Showing papers in "Stem Cells in 1983"
TL;DR: Results indicate that the KCL-22 cells were derived from chronic myelogenous leukemia cells, a cell line that possessed receptors for the Fc portion of IgG, but lacked lymphoid cell characteristics and the Epstein-Barr virus-associated nuclear antigen.
Abstract: A new hematopoietic cell line, designated KCL-22, was established in vitro by cultivation of pleural effusion cells obtained from a woman with chronic myelogenous leukemia in blast crisis. KCL-22 grew in suspension culture with a doubling time of 24 h and consisted of immature undifferentiated cells which were positive for periodic acid-Schiff and acid phosphatase staining. Chromosome analysis of the KCL-22 line showed a female karyotype with double Ph1 chromosomes and additional chromosome abnormalities. Its representative karyotype was 52,XX, + 1p-,+6,+8,+8,+8, t(9q+;22q-), +22q-. This cell line possessed receptors for the Fc portion of IgG, but lacked lymphoid cell characteristics and the Epstein-Barr virus-associated nuclear antigen. These results indicate that the KCL-22 cells were derived from chronic myelogenous leukemia cells. This cell line should prove useful for research involving various aspects of chronic myelogenous leukemia.
114 citations
TL;DR: A murine monoclonal antibody (anti-MY7) has been developed that detects an antigen expressed by 6% of normal human bone marrow cells, including approximately 40% of myeloid colony-forming cells (CFU-C).
Abstract: A murine monoclonal antibody (anti-MY7) has been developed that detects an antigen expressed by 6% of normal human bone marrow cells, including approximately 40% of myeloid colony-forming cells (CFU-C). The number of bone marrow cells and CFU-C expressing MY7 is significantly increased in regenerating bone marrow, but less than 5% of peripheral blood CFU-C express the MY7 antigen. Erythroid precursors are MY7 negative from peripheral blood and bone marrow. Thymidine suicide studies indicate that CFU-C in S-phase tend to be MY7 positive while CFU-C not in S-phase are MY7 negative. MY7 expression thus appears to identify a fraction of CFU-C that is actively proliferating.
89 citations
TL;DR: Findings may suggest that there are abnormalities in the pluripotent hemopoietic stem cells at least in some cases of PASA.
Abstract: In order to study the pathogenesis of primary acquired sideroblastic anemia (PASA), bone marrow and/or peripheral blood specimens obtained from four patients with PASA were cultured for erythroid colony-forming units (CFU-E), erythroid burst-forming units (BFU-E), and granulocyte/macrophage colony-forming units (GM-CFU). The number of CFU-E was markedly decreased in all four patients. CFU-E colonies consisted exclusively of normal-appearing erythroblasts, while ringed sideroblasts were observed only in scattered single erythroblasts or in small erythroblast aggregates. In one case, very few BFU-E colonies containing both normal-appearing erythroblasts and ringed sideroblasts were detected. In addition, the number of GM-CFU was significantly decreased in three out of the four cases. These findings may suggest that there are abnormalities in the pluripotent hemopoietic stem cells at least in some cases of PASA.
50 citations
TL;DR: This work has shown that there exists in the white adipose tissue, a population of stem cells which under appropriate conditions can differentiate and mature into adipocytes containing lipid inclusions.
Abstract: Our fundamental understanding of adipose tissue kinetics has, in recent years, been advanced by the finding that there exists in the white adipose tissue, a population of stem cells which under appropriate conditions can differentiate and mature into adipocytes containing lipid inclusions The evidence for the presence of this stem cell population is derived from both in vivo and in vitro studies
44 citations
TL;DR: The depressive effects of benzene inhalation on erythropoiesis may be compensated by the rapid proliferation of CFU-e, which is not ascribable to migration of stem cells between femur and spleen.
Abstract: Male B6D2F1 mice were exposed by inhalation to 4000 ppm of benzene for 8 h/day for 1, 2, 3, 5, 7 or 14 days and then sacrificed at 18 h following the last exposure. The cellularities of both femur and spleen were depressed for the duration of benzene exposure. BFU-e/femur declined for 3 days, rebounded to control levels by day 5, and were again depressed by day 7. CFU-e/femur were initially depressed, but by day 7, the concentrations of CFU-e were so elevated that the total number had actually rebounded to slightly higher than control values. CFU-gm/femur remained depressed for the duration of the exposure periods. CFU-e, BFU-e and CFU-gm/spleen were depressed following all exposures. The toxic effects of benzene on hematopoiesis that are immediate are not ascribable to migration of stem cells between femur and spleen. The depressive effects of benzene inhalation on erythropoiesis may be compensated by the rapid proliferation of CFU-e.
43 citations
TL;DR: The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.
Abstract: The factors controlling the growth of human tumor cells in soft agar are poorly understood. However, it has been demonstrated that serum provides factors which promote anchorage-independent growth. We tested 58 tumor specimens, which were obtained from patients with adenocarcinoma of the lung, colon, ovary or squamous cell carcinoma, for their ability to form colonies in soft agar in serum-free or serum-supplemented media. The cells were unable to replicate, and none of the hormones or growth factors tested: insulin (I), transferrin (T), selenium (S), estradiol (E), hydrocortisone (H) or epidermal growth factor (EGF) could substitute for serum. Examination of the serum dose-response curves indicated that growth factors reduced the serum concentrations needed to support anchorage-independent growth. The addition of the supplements and the lowering of serum concentrations increased cloning efficiencies (C.E.) in 38/51 trials, when cells were able to grow initially. The addition of ITS increased C.E. in 18/21 cases, HITES in 15/17 cases and EGF in 12/18 cases as compared to controls. ITS and HITES increased the number of colonies only when serum was the limiting factor. EGF, however, increased the number of colonies even when serum was not the limiting factor. The ability of the supplements to enhance growth could not be correlated to tumor type or initial cloning efficiencies. However, in only 1/25 cases were cells that were unable to form colonies under standard conditions induced to form colonies in the presence of the growth factors. Normal and tumor-derived human fibroblasts did not form colonies in soft agar in the presence of these growth factors. The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.
28 citations
TL;DR: The addition of the hypo-osmolar procedure to the standard methods for disaggregation increased the viable single cell yield in solid tumors and in malignant effusions and the increased total yield of clonogenic cells obtained with this procedure enhances the opportunity for experimental versatility and in vitro drug testing.
Abstract: A hypo-osmolar medium and tissue processing technique is described which is useful for disaggregation of residual human tumor cell clumps persisting after mechanical or enzymatic treatment of solid tumors and malignant effusions. The addition of the hypo-osmolar procedure to the standard methods for disaggregation increased the viable single cell yield in solid tumors by 47% and in malignant effusions by 67%. In 5 of the 26 solid tumor specimens tested in the human tumor stem cell assay, clonogenic single cells were obtained with the hypo-osmolar procedure, whereas no growth was observed using standard methods. Overall, the success rate for clonogenicity increased from 46% to 65% for the 26 solid tumors, with the major improvement occurring in ovarian cancer. Clonogenicity was obtained in 80% of malignant effusions both by standard methods and the hypo-osmolar techniques. The increased total yield of clonogenic cells obtained with this procedure enhances the opportunity for experimental versatility and in vitro drug testing.
24 citations
TL;DR: Mouse bone marrow, obtained from donors three days after treatment with 5-fluorouracil, had a very low ability to form macroscopic spleen colonies in irradiated mice at 10 days after transplantation of the cells (CFU-S10); such marrow also had no detectable erythropoiesis repopulating ability but did have near normal marrow repopulate ability and spleen megakaryocyte repopulated ability.
Abstract: Mouse bone marrow, obtained from donors three days after treatment with 5-fluorouracil, had a very low ability to form macroscopic spleen colonies in irradiated mice at 10 days after transplantation of the cells (CFU-S10); such marrow also had no detectable erythropoiesis repopulating ability but did have near normal marrow repopulating ability and spleen megakaryocyte repopulating ability. Incubation of this marrow in vitro for 7 days with medium containing growth factor preparations (a) pregnant mouse uterus extract plus human spleen conditioned medium or (b) mouse spleen conditioned medium, resulted in marked increases in CFU-S10 and in cells with erythropoietic repopulating ability together with maintenance of cells with marrow repopulating ability. These responses were not observed in cultures with control medium alone. Spleen megakaryocyte repopulating ability was also maintained in the presence of the factor preparations.
21 citations
TL;DR: Two methods for activating DTIC are explored so it may be used in vitro in a human tumor cloning system and the activation of DTIC by white light was found to be a viable alternative to utilizing a microsome plus cofactor system for bioactivation.
Abstract: Dacarbazine (DTIC) is an agent with clinical activity against human malignant melanoma. We have explored two methods for activating DTIC so it may be used in vitro in a human tumor cloning system. The activation of DTIC by white light was found to be a viable alternative to utilizing a microsome plus cofactor system for bioactivation. The microsome plus cofactor system itself actually caused some inhibition of tumor colony formation. Light activation of DTIC appears to be a reliable and simple method which allows testing of DTIC in an in vitro soft agar culture system.
21 citations
TL;DR: An increased proliferative rate of pluripotent hemopoietic progenitors is a consistent feature of CML patients and at least in some patients, different requirements for erythropoietin or PHA-LCM were observed when compared to normal culture conditions.
Abstract: Human hemopoietic pluripotent progenitors form multilineage colonies when cultured in methylcellulose with medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) and erythropoietin (EPO). We have examined their frequency, culture requirements and proliferative activity in 20 peripheral blood and 29 bone marrow specimens from patients with CML in chronic phase. Multilineage colonies developed under regular culture conditions in approximately 50% of all samples. The frequency ranged from 1-36 per 2 X 10(5) mononuclear cells of density less than 1.077 gm/ml. The requirements for PHA-LCM and EPO varied for patients with CML when compared to normal individuals; i.e., cells from some patients gave rise to mixed colonies with substantial erythroid components in the absence of PHA-LCM or without addition of the usually required EPO concentrations. The proliferative activity of CFU-GEMM was assessed using a short-term exposure to tritiated thymidine (3HTdR) prior to plating. The plating efficiency in all bone marrow and peripheral blood samples was reduced to 40-70% of the unexposed controls. In contrast, the plating efficiency after exposure to 3HTdR in normal individuals usually ranged from 70-90% of controls for bone marrow samples and from 85-100% of controls for peripheral blood samples. Thus, an increased proliferative rate of pluripotent hemopoietic progenitors is a consistent feature of CML patients. In addition, at least in some patients, different requirements for erythropoietin or PHA-LCM were observed when compared to normal culture conditions.
19 citations
TL;DR: A simple and reproducible micro-agar culture technique for cloning human CFU-M and cytochemical stainings and immunofluorescence tests with rabbit-antihuman factor VIII-related antigen were performed on the whole agar layers.
Abstract: A simple and reproducible micro-agar culture technique for cloning human CFU-M is described. Human bone marrow mononuclear cells were suspended in agar and incubated for 12 days. Stimulation was provided by the direct addition of phytohemagglutinin-P (PHA-P), erythropoietin (Epo) and 2-mercaptoethanol (2-ME) to the liquid overlayer. A shift from BFU-E and CFU-C proliferation to CFU-M and CFU-TL was observed with increasing PHA concentrations. Under optimal conditions (PHA 50 micrograms, Epo 1.2 IU, 2-ME 2 x 10(-4) M, 1% purified BSA, 0.04% human transferrin, saturated with Fe C13) a linear relationship between colonies formed and plated cell number were observed. For the routine morphological analysis, the whole agar layers were stained using the Pappenheim method. For further characterization of CFU-M, cytochemical stainings and immunofluorescence tests with rabbit-antihuman factor VIII-related antigen were performed on the whole agar layers.
TL;DR: Fibroblasts, fat cells, and macrophages appear to be necessary for the maintenance of human hemopoiesis in vitro, and fat cells may permit repopulation of marrow stroma by transplanted hemopOietic stem cells.
Abstract: The aims of the present study were to determine which stromal elements are important for the proliferation of human hemopoietic precursor cells in vitro and to develop a model for human bone marrow transplantation. First, we incubated bone marrow mononuclear cells in liquid culture under different conditions to obtain different proportions of fibroblasts, fat cells, and macrophages. We also looked for persistent hemopoiesis in association with these stromal cells. Second, we seeded nonadherent bone marrow mononuclear cells onto established stromal monolayers by incubating them together for 2 h and then washed off the unattached cells. The cells remaining on the monolayer were then stimulated by granulocyte-macrophage colony-stimulating activity (GM-CSA). We found that persistent hemopoiesis was maintained only in the presence of fibroblasts, fat cells, and macrophages. We also found that hemopoietic precursor cells attached to monolayers containing fibroblasts and fat cells, but not to monolayers containing fibroblasts or macrophages alone. Therefore, fibroblasts, fat cells, and macrophages appear to be necessary for the maintenance of human hemopoiesis in vitro, and fat cells may permit repopulation of marrow stroma by transplanted hemopoietic stem cells. This in vitro model might reflect features of human bone marrow transplantation in vivo.
TL;DR: The kinetics and pattern of hemopoietic recovery after supralethal total-body irradiation (TBI) were compared after transfusion of cryopreserved autografts derived from peripheral blood and bone marrow, and group A dogs showed a significant regeneratory advantage over group B dogs, in agreement with the hypothesis that there are differences in the relationship between CFU-GM content and hemopOietic potential of autografteds from different sources.
Abstract: The kinetics and pattern of hemopoietic recovery after supralethal total-body irradiation (TBI) were compared after transfusion of cryopreserved autografts derived from peripheral blood and bone marrow. Fractionated TBI was given in three doses of 6 Gy each at intervals of 48 h. Grafts of peripheral blood mononuclear cells (MNC) were collected by means of continuous-flow centrifugation and by using the mobilizing agent, dextran sulphate. Autografts were adjusted to contain equal numbers of committed progenitor cells (CFU-GM). Dogs grafted with blood-derived MNC (group A) and with MNC from bone marrow (group B) all received about 1 X 10(5) CFU-GM per kg body weight. In all dogs consistent hemopoietic engraftment was achieved. Comparing the pattern of regeneration of the granulocytes, group A dogs showed a significant regeneratory advantage over group B dogs, particularly during the first 20 days after transplantation. Lymphoid recovery was more rapid in group A until day 14. In both groups, blood lymphocytes remained below normal values beyond day 100. The regeneration patterns of the platelets and reticulocytes revealed no significant differences. These results are in agreement with the hypothesis that there are differences in the relationship between CFU-GM content and hemopoietic potential of autografts from different sources.
TL;DR: Human bone marrow cells were grown in a micro-agar culture system in the presence of human placenta and giant cell tumor conditioned media and both sources of colony stimulating activitites (CSA) employed had no significant difference in their ability to stimulate myelopoiesis.
Abstract: Human bone marrow cells were grown in a micro-agar culture system in the presence of human placenta (HPCM) and giant cell tumor conditioned media (GCT). The effects of HPCM and GCT conditioned media on linearity, growth dynamics, and morphological composition of colonies were studied after 7 and 14 days of incubation. Under the described conditions the dose-response curves for HPCM and GCT were different: on day 7 maximal stimulation was obtained with 2.5% HPCM and 20% GCT; on day 14 a maximal response was reached with 1.25% HPCM and 5% GCT. Both stimuli produced maximal growth after 7 days of incubation, followed by a rapid decrease in the number of formed colonies up to day 14. The morphological study of aggregates showed that after 7 days of incubation 80% pure granulocyte and 20% mixed granulocyte-macrophage colonies were found in the presence of both stimuli. However, on day 14 the incidence of granulocyte-macrophage colonies increased to 60%, whereas the percentage of pure granulocyte colonies decreased to 20%. The frequency of eosino-phil colonies was relatively low (median 15%) with both stimuli. The described system can be applied successfully for studies of myelopoiesis in vitro. Both sources of colony stimulating activities (CSA) employed had no significant difference in their ability to stimulate myelopoiesis.
TL;DR: The results may indicate that some CFU-MIX proliferate and differentiate along B-cell lineage to sIGM or cIgM bearing cells in vitro.
Abstract: Recent progress in clonal cell culture techniques makes it possible to detect pluripotent hemopoietic precursors from murine marrow cells. The precursors can proliferate, differentiate and form mixed colonies containing eryth-roblasts, granulocytes, macrophages and often megakaryocytes in viscid culture medium. In the present investigation, the presence of cells of B-cell lineage in mixed colonies was investigated.
Experiments on colonies containing clgM, clgG, slgM and slgG bearing cells using goat IgG fluorescein-conjugated anti-mouse IgM, goat F(ab′)2 fraction fluo-rescein-conjugated anti-mouse IgG and immunobeads revealed the presence of cytoplasmic IgM bearing cells in 47% of the colonies and surface IgM bearing cells in 74-84% of the colonies. Mixed colonies, however, did not contain either clgG bearing cells or slgG bearing cells. The results may indicate that some CFU-MIX proliferate and differentiate along B-cell lineage to slgM or clgM bearing cells in vitro.
TL;DR: Based on the quantity of CFU-Gm obtained, it appears feasible to use fetal liver cells as a source of progenitor cells for transplantation, and certain fetal tissues contain substantial numbers of hemopoietic progenitors.
Abstract: The CFU-Gm content of liver, spleen and bone marrow of human fetuses was determined from the 8th to 28th week of gestational age. The study of progenitor cells from 43 fetal livers revealed that the CFU-Gm increased from 8 to 12 weeks, was relatively stable through 16 weeks and thereafter declined by 22 to 24 weeks. The total number of liver CFU-Gm rose from 10(4) to greater than 10(6) CFU-Gm by the 16th week of gestation and declined to approximately 4 X 10(5) progenitor cells by 22 to 24 weeks. Peak values of bone marrow CFU-Gm were noted at 10 to 14 weeks of gestation. Generally, the numbers of these progenitor cells declined by 16 to 18 weeks; however, several high values were obtained at later times. Splenic CFU-Gm were relatively constant in number between 12 and 28 weeks of gestation with no correlation with fetal age. Thus, these studies indicate that certain fetal tissues contain substantial numbers of hemopoietic progenitor cells. Based on the quantity of CFU-Gm obtained, it appears feasible to use fetal liver cells as a source of progenitor cells for transplantation.
TL;DR: The growth kinetics of spleen colony forming units (CFU-S) in bone marrow transplanted into WBB6FI-W/WV mice in which one leg had been exposed to 10-30 Gy of X rays 4-5 months previously are characterized to reflect the highly localized nature of delayed radiation damage to the marrow microenvironment.
Abstract: Although the hematopoietic integrity of locally X-irradiated sites can be restored for a time even after fairly large doses, a secondary aplasia often occurs some months later. To gain further insight into this delayed effect within the framework of the stem cell regulatory domain hypothesis, we characterized the growth kinetics of spleen colony forming units (CFU-S) in WBB6FI-+/+ bone marrow transplanted into WBB6FI-W/WV mice in which one leg had been exposed to 10-30 Gy of X rays 4-5 months previously. Compared to unirradiated contralateral marrow, fewer CFU-S either reached the previously irradiated marrow or were seeded into sites that could support growth. The initial exponential growth of effectively seeded CFU-S was unchanged, but growth deceleration (inflection point) occurred at a lower level of CFU-S in marrow previously irradiated with 20-30 Gy. This change in the inflection point indicates a radiation dose-dependent decrease consistent with the decrease in bone marrow cellularity. The decrease in effective stem cell domains after 20 Gy was calculated to be about 35%. We interpret these results to reflect the highly localized nature of delayed radiation damage to the marrow microenvironment.
TL;DR: Results suggested that T cell subsets played an important role in the regulation of steady-state granulopoiesis through the stimulation of CSA production in peripheral blood mononuclear cells.
Abstract: The present study was undertaken to elucidate the role of T cell subsets in regulating the in vitro growth of human granulopoietic progenitor cells (CFU-C). Prior to CFU-C assay, mononuclear cells obtained from bone marrow and cord blood were depleted of T cells or functionally distinct T cell subsets by the method of complement-mediated cytolysis with the use of monoclonal antibodies, OKT3, OKT4 and OKT8. The depletion of any T cell subset of OKT3+, OKT4+ or OKT8+ cells from bone marrow and cord blood cells showed no significant alterations in the generation of CFU-C. The supplementation to the in vitro culture system of OKT4+ or OKT8+ cells, which had been negatively selected by complement-mediated cytolysis using the mutually exclusive monoclonal OKT8 or OKT4 antibody, respectively, did not alter the growth of CFU-C-derived colonies without mitogenic stimulation. In contrast, the production of colony-stimulating activity (CSA) in peripheral blood mononuclear cells was significantly reduced by removing not only OKT3+ cells but also OKT4+ or OKT8+ cell subsets. There were no significant differences in the degree of reduction between the different procedures. These results suggested that T cell subsets played an important role in the regulation of steady-state granulopoiesis through the stimulation of CSA production. The presence of a specific subset of T cells in CSA production was not demonstrated.
TL;DR: In mice without leukemia and mice with leukemic cells in only one organ, there was a general tendency to normal CFU-S numbers and T- and B-cell responses with time after BNU, although control levels were reached in only a few of the mice.
Abstract: T-cell leukemias have been induced in adult BDF1 mice by 12 or 15 weeks of exposure to butylnitrosourea (BNU) in the drinking water. This led to a depression of CFU-S numbers and reduced T- and B-cell responses to mitogens. These parameters were then studied during the BNU-free preleukemic latency period in individual mice. At the same time, leukemic cells were traced in the thymus, the spleen, and the bone marrow by transplantation. In mice without leukemia and mice with leukemic cells in only one organ, there was a general tendency to normal CFU-S numbers and T- and B-cell responses with time after BNU, although control levels were reached in only a few of the mice. The reaction of mixed lymphocyte cultures (MLC) remained low during the latency period. In the thymus an imbalance of the Con A, PHA, and MLC responses was observed. Out of 25 mice with induced leukemia, 8 had leukemic cells in the thymus only and 2 in the marrow only. In mice with leukemic cells in all 3 he-mopoietic organs and an enlargement of the spleen, a shift of CFU-S from the marrow to the spleen was observed.
TL;DR: Testicular cancer can be cloned directly in soft agar; a heterogeneous tumor cell population exists in metastatic testicular cancer specimens; and a dose response exists for VP-16 in relapsed testicularcancer which suggests that increasing the dose ofVP-16 may be clinically beneficial.
Abstract: Sixteen histologically documented testicular cancer specimens obtained at diagnostic procedures following induction chemotherapy with cis-plati-num containing regimens were cloned in soft agar. Seven (44%) of the specimens cultured formed colonies with a mean cloning efficiency of .021%. Colony formation was observed with all the common histologic subtypes of testicular cancer (seminoma, embryonal carcinoma, choriocarcinoma and mixed tumors).
In vitro drug sensitivity tests were performed using cis-platinum, vinblastine and VP-16. Three of four specimens demonstrated a decrease in colony formation to less than 50% of controls after a 1 h exposure to VP-16 at 300 μg/ml. Two of these patients had a response to treatment with a VP-16 based salvage regimen.
Immunoperoxidase staining of the colonies for alpha feto protein and human chorionic gonadotropin were correlated with the serum levels of these tumor markers determined at the time the specimen was obtained. In three instances the same markers were elevated in the serum as detected within cells which formed the colonies; however, in two other cases the marker(s) that was elevated in the serum was not expressed in the colonies. In one case a biopsy of a residual retro-peritoneal mass following chemotherapy histologically was a teratoma, but it formed colonies in the assay which stained positive for alpha feto protein. This patient subsequently developed an elevated serum alpha feto protein.
These studies have demonstrated that (a) testicular cancer can be cloned directly in soft agar; (b) a heterogeneous tumor cell population exists in meta-static testicular cancer specimens; and (c) a dose response exists for VP-16 in relapsed testicular cancer which suggests that increasing the dose of VP-16 may be clinically beneficial.
TL;DR: Fibroblastoid colonies produced in vitro by CFU-F of Ara-C-treated marrow were significantly larger than colonies from control marrow, and they markedly inhibited G/M colonies in split-phase agar cultures, by contrast, fibroblastoids produced by PHZ- treated marrow were of regular size and did not inhibit G-M colonies from test bone marrow.
Abstract: A study of murine adherent marrow cells (AMC) under conditions of high and low concentrations of hematopoietic stem cells and progenitors was carried out. In one group of weanling mice, decreased marrow cellularity and increased concentrations of CFU-S, CFU-D, and CFU-C were observed two days after administration of two consecutive intraperitoneal (i.p.) cytosine arabinoside (Ara-C) injections (2 × 200 mg/kg, at 6 h interval). Fibroblast colony-forming units (CFU-F) of this marrow were studied. In a second group of mice, which were given three i.p. phenylhydrazine (PHZ) injections (3 × 60 mg/kg on days 0, 1, and 3), CFU-S and CFU-C levels were unchanged or lowered 6 days after the start of PHZ administration. In these animals, however, the number of CFU-D was four times higher than in controls. The study of CFU-F in experiments of groups 1 and 2 indicated a high concentration of these progenitors in group 1 and lower concentration in group 2. Furthermore, fibroblastoid colonies produced in vitro by CFU-F of Ara-C-treated marrow were significantly larger than colonies from control marrow, and they markedly inhibited G/M colonies in split-phase agar cultures. By contrast, fibroblastoid colonies produced by PHZ-treated marrow were of regular size and did not inhibit G/M colonies from test bone marrow.
TL;DR: Morphologic analysis of colonies revealed that preferential inhibition of monocytic colony formation by PGE1, characteristic of normal bone marrow cultures, was not always observed in leukemia cell cultures.
Abstract: The effects of prostaglandin E1 (PGE1) on leukemic colony formation were investigated in seven cases of acute nonlymphocytic leukemia. In contrast to the almost constant results observed for normal myeloid colony formation, in patients there were apparent individual variations. Strong inhibition of colony formation by PGE1 was observed in three cases. In one case, enhancement rather than inhibition was observed. These in vitro inhibitory or stimulatory effects of PGEi were not related to the degree of the in vivo proliferation of leukemia cells. Morphologic analysis of colonies revealed that preferential inhibition of mono-cytic colony formation by PGEl, characteristic of normal bone marrow cultures, was not always observed in leukemia cell cultures. These abnormal responses to PGE1 suggest that the proliferation and differentiation of leukemic progenitor cells are regulated in a different way from normal hemopoiesis.
TL;DR: Human myeloid stem cell growth in agar was enhanced by the addition of human plasma, red-cell lysate, hemin, and beta-mercaptoethanol to the culture system and the number of colonies was increased due to enhancement by an average of 54%.
Abstract: Human myeloid stem cell growth in agar was enhanced by the addition of human plasma, red-cell lysate, hemin, and beta-mercaptoethanol to the culture system. Using conditioned media from a human T-cell or leukocyte-conditioned media as a source of colony-stimulating activity, the number of colonies was increased due to enhancement by an average of 54%. Analysis of colony types indicated that over 80% of the increase in colony number at eight days was due to granulocytic colony growth. Light density marrow cells cultured with enhancing agents produced colony levels of approximately 12-27 CFU-C/2 x 10(3) cells.
TL;DR: These studies document the ability of choline to influence hematopoiesis, and provide further evidence that cholinergic mechanisms may be important in the control of steady-state hematocerosis.
Abstract: We report here studies to further document the ability of cholinergic agonists to influence murine hematopoiesis. B6C3F1 mice were administered ultrapure choline (0.14, 1.4, and 7.0 mg/animal) i.p. for three days, then randomly sacrificed for assessment of hematological parameters. No significant change was observed in the packed red cell volume; however, the WBC was elevated significantly (85-400%) over noncholine administered controls during all days examined [1-12]. In vitro choline (10 mM) increased heterogeneous marrow-derived CFU-GM colony formation 210% above controls. Nonadherent marrow cell-derived CFU-GM produced a significant elevation in colony formation (36%), suggesting choline may influence directly a portion of the CFU-GM population. A twofold increase in the CFU-GM kill after 16 mM hydroxyurea exposure suggests choline can stimulate a subpopulation of CFU-GM not normally in cell cycle. In addition, choline (10 mM) was effective in stimulating CSF activity from marrow-derived conditioned medium. Dose-response studies demonstrated that choline was also stimulatory for megakaryocyte colony formation (CFU-Mk), 26% at 5 mM. Erythroid precursor stem cells, CFU-E/BFU-E, were reduced significantly (10-50%) at all choline concentrations tested (1-50 mM). These studies document the ability of choline to influence hematopoiesis, and provide further evidence that cholinergic mechanisms may be important in the control of steady-state hematopoiesis.
TL;DR: The studies show that short-term exposure of marrow cells to VDS causes a dose-dependent inhibition of in vitro colony formation; this inhibition is prevented by increasing doses of CSF in agar culture or by simultaneous preincubation with CSF.
Abstract: Vindesine (VDS) is a new vinca-alkaloid related to vinblastine and vincristine that blocks production of the microtubules in the mitotic phase of the cell cycle. Studies were undertaken to investigate the inhibitory effect of VDS on normal murine bone marrow cell proliferation and the possible interactions between this compound and L-cell derived colony stimulating factor (CSF). One X 10(7) murine bone marrow cells were exposed to various concentrations of VDS, ranging from 0.1 to 1.5 micrograms/ml for 1 h at 37 degrees C. Following this period, the cells were plated in agar in the presence of 100 units of CSF. A dose-dependent inhibition of colony formation was noted with increasing doses of the drugs. To determine whether an increased dose of CSF could overcome the inhibitory effect of VDS, further studies compared colony growth in response to 100 and 200 units of CSF. Virtually no inhibition of colony growth was detected in VDS-treated cells exposed to this higher dose of CSF while a dose-dependent reduction in CFU-C was noted with 100 units of CSF. Preincubation of cells with VDS and CSF prevented the inhibition that occurred with VDS alone. The addition of anti-CSF serum during the preincubation phase abolished the protective effect of CSF. The studies show that short-term exposure of marrow cells to VDS causes a dose-dependent inhibition of in vitro colony formation; this inhibition is prevented by increasing doses of CSF in agar culture or by simultaneous preincubation with CSF. The CSF action appears specific as its protective effect is neutralized by antibody to CSF, suggesting a potential role for CSF in preventing the antimitotic activity of VDS.
TL;DR: It is suggested that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.
Abstract: The effect of agar sterilized by either boiling or autoclaving on human melanoma colony formation in soft agar was compared using cells from 17 biopsies of metastatic malignant melanoma. The frequency of colony formation was significantly increased for cells grown in boiled agar in 8 samples (47%), unchanged in 8 samples (47%), and decreased in only one sample (6%). There were increases in both cluster and colony formation for the melanomas which had augmented colony formation when grown in boiled agar. There was also qualitative morphological improvement, including rounder, smoother cells and less extracellular debris surrounding the colonies. These data suggest that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.
TL;DR: The data emphasizing the importance of identifying colonies of all cell types in histological sections of the spleen and evaluating spleen sections at least at two levels, one adjacent to the surface and the other in the midzone area suggests a localized microenvironment conducive for megakaryocytopoiesis.
Abstract: This study is the first report on the utilization of specific cell function to identify splenic megakaryocytic colonies. Stem cell differentiation into megakaryocytes was studied by injecting each irradiated murine syngeneic recipient with 1 x 10(6) spleen cells. Morphological identification of erythroid, granulocytic, megakaryocytic, and mixed and undifferentiated colonies was done by staining consecutive cryostat sections with hemotoxylin and eosin, benzidine, myeloperoxidase, and acetylcholinesterase. The variation in the distribution of hemopoietic colonies within the spleen was reflected in the different ratio values derived for erythroid, granulocytic, and megakaryocytic colonies at varying depth within the spleen. An increase by 50% of megakaryocyte colonies was seen within the splenic pulp in the midzone region, compared with the surface. This suggests a localized microenvironment conducive for megakaryocytopoiesis. The data emphasizes the importance of identifying colonies of all cell types in histological sections of the spleen and evaluating spleen sections at least at two levels, one adjacent to the surface and the other in the midzone area.
TL;DR: It is demonstrated that the addition of 2-mercaptoethanol to rat bone marrow cultures enhances bone marrow fibroblast proliferation as evidenced by a 2-fold increase in the total number of fibro Blast colonies and a 5-fold rise in the number of these colonies which contain more than 80 cells.
Abstract: The definition of the function of bone marrow strornal cells in the regulation of hematopoiesis has been complicated by the limited growth of these cells in vitro. We have demonstrated that the addition of 2-mercaptoethanol to rat bone marrow cultures enhances bone marrow fibroblast proliferation as evidenced by a 2-fold increase in the total number of fibroblast colonies and a 5-fold increase in the number of these colonies which contain more than 80 cells. We also present evidence suggesting that enhancement of fibroblast growth may not be due to direct action of 2-mercaptoethanol, but may result from the activation of a serum component. The results from this study should facilitate further research into the function of bone marrow fibroblasts in the regulation of hema-topoietic cell differentiation.
TL;DR: The effects of intralesional chemotherapy with 7 different drugs on line 10 hepatoma grown in strain 2 guinea pigs were compared with the sensitivity of line 10 tumor cells in vitro, using a micro modification of the tumor stem assay with capillary tubes.
Abstract: The effects of intralesional chemotherapy with 7 different drugs on line 10 hepatoma grown in strain 2 guinea pigs were compared with the sensitivity of line 10 tumor cells in vitro, using a micro modification of the tumor stem cell assay with capillary tubes. A modified method was used to evaluate the in vitro dose-response curves. The correlation for in vivo/in vitro resistance was found to be 100% and for in vivo/in vitro sensitivity it was 80%.