Human bone marrow colony growth in agar-gel

A technique for growing human bone marrow cell colonies in agar-gel medium is described. “Feeder layers” containing 1 × 10⁶ normal human peripheral white blood cells are used as the stimulus for colony growth. Human bone marrow aspirates are collected in heparinized syringes and plated as 2 × 10⁵ cells on “feeder layers.” Normal human bone marrow yields 32–102 colonies per 2 × 10⁵ cells plated. Colonies are almost exclusively granulocytic. Growth rate of colonies is slower than with mouse bone marrow but colonies reach a comparable size (500–1500 cells) at days 12–16.     

Retinoic acid enhances colony-stimulating factor-induced clonal growth of normal human myeloid progenitor cells in vitro

We studied the effect of vitamin A and its analogues (retinoids) on the clonal growth in vitro of normal human myeloid progenitor cells. Normal human bone marrow cells were cultured in soft gel in the presence of a source of colony-stimulating factor (CSF), and various retinoids, and the number of granulocyte-macrophage colonies (CFU-GM) were scored. The addition of 3 × 10^?8 to 3 × 10^?6 M retinoic acid to culture plates containing CSF markedly increased the number of myeloid colonies as compared with culture plates containing CSF alone. Maximal stimulation occurred at a concentration of 3 × 10^?7 M retinoic acid which increased the mean number of colonies by 213 ± 8 % (±S.E.) over plates containing CSF alone. Retinal or retinyl acetate was less potent than retinoic acid, and retinol (vitamin A) had no effect on colony growth. Retinoic acid had no direct CSF activity nor did it stimulate CSF production by the cultured bone marrow cells. Our studies show for the first time that retinoids can stimulate granulopoiesis in vitro and we suggest that this stimulation may be mediated by increased responsiveness of the granulocyte-macrophage progenitors to the action of CSF.     

CFU-F from dog marrow: a colony assay and its significance

A colony assay method is described for studying dog fibroblast colony development in marrow cells derived from resected ribs. The assay showed an increased number of fibroblast colony forming units (CFU-F) in cell suspensions prepared from resected ribs compared to cell suspensions prepared from bone marrow aspirates or from peripheral blood. A linear relationship between the number of cells plated and the number of fibroblastoid colonies was demonstrated in each case. The proportion of phagocytic cells was lower in cultures prepared from resected ribs than in those prepared from bone marrow aspirates. Staining for acid phosphatase and with sudan black showed differences between phagocytic cells and non-phagocytic fibroblasts. When left in plastic dishes for 2 hrs, 81% +/- 10% of the CFU-F adhered to the plastic dishes. Velocity sedimentation separation showed a modal sedimentation rate of 6.49 mm/h.     

THE DEVELOPMENT OF FIBROBLAST COLONIES IN MONOLAYER CULTURES OF GUINEA-PIG BONE MARROW AND SPLEEN CELLS

In monolayer cultures of guinea-pig bone marrow and spleen the development of discrete fibroblast colonies takes place on days 9–12. The linear increase in the number of colonies with increasing numbers of explanted cells and the distribution of male and female cells in mixed cultures support the view that fibroblast colonies are clones. The concentration of colony-forming cells in bone marrow and spleen is approximately 10^-5. Bone marrow culture (but not spleen culture) fibroblasts are capable of spontaneous bone formation in diffusion chambers. Fibroblasts from both bone marrow and spleen cultures are inducible to osteogenesis in diffusion chambers in the presence of transitional epithelium.     

A comparison of barley isolated microspore and anther culture and the influence of cell culture density

Comparisons were made between the efficiency of barley plant regeneration from anther culture (AC) and isolated microspore culture (IMC) for the European winter cultivar `Igri' and the spring F<sub>1</sub> Australian breeder's hybrid Amagi Nijo×WI2585. In both cases, IMC produced a higher number of green regenerant plantlets per anther than AC. For `Igri' there was a 100- to 200-fold improvement and for Amagi Nijo×WI2585 there was a five- to ninefold improvement of IMC over AC. To improve the consistency and reliability of the IMC method, we investigated several parameters, including maltose concentration, subculture protocol, microspore plating density and colony plating density. Subculturing during the liquid culture phase produced no significant improvement in the number of microspores developing into colonies. The optimal concentration of maltose in the liquid induction medium was found to be 90 g l^–1. Both microspore plating density and colony plating density were found to influence plant regeneration. Microspores produced the highest numbers of colonies when plated at densities greater than 5×10⁴ ml^–1, and colonies produced optimal numbers of green plantlets when plated at 12.5–25 colonies/cm². Received: 23 March 1997 / Revision received: 29 May 1997 / Accepted: 25 June 1997     

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells

Summary The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5×10³/cm² in the presence of 2×10⁴/cm² lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10⁻¹⁰ M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.     

Effect of opioid peptides on proliferation and differentiation of skeletogenic cells of rabbit bone marrow in vitro

The influence of opioid peptides DSLET and DAGO in doses 10(-5), 10(-7) or 10(-10) mg per 1 ml of the medium on colony formation in the culture of stromal bone marrow fibroblast precursors was investigated 5. 10(-6) bone marrow cells were placed in plastic containers (Costar). 12 day old cell cultures were fixed with ethanol and stained with hematoxyline-eosin. Effectiveness of fibroblast colony formation (EFFC) was detected. Grown fibroblast colonies were stained after Gomory for alkaline phosphatase. Opioid peptides DSLET and DAGO in the used doses exerted no influence on EFFC and percentage phosphatase-positive colonies, which casts doubt on a presumable direct action of opioid peptides on stromal bone marrow cell-precursors. But it does not seem unlikely that opioid peptides may affect stromal bone marrow precursors of fibroblasts through the cell environment, particularly, via macrophages.     

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     

DIFFERENTIATION OF MURINE MARROW MEGAKARYOCYTE PROGENITORS (CFUM): HUMORAL CONTROL IN VITRO

Differentiation of mouse marrow megakaryocyte progenitors (CFUm) was studied in vitro by a colony assay using a plasma clot system. Erythropoietin (EPO) from sheep plasma (6 units/mg protein) in doses from 1 to 5 units/ml induced a linear increase in CFUm to a maximum of 20 colonies/10⁵ cells plated. Human urinary EPO also induced a dose-responsive increase in CFUm, but the maximum was 9 colonies/10⁵ with 2·0 units/ml of EPO and there was a decrease in colonies above that concentration. Thrombocytopoiesis-stimulating factor (TSF) derived from human embryonic kidney culture supernatant fluids induced a dose-responsive increase in CFUm in concentrations from 0·01 to 0·32 mg protein/ml in the absence of added EPO. TSF did not support the growth in vitro of erythroid colonies from mouse marrow (CFUe and BFUe) indicating an absence of EPO activity. In these studies sheep EPO appeared more effective in supporting CFUe growth than human EPO. TSF also had a stimulatory function in megakaryocyte differentiation at a precursor level. Multiple humoral factors play a role in megakaryocytopoiesis in vitro.     

A quantitative assay for mouse granulocyte (CFU-g) and macrophage (CFU-m) precursors using plasma clots

Cytochemical procedures were used to identify and quantitate granulocyte and macrophage precursors from mouse bone marrow cells in plasma clot cultures. Excellent clonal morphology and cellular enzyme activity were obtained when using plasma clots as the support matrix and buffered formalin acetone as the fixative. For cytochemical identification, naphthol AS acetate esterase staining was used for macrophages and peroxidase for granulocytes. These enzyme properties were confirmed by inactivation studies with a variety of inhibitors, group specific chemical modifications, and pinocytotic affinity for horseradish peroxidase. When mouse bone marrow cells (3 X 10(4) cells/dish) were cultured in plasma clots with human placental or L-cell-conditioned medium, 70 to 110 colonies were produced. Both pure granulocyte (CFU-g) and pure macrophage colonies (CFU-m) were observed, but approximately 5% of the total colony number was composed of mixed granulocyte/macrophage colonies (CFU-gm). The number of plated cells correlated strongly with the colony number (0.990 less than r less than 0.999).     

Pre CFU-F in bone marrow cultures from children with hematopoietic disease including acute leukemia

Stromal precursor cells from bone marrow aspirates of children have been studied in culture. In 7 day liquid cultures normal individuals and patients with acute leukemia in remission grew 110 +/- 50 CFU-F and 100 +/- 40 CFU-F (colony forming unit--fibroblasts) respectively, per 6 X 10(5) buffy coat mononuclear cells. Staining with monoclonal antibodies suggests that stromal cells from CFU-F colonies are fibroblasts. CFU-F colony growth from the bone marrow of patients with active leukemia was low. After cultivation periods of more than 21 days, we observed, in addition, still more immature, clonogenic fibroblast precursor cells, "pre CFU-F", and round cells attached to stromal cells from pre CFU-F colonies. From the round cells, we have passaged pre CFU-F and CFU-GM (colony forming unit--granulocytic, monocytic) in secondary cultures. Our observations are in agreement with the concept that the bone marrow stromal cell matrix serves as a sanctuary for reversibly attached clonogenic cells of both the hematopoietic and fibroblast lineages.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization

Marrow culture from mice has been reported to be overgrown by non-mesenchymal cells. In almost all protocols for isolation of murine mesenchymal stem cells (MSCs), high density culture systems have been employed. Since MSCs are colonogenic cells, the initiating cell seeding density may have significant impact on their cultures. This subject was explored in this study. For this purpose, the bone marrow cells from NMRI mice were plated at 2.5 × 10⁶ cells/cm² and upon confluency were reseeded as either low density (50 cells/cm²) or high density (8 × 10⁴ cells/cm²) cultures. The cells were expanded through an additional subculture and the passage 2 cells as a product of two culture systems were statistically compared with respect to their surface antigen profiles and osteogenic culture mineralization. While low density culture grew with multiple colony formation, there were no distinct colonies in high density cultures. In contrast to high density cultures, passage 2 cells from low density system possessed typical homogenous fibroblastic morphology. Some cells from high density system but not the low density cultures expressed hematopoietic and endothelial cell markers including CD135, CD34, CD31, and Vcam surface antigens. Furthermore, osteogenic cultures from low density system displayed significantly more mineralization than those from high density system. Taken together, it seems that low density culture system resulted in more purified MSC culture than its counterpart as high density culture system.     

Detection of lymphoid leukemia colony-forming cells in abelson virus infected mice: Differences in inbred strains

BALB/c or DBA/2 mice were infected with Abelson murine leukemia virus (A-MuLV), pseudotype Molony murine leukemia virus (M-MuLV). Infection of these mice with 10⁴ focus-forming units of A-MuLV (M-MuLV) induced overt leukemia, detectable grossly or microscopically in 90% of the mice at 20–38 days. However, these methods did not detect leukemia at 17 days or before. Bone marrow cells from A-MuLV-infected leukemic or preleukemic mice were placed in tissue culture in a soft agarose gel. Cells from leukemic or preleukemic BALB/c mice grew to form colonies of 10³ cells or more, composed of lymphoblasts, whereas marrow cells from normal uninfected mice did not. Cells from these colonies grew to form ascitic tumors after intraperitoneal inoculation into pristane-primed BALB/c recipient. Colony-forming leukemia cells could be detected in the marrow of A-MuLV-infected mice as early as 8 days after virus incoluation. The number of colony-forming leukemia cells increased as a function of time after virus inoculation. Colony-forming leukemia cells require other cells in order to replicate in tissue culture. Normal bone marrow cells, untreated or after treatment with mitomycin-C, provide this “helper” function. Only in the presence of untreated or mitomycin-C treated helper cells was the number of colonies approximately proportional to the number of leukemia cells plated. Marrow cells from leukemic BALB/c mice form more colonies than those from leukemic DBA/2 mice. The number of colonies formed per 10³ microscopically identifiable leukemia cells plated was determined to be 2–3 for leukemic BALB/c mice and 0.3 for DBA/2 mice. Cocultivation of leukemic DBA/2 marrow cells with mitomycin-C treated normal BALB/c cells did not increase the number of colonies formed by the DBA/2 leukemic cells. Thus, the decreased ability of DBA/2 leukemia cells to form colonies appears to be a property of the leukemia cell population.     

An in vitro assay for T lymphocyte progenitors (CFU-preT)

Thy-1.2 negative progenitors give rise to Thy-1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen-stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy-1 positive, peanut agglutinin-positive, and active in a pre-T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU-preT.     

In vitro colony assay for a new class of megakaryocyte precursor: colony-forming unit megakaryocyte (CFU-M).

A plasma culture system has been used successfully to grow and quantitate megakaryocyte colonies from mouse bone marrow following their staining for acetylcholinesterase activity in situ. Colonies averaging about six acetylcholinesterase-positive cells appear with a peak incidence after 4 days in culture with a plating efficiency of one colony formed for every 10(4) nucleated cells plated.     

Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells

Abstract. The kinetics of megakaryocyte formation from mouse bone marrow cells in semi-solid medium was studied directly in the culture dish by staining the cells for acetylcholinesterase after drying the cultures. A WEHI-3 cell-conditioned medium (WEHI-3 CM) was used as a general source of stimulus for megakaryocyte colony formation. The addition of peritoneal exudate supernatant as well as WEHI-3 CM increased the frequency of megakaryocyte colonies detected. Colonies containing acetylcholinesterase-positive cells were first detected at day 3. Maximum numbers of 25–40 megakaryocyte colonies per 10⁵ nucleaet mouse bone marrow cells were observed from days 7 to 11. The mean number of cells within each colony increased progressively with time of culture, and a modal range of 11–20 cells was obtained by day 7. Between 3 and 200 cells per colony were generally detected. A continuous distribution of the number of megakaryocytes per colony suggests that the clonable precursor cells are not synchronized either with respect to maturation stage or with respect to their capability to undergo nuclear endoreduplication. The addition of peritoneal exudate supernatant to the cell cultures increased the DNA levels of megakaryocytes grown in the presence of WEHI-3 CM but did not affect the number of cells per colony. The DNA content of colony megakaryocytes was measured after staining the cells with Feulgen reagent. A modal DNA value of 8 N was observed between days 4 and 7 for megakaryocytes stimulated with WEHI-3 CM. In the presence of both WEHI-3 CM and peritoneal exudate supernatant, the DNA content of megakaryocytes increased with the time of cell culture. Modal DNA values increased from 8 N at days 4 and 5, to 16 N by day 6. In these optimally stimulated cultures, 44% of colony megakaryocytes were 32 N or greater, a proportion higher than in normal bone marrow, but similar to that seen in the marrow of acutely thrombocytopenic animals. It is concluded that megakaryocytopoiesis in cell cultures is not a strictly controlled process with respect to cell division and endomitosis and that when certain culture conditions are employed, megakaryocyte development in vitro might reflect that seen in a stressed animal condition.     

Kinetic studies of pyruvate kinase duringin vitro differentiation of GM-CFC haemopoietic precursor and bone marrow cells in mice

Pyruvate kinase studies in the granulocyte-macrophage lineage duringin vitro differentiation have been performed using culture techniques on GM-CFC cells and a study has also been done in bone marrow cells.The enzyme exhibits biphasic behaviour with respect to both of its substrates in cells derived fromin vitro cultures at 5 and 7 days of incubation period. However in bone marrow cells these kinetics are only observed for ADP.The different kinetic behaviour of pyruvate kinase toward Fru-1,6-P₂, Ala, Phe and ATP in the three cellular populations allows us to conclude that the expression of pyruvate kinase is associated with the differentiation of these cells.Abbreviations GM-CFC granulocyte-macrophage colony forming cells - PK pyruvate kinase - CFU-E Colony Forming Units Erythroid - E_w Error weight - PEP phosphoenolpyruvate - Fru-1,6-P₂ fructose 1,6-bisphosphate - Ala L-alanine - Phe L-phenylanine - 5 GM granulocytemacrophage colonies obtained after 5 days incubation - 7 GM granulocyte-macrophage colonies obtained after 7 days incubation - h Hill coefficient - S_0,5 substrate concentration that yields half-maximal velocity     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Enhancement of rat ACT-1 tumor clonogenicity by xenogeneic mouse macrophages

Summary In vitro growth of rat atriocaval epithelial tumor cells (ACT-1) was enhanced by the inclusion of xenogeneic mouse adherent peritoneal exudate cells (PECs) in a two-layer soft agar system. A linear relationship was found between the number of cells plated and the number of colonies when ACT-1 tumor cells were plated at plating densities of between 1 and 5×10⁵ cell/60 mm plate (r=0.9,P<0.001). Inclusion of irradiated PECs in the bioassay for tumor stem cells resulted in a two and a half-fold increase in colony formation in three separate experiments (P<0.001). This work was supported by grants from the Cancer Research Trust, the University of Otago Cancer Research Fund and by the Medical Research Committee (Golden Kiwi).