Biomedical subjects

J F Eliason

Publications and source records attributed to J F Eliason.

At least 55 records · Page 3Linked to original sources

PubMed1985

Colony formation by primitive hemopoietic progenitor cells in serum-free medium.

A serum-free medium has been developed for clonal growth of murine hemopoietic progenitor cells. In this medium, the number of nonerythroid colonies induced by factors produced by cloned T lymphocytes was 90 +/- 10% of the number generated in serum-containing medium. Erythroid colony number in serum-free cultures containing the T-cell factors but no exogenous erythropoietin was significantly higher than that in cultures with serum, and the cloning efficiency was independent of cell concentration. Further addition of erythropoietin increased erythroid colony number approximately equal to 4-fold. Pure erythropoietin alone stimulated erythroid colony formation, but the cloning efficiency was highly dependent on cell concentration. Analysis of individual colonies generated in serum-free cultures containing the T-cell factors indicated that some contained cells of several hemopoietic lineages, demonstrating that multipotential progenitors can give rise to colonies in this system.

Animals↗

PubMed1985

Direct morphological and functional examination of murine pluripotent hemopoietic stem cells.

Pluripotent hemopoietic stem cells (PHSC) were isolated from adult mouse bone marrow by a combination of equilibrium density centrifugation and light-activated cell sorting for WGA-positive and H-2k antigen-positive cells. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 days and 6.6 colonies per 100 cells at 12 days after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony forming unit-spleen) equalled 135 (range, 90-230; n = 15). Enrichment for the cell type that provides radioprotection was equal to 180 +/- 70, indicating that PHSC and CFU-S are identical. Evidence is provided that the spleen seeding efficiency (the f-factor) of these cells was 0.10 and, therefore, that the average purity of the sorted PHSC was 65% (range in 15 experiments, 35-110%). The sorted cells were all in the G0 or G1 phase of the cycle. They appeared to be undifferentiated blasts by morphological criteria, the majority of the cells being similar in structure to the PHSC previously identified in less purified concentrates. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. Thirty percent of the sorted cells could be induced to form progeny in vitro. This procedure facilitates direct examination of the first events of hemopoietic regulation.

Animals↗

PubMed1985

Phenotypic and functional heterogeneity of human peripheral blood T lymphocytes producing colony stimulating factor. A clonal and precursor frequency analysis.

In this report we describe the precursor frequency and the subset distribution of peripheral blood human T cells producing lymphokine(s) acting on the proliferation and differentiation of bone marrow precursors of the granulocyte/macrophage series, as assessed in a liquid microculture assay. Because the sensitivity of this system was similar to that of the classic colony formation assay in semi-solid (methylcellulose) medium, it is likely that the lymphokine activity measured in this assay corresponds to the colony-stimulating factor (CSF) activity. Single human T lymphocytes, isolated from peripheral blood by E rosetting and Ficoll-Hypaque gradients, were seeded into microculture wells by limiting dilution or micromanipulation techniques and were incubated under culture conditions that allow clonal expansion of essentially all T cells. After 15 to 20 days, microcultures were stimulated with PHA and CSF activity was assayed in culture supernatants 24 hr thereafter. About 45% (1/2.3) of peripheral blood T cells were found to give rise to CSF-producing progenies. Moreover, when fluorescence-activated cell sorter-purified T4+ and T4- (or T8- and T8+) were analyzed, the frequency of the precursors of CSF-producing cells was 1/1.5 in the T4+ subset, whereas approximately one-third of the T8+ cells had this functional potential. To additionally characterize T cells responsible for CSF production, unfractionated T cells as well as T4+ and T4- cells were cloned by single cell micromanipulation. The resulting clones were analyzed simultaneously for the production of IL 2, production of CSF and for cytolytic activity in a lectin-dependent assay. It was found that 25/48 clones obtained from unfractionated T cells produced CSF, whereas 23/48 and 19/48 produced IL 2 or had cytolytic activity respectively. Six of the 25 CSF-producing clones had only this functional capability, whereas the remaining clones in addition displayed cytolytic activity (4/25), IL 2 production (10/25), or both (5/25). A similar functional heterogeneity was observed among T4+ and T4- clones, thus indicating that T cells producing CSF are functionally heterogeneous within both the T4+ and T8+ subsets.

Cell Differentiation↗

PubMed1984

Isolation of murine pluripotent hemopoietic stem cells.

A method described to purify pluripotent hemopoietic stem cells ( PHSC ) from adult mouse bone marrow. The method consists of three separation steps. First, bone marrow cells are centrifuged in a discontinuous metrizamide gradient and simultaneously labeled with wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC). Second, the low density cells are analyzed by a fluorescence-activated cell sorter (FACS) and the WGA-positive cells with medium forward and low perpendicular light scatter intensities are sorted. The WGA-FITC is removed from the cells by incubation with N-acetyl-D-glucosamine. Finally, the sorted cells are incubated with anti-H-2K-biotin and avidin-FITC and sorted a second time to enrich cells with high H-2K density. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 d and 6.6 colonies per 100 cells at 12 d after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony-forming unit/spleen) was 135 (range, 90--230; n = 15) and was similar to that for the cell type that provides radioprotection (180 +/- 70), indicating that these functional properties were copurified. Indirect evidence suggests that the spleen-seeding efficiency (f factor) of these cells is 0.10 and, therefore, the average purity of the sorted PHSC was 65% (range in 15 experiments, 35--110%). The sorted cells were all in the G1 or G0 phase of the cell cycle. They appeared to be undifferentiated blasts by morphological criteria. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. 32% of the sorted cells could be induced to form myeloid progeny in vitro. This procedure should be useful for direct studies on the regulation of hemopoietic cell differentiation.

Animals↗

PubMed1984

Improving techniques for clonogenic assays.

A serum-free medium has been developed which supports colony formation by cells from several human tumor cell lines, one colon adenocarcinoma (WiDr) and four melanoma (Me43, Me85, MP6, MeIuso). This medium consists of a 1:1 mixture of an enriched Dulbecco's modified Eagle's medium (EMED) and a modified Ham's F-12 nutrient mixture (FMED) supplemented with 0.9% methylcellulose, 1% bovine serum albumin, 80 micrograms/ml human transferrin, 3 micrograms/ml insulin, 2.8 micrograms/ml linoleic acid, 2.6 micrograms/ml cholesterol, 20 microM ethanolamine, and trace elements. Colony formation by WiDr cells is linear with the numbers of cells plated, having a plating efficiency (PE) of 34%, as compared to 26% in serum-containing medium. Two of the melanoma cell lines. MP6 and MeIuso, exhibit linear relationships between colony numbers and cell concentration with PEs of 21% and 70% respectively. Colony formation by the other two melanoma cell lines appears to be nonlinear. This work represents a step toward standardizing culture conditions for human tumor clonogenic cell assays.

Adenocarcinoma↗

PubMed1984

The role of prothymocytes in radiation-induced leukemogenesis in C57BL/Rij mice.

Following transplantation of bone marrow into recipient mice subjected to irradiation with four weekly fractions of 1.8 Gy, donor progenitor cells were found to compete with host elements for colonization of the thymus. The capacity for thymus repopulation of bone marrow cells from 4 X 1.8 Gy irradiated mice is decreased to a similar low level as that of regenerating bone marrow derived from lethally irradiated mice reconstituted with isogeneic marrow. It was calculated that the prothymocyte content of both types of regenerating marrow was less than 0.1% of that of normal marrow. Long-term cultures of C57BL bone marrow were found to contain the same numbers of prothymocytes per CFU-S as normal bone marrow, in contrast to cultured marrow from (C3H X AKR)F1 mice, that shows a striking decrease of prothymocytes. Normal bone marrow cells and long-term cultured bone marrow cells of C57BL mice were about equally effective on the basis of numbers of CFU-S injected in protecting 4 X 1.8 Gy irradiated syngeneic recipients from developing thymic lymphomas, while regenerating bone marrow was virtually nonprotective. These data are interpreted as supporting the notion that prothymocytes from the bone marrow play a crucial role in the prevention of radiation-induced thymic lymphomas.

Animals↗

PubMed1984

Long-term production of hemopoietic progenitors in cultures containing low levels of serum.

A new low-serum-containing medium is described for long-term murine bone marrow cell cultures. This medium consists of a 1:1 mixture of an enriched Dulbecco's modified Eagle's medium (EMED) and a modified Ham's nutrient mixture F-12 (FMED) supplemented with fetal calf serum (1% vol/vol), bovine serum albumin (1 mg/ml), human transferrin (78 micrograms/ml), linoleic acid (2.1 micrograms/ml), cholesterol (2.6 micrograms/ml), hydrocortisone (10(-5) M), and alpha-thioglycerol (7.5 X 10(-5) M). Hemopoietic progenitor cell production was maintained for at least nine weeks in cultures established with a single inoculum of marrow cells and fed weekly by removal of all nonadherent cells and growth medium. The kinetics of hemopoiesis in these cultures was highly reproducible, facilitating quantitative comparison between experimental groups. Coating the flasks with serum proteins enhanced adherent layer formation and increased progenitor cell production compared to cultures established in untreated flasks. Hemopoietic cell production was, in fact, higher in this medium than in the mixture of EMED and FMED supplemented with 20% serum.

Animals↗

PubMed1983

Haemopoietic colony formation (BFU-E, GM-CFC) during the development of pure red cell hypoplasia induced in the cat by feline leukaemia virus.

The GM-CFC assay for granulocyte-macrophage progenitors and the BFU-E and CFU-E assay for early and late erythroid progenitors from cat bone marrow were characterized. GM-CFC gave 59 +/- 4 to 118 +/- 6 colonies per 10(5) bone marrow cells using colony stimulating factors (CSF) from cat, mouse or human sources. The CFU-E and BFU-E assays gave 114 +/- 7 and 58 +/- 7 colonies respectively with optimum doses of erythropoietin. Irradiated cat bone marrow cells were good sources of CSF and of burst promoting activity for these assays. Kittens infected with feline leukaemia virus, subgroup C (FeLV-C), which induces pure red cell hypoplasia, showed the incidence of BFU-E decreased to 25-35% of controls as early as one week postinfection, and even lower values at later times. In contrast, the incidence of GM-CFC remained normal for several weeks. No evidence of inhibitory cells or of lack of stimulatory cells in the infected marrows was seen when they were cultured together with normal marrow in the BFU-E assay. Conversely, normal marrow cells were not able to restore BFU-E growth from infected marrow. This suggests a direct action of FeLV-C on early erythroid precursors. Infection with FeLV, subgroup A, which induces only a mild transitory anaemia, produces only a moderate decrease in the incidence of BFU-E.

Anemia, Aplastic↗

PubMed1983

In vivo studies on the regeneration kinetics of haemopoietic spleen colony-forming cells from long-term bone marrow cultures.

Production of murine haemopoietic progenitor cells, capable of forming colonies on the spleens of irradiated recipients (CFU-s), can be maintained in vitro for up to several months. We have examined the properties of the suspension (SUSP) and adherent (ADH) CFU-s from these long-term marrow cell cultures in the spleen colony assay system. The numbers of colonies formed was linear with the numbers of SUSP or ADH cells injected, although the concentrations were only 20-25% of that found in normal bone marrow (NBM). The seeding efficiencies for SUSP and ADH CFU-s, measured in spleen and femur 24 hr after transplantation, were not significantly different from those for CFU-s from NBM. The results demonstrate that the CFU-s assay can give valid estimates of colony-forming cell numbers in the cultured cell populations. We therefore used the CFU-s assay to study the regeneration of SUSP, ADH and NBM CFU-s in spleen and femur at various times after transplantation. The growth of the cultured cells appeared to be delayed by 36-48 hr compared to the regeneration of CFU-s from NBM. Thereafter, ADH CFU-s grew at the same rate as NBM CFU-s, whereas the rate of SUSP CFU-s regeneration was slightly, but not significantly, slower. This similarity in growth rates indicates that the CFU-s from long-term marrow cultures have similar self-renewal probabilities to normal CFU-s.

Animals↗

PubMed1983

Erythropoietin-dependent and erythropoietin-independent enhancement of colony formation by immature erythroid progenitors (BFUe).

The erythropoietin (epo)-dependent burst forming activities (BFA) of foetal calf serum (FCS), mouse bone marrow cells (BFA-cell) and lectin-stimulated mouse spleen conditioned medium (BFA-mscm) were investigated. Burst enhancement by BFA-mscm was independent of FCS concentration above 5% FCS. The activity of BFA-cell was directly proportional to FCS concentration. There was a high correlation for the relationship between the logarithm of total cell concentration and the logarithm of burst number for each concentration of FCS. Burst formation in the presence of BFA-cell showed a high epo requirement whereas plateau numbers of bursts were evident at concentrations of 1 to 2 units of epo/ml in cultures containing BFA-mscm. The epo-independent developmental activities (EIDA) of BFA-cell and BFA-mscm were examined in methylcellulose cultures to which the addition of epo was delayed for up to four days. Under all conditions examined, the number of bursts formed in the presence of BFA-cell declined with increasing delay of epo addition. Studies on the kinetics of haemoglobin synthesis in such experiments demonstrated that this decline in colony number was associated with a delay in haemoglobin synthesis. Burst formation in cultures containing various levels of BFA-mscm was independent of the time of epo addition. These results indicate that the burst enhancing ability of BFA-mscm is directly related to its EIDA whereas bone marrow cells appear to lack this activity.

Animals↗

PubMed1983

Studies on the role of serum in long-term bone marrow cell cultures.

The effect of fetal calf serum (FCS) concentration on hemopoiesis in long-term bone marrow cell cultures (supplemented with 10(-6) M hydrocortisone) was investigated. The duration of hemopoiesis was directly related to the concentration of serum in the cultures. At each concentration, marked differences were seen between different batches of FCS. The longevity of cultures established with a suboptimum concentration of serum was increased by the addition of HEPES buffer. Removal of all non-adherent cells and growth medium for the weekly feedings increased the overall production of hemopoietic cells when compared to the standard feeding procedure of replacing one-half of the growth medium.

Animals↗

PubMed1983

In vivo studies on the regeneration kinetics of enriched populations of haemopoietic spleen colony-forming cells from normal bone marrow.

Functional properties of mouse haemopoietic spleen colony-forming cells, enriched 40- to 80-fold, from normal bone marrow were studied. It was found that: (1) the number of partially purified CFU-s (colony forming unit-spleen) required to rescue lethally irradiated mice was similar to the number of normal unfractionated bone marrow CFU-s giving the same level of protection; (2) the homing of partially purified CFU-s was similar to that of CFU-s from unfractionated bone marrow; (3) the regeneration of CFU-s in spleen was similar for enriched and unfractionated cell populations between 4 and 11 days after transplantation. In contrast, the rate of regeneration of CFU-s in femur was slower with enriched progenitor cells than with unfractionated bone marrow. The growth rate in femur, however, could be restored to normal by injecting freshly isolated syngeneic thymocytes with the enriched CFU-s population. The results indicate that the partially purified CFU-s are by themselves functionally normal and show that the rate of CFU-s repopulation in bone marrow can be affected by cell types other than spleen colony-forming cells.

Animals↗

PubMed1982

Quantification of prothymocytes in long-term bone marrow cultures.

Prothymocytes were present in long-term marrow cell cultures for 17 weeks, although at very low levels. The prothymocytes from the cultures appeared normal in their thymus repopulation ability since the growth curves for thymocytes derived from cultured cells, measured in thymuses of irradiated mice, were parallel to those for thymocytes derived from fresh bone marrow prothymocytes. The buoyant density distribution for prothymocytes from the cultures appeared normal as well, with a modal density of 1,069 g/cm3. The prothymocyte density profile was similar to that for CFUS from the cultures which had a modal density of 1.070 g/cm3. Prothymocytes were lost from culture before CFUS and a decreased ratio of prothymocytes to CFUS was evident within the first 24 hours of culture. The kinetic differences between prothymocytes and CFUS in vitro suggest that these two functional assays detect different cells.

Animals↗

PubMed1982

The regulation of hemopoiesis in long-term bone marrow cultures. III. The role of burst forming activity.

The addition of burst forming activity (BFA) from mouse spleen conditioned medium (MSCM) to serum-free long-term cultures induced the formation of day 4 BFUE demonstrating that MSCM has an erythropoietin-independent activity on erythroid development. Conditioned medium (CM) from long-term mouse marrow cell cultures was inhibitory to burst formation at concentrations as low as 0.01% when bone marrow cells were used as the external source of BFA, whereas significant levels of inhibition were apparent only at 100-fold greater concentrations of CM in cultures containing MSCM. CM did not inhibit erythropoietin-stimulated erythropoiesis in liquid subcultures of long-term culture cells nor did it stimulate erythroid maturation in the absence of erythropoietin. Adherent cells from the long-term cultures, used as feeder cells, were more active than normal bone marrow cells in enhancing erythroid burst formation.

Animals↗

PubMed1980

Evidence for cellular cooperativity in hemoglobin synthesis by erythroid bursts.

We have studied hemoglobin synthesis associated with erythroid bursts under various conditions. The slope of the curve relating log cell number to log hemoglobin synthesis is consistently greater than the slope of the curve relating log cell number to log bursts, suggesting that cellular interactions may be involved in maximal hemoglobin synthesis by the cells comprising the bursts. This suggestion if strengthened by experiments which show that gentle disruption of burst structure is accompanied by a decline in hemoglobin synthesis.

Animals↗

PubMed1980

Role of purified erythropoietin in the amplification of the erythroid compartment.

A single dose of Myleran induced a prolonged depletion of multi-potential haemopoietic cells and of early erythroid precursors to levels between 2 and 6 of control. In these mice, a priming dose of highly purified erythropoietin (Epo) or of any of 3 Epo preparations from different sources, and having different specific activities, increased the magnitude of the response to a test dose of Epo by erythropoietin responsive cells (ERC) in the hypertransfused mouse assay. As significant feeding into the ERC compartment from the most immature cells is unlikely because of the depletion induced by the Myleran, it may be concluded that highly purified Epo, not contaminated with other biologically active molecules, has a dual effect on the ERC: it causes increased amplification besides inducing differentiation into cells with the capacity to synthesize haemoglobin.

Animals↗

PubMed1979

Erythropoietin-stimulated erythropoiesis in long-term bone marrow culture.

The proliferation of multipotential haematopoietic stem cells (CFU-S) is possible in some long-term bone marrow cultures. Granulocyte and macrophage progenitor (CFU-C) and megakaryocyte precursor cells (CFU-M) are present in these cultures and undergo full development into mature cells. In contrast, while immature erythroid progenitors ('early' BFU-E) are maintained in long-term culture, none of the more differentiated progeny (CFU-E) have been detected, and no morphologically recognisable erythroid cells have been observed. We now describe a modified culture system in which the 'early' BFU-E develop into 'late' BFU-E in response to added erythropoietin. Further maturation of these cells into CFU-E and non-nucleated erythrocytes can be achieved by mechanical agitation of the long-term cultures or by transferring the cells into dishes which do not allow cell attachment to occur.

Animals↗

PubMed1979

The relationship of hemoglobin synthesis to erythroid colony and burst formation.

We have demonstrated that the cyclohexanone method for the extraction of hematin can be used to measure hemoglobin synthesis induced by erythropoietin (epo) in mouse bone marrow cells cultured in medium containing methyl cellulose. The time course of hemoglobin synthesis by mouse marrow cells showed two effects due to epo: an increase in hemoglobin synthesis at day 2, which corresponded to the formation of small erythroid colonies resulting from the CFU-E (colony-forming unit, erythroid), and a very large increase in hemoglobin synthesis, which was maximal at days 7-8 and corresponded to the formation of large erythroid colonies (bursts) resulting from the BFU-E (burst-forming unit, erythroid). The epo dose-response curves for CFU-E colony counts and day-2 hemoglobin synthesis were similar, and the cell-number-response curves for these two paramaters were parallel. The epo dose-response curve for BFU-E colony counts reached a plateau at an epo concentration between 3 and 5 units/ml, whereas the dose-response curve for 6-8-day hemoglobin synthesis did not reach a plateau even at an epo dose of 10 units/ml.

Animals↗