cultures, each of them brings some advantages within individual steps of the entire process. We suggest that colony isolation performed on fibronectin followed by cell expansion performed on collagen may represent a novel and the most efficient strategy to obtain ECFCs from adult peripheral blood samples.     

Hemopoietic progenitor cells of W anemic mice studied in vivo and in vitro

The proliferation and differentiation of hemopoietic cells from genetically anemic W^v/W^x,W/W^v, and W^v/W^v mice, and from nonanemic carrier W/+, W^b/+, and W^v/+ mice have been evaluated in vivo by transplantation techniques and in vitro by the agar gel culture method. Marrow from anemic and carrier mice contained progenitor cells which were decreased in number and formed small, often rudimentary, colonies in the spleens of irradiated recipient mice. Proliferation and differentiation of both erythropoietic and leukopoietic progenitor cells were delayed and reduced, but erythropoiesis was more severely affected than leukopoiesis. The severity of the hemopoietic impairment was gene-dose dependent. The W gene effect on leukopoietic progenitor cells was not secondary to anemia or to abnormal erythropoiesis. The marrow cells of anemic and carrier mice which form colonies of granulocytic and mononuclear cells in vitro were neither decreased in number nor impaired in proliferation and differentiation. Hypertransfusion of red blood cells increased the frequency of in vitro colony-forming cells, but not that of in vivo progenitor cells. The data demonstrate that colony-forming cells which proliferate in the agar gel cultures in vitro are distinct from the in vivo colony-forming cells and suggest that the former are primitive members of the granulocytic cell line. Perhaps in vitro CFU are in an intermediate stage of differentiation between in vivo CFU and myeloblasts, analogous to that which has been suggested for the erythropoietin-sensitive cell in the red cell series. W mutant alleles appear to act, therefore, at or very near the beginning of hemopoietic differentiation.