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G Wagemaker

Publications and source records attributed to G Wagemaker.

At least 91 records · Page 5Linked to original sources
PubMed1986

A fractionation procedure of mouse bone marrow cells yielding exclusively pluripotent stem cells and committed progenitors.

The cell surface phenotype of pluripotent hemopoietic stem cells (CFU-S) and committed progenitors (CFU-C1, CFU-C2, BFU-E) of mouse bone marrow was analyzed with respect to their binding of wheat germ agglutinin (WGA) and two monoclonal antibodies, anti-GM-1.2 and anti-PGP-1. Stained cells were fractionated on the basis of differences in fluorescence and light scatter intensity using a light-activated cell sorter. The 6% of the cells that bound most WGA and that also had a relatively high forward light scatter (FLS) and low perpendicular light scatter (PLS) contained nearly all stem cells (CFU-S) and progenitors. Anti-GM-1.2 stained only mature myeloid cells, not CFU-S or the in vitro colony-forming cells. Anti-PGP-1 stained all bone marrow cells in varying intensities: lymphoid cells were dull, CFU-S were intermediate, CFU-C2 were brighter, and mature myeloid cells very bright. Enrichment of progenitor cells was performed by a two-step sorting procedure. First, the 6% most WGA-binding cells with high FLS and low PLS were sorted out. A 10-15-fold enrichment of progenitors and CFU-S was obtained. Next, these cells were restained with anti-GM-1.2 or anti-PGP-1 and again fractionated on the FACS. The GM-1.2-negative cells were then another four- to sevenfold more enriched for stem cells and progenitors. Of the cells in this fraction, 95% could be assigned to a colony-forming unit. With anti-PGP-1, CFU-C2 could be partly separated from more early cells such as CFU-S and BFU-E.

Animals↗
PubMed1986

Cure of murine thalassemia by bone marrow transplantation without eradication of endogenous stem cells.

alpha-Thalassemic heterozygous (Hbath/+) mice were used to investigate the possible selective advantage of transplanted normal (+/+) hemopoietic cells. Without conditioning by total-body irradiation (TBI), infusion of large numbers of normal bone marrow cells failed to correct the thalassemic peripheral blood phenotype. Since the recipients' stem cells are normal with respect to number and differentiation capacity, it was thought that the transplanted stem cells were not able to lodge, or that they were not stimulated to proliferate. Therefore, a nonlethal dose of TBI was given to temporarily reduce endogenous stem cell numbers and hemopoiesis. TBI doses of 2 or 3 Gy followed by infusion of normal bone marrow cells proved to be effective in replacing the thalassemic red cells by normal red cells, whereas a dose of 1 Gy was ineffective. It is concluded that cure of thalassemia by bone marrow transplantation does not necessarily require eradication of thalassemic stem cells. Consequently, the objectives of conditioning regimens for bone marrow transplantation of thalassemic patients (and possibly other nonmalignant hemopoietic disorders) should be reconsidered.

Animals↗
PubMed1986

Graft-versus-host disease following transplantation of 'one log' versus 'two log' T-lymphocyte-depleted bone marrow from HLA-identical donors.

Prevention of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation, requires the depletion of mature T-lymphocytes from bone marrow grafts. The optimal degree of T-cell reduction is still an open question. We compared two procedures of T-cell separation in 18 consecutive recipients of genotypically HLA-matched bone marrow, who also received cyclosporin A for 6 months. The first method (A) was based on a discontinuous albumin gradient fractionation and resulted in an average T-lymphocyte content of 50 X 10(5)/kg body weight (n = 9 patients); the second method (B) was based on E-rosette sedimentation and reduced the contamination to 15 X 10(4) grafted T-lymphocytes/kg body weight on the average (n = 9 patients). Thus, approximately 90 and 99% of the original T-lymphocytes were removed from the marrow grafts respectively. Of the seven patients of the first group who were at risk of GVHD (excluding two cases of early death), five developed a minimal-to-moderately severe acute GVHD and in two cases chronic GVHD ensued. Lethal GVHD was not seen. Of group B, all recipients engrafted and none developed GVHD (0/9). The difference in the frequency of GVHD between the two groups was highly significant (P less than 0.0025). These data confirm our preclinical studies. They demonstrate that a one-log T-lymphocyte reduction of the marrow inoculum, when combined with cyclosporin A prophylaxis after major histocompatibility complex (MHC)-matched transplantation, is still associated with a considerable incidence of GVHD, whereas a two-log reduction of T-lymphocytes may provide full protection against acute GVHD.

Adolescent↗
PubMed1986

Enumeration of stem cells and progenitor cells in alpha-thalassemic mice reveals lack of specific regulation of stem cell differentiation.

Heterozygous alpha-thalassemic (Hbath/+) female mice were investigated for the effect of persistent erythropoietic stress on the number of stem cells and progenitor cells along the the erythroid (E), granulocyte-macrophage (GM), and megakaryocyte (Meg) pathways. At the progenitor cell level, compensatory erythropoiesis was demonstrated in the spleen but not in the bone marrow. In the spleen, developmentally early progenitor cells (BFU-E) were expanded two- to threefold and late progenitor cells (CFU-E) five- to sixfold. A comparable expansion of progenitor cells was observed along the GM and Meg pathways. CFU-S numbers were increased in the spleen, but not in the bone marrow. The increases in GM and Meg progenitor cells appeared to result in an inappropriate hemopoiesis: peripheral thrombocyte and monocyte numbers were elevated. However, granulocyte numbers were not significantly increased. It is concluded that the persistently increased erythropoietic demand results in inappropriate production of other hemopoietic cells, most likely because pathway-specific regulatory mechanisms do not influence differentiation at the stem cell level.

Animals↗
PubMed1985

Failure to demonstrate pluripotential hemopoietic stem cells in mouse brains.

Hemopoietic stem cells as defined by the capacity to produce spleen colonies in lethally irradiated recipients were reported by P. F. Bartlett [(1982) Proc. Natl. Acad. Sci. USA 79, 2722-2725] to be present in high frequencies in mouse brain. He also reported similar numbers of colony-forming units, spleen (CFU-s), in the brains of Wf/Wf mice, the bone marrow of which lacks detectable spleen colony-forming cells. To verify these observations, single cell suspensions were produced from murine brains by incubation with trypsin and DNase, followed by removal of myelin by Percoll gradient centrifugation. Two to 13 CFU-s were detected per brain. This low number suggested contamination of the brains by either blood or bone marrow leaking from the skull bones during dissection. When the isolated, intact brains were washed carefully in balanced salt solution, the recovered number of CFU-s decreased to 0.1-0.4 per brain. No CFU-s could be detected in the brains of W/Wv mice. It is concluded that the CFU-s observed by Bartlett in preparations of mouse brain did not originate from the brain tissue.

Animals↗
PubMed1985

Biochemical fractionation of conditioned media containing factors which support in vitro proliferation of high proliferation potential colony forming cells.

Synergistic activity (SA), which is the capacity to support the in vitro proliferation of High Proliferation Potential Colony Forming Cells (HPP-CFC's), has been fractionated from conditioned media by standard biochemical techniques. On fractionation of both Wehi conditioned medium (Wehi CM) or human placental conditioned medium (HPCM) by several techniques, multiple peaks of SA were observed. Protein from HPCM expressing SA, ranged from 9,000-28,000 in molecular weight as determined by gel filtration. Chromatofocusing crude HPCM separated two activities possessing isoelectric points of 5.7 and 5.3. In crude Wehi CM, inhibitors or toxicity occasionally masked SA which partially adsorbed to Con-A sepharose and could be eluted with competing sugar. Further fractionation of this material on DEAE-sepharose separated three peaks of activity. Pooling the active fractions from the first two peaks gave a preparation that was up to 179 fold more active than the crude material.

Animals↗
PubMed1985

Reconstitution of the W/Wv stem cell differentiation defect by infection with Rauscher leukemia virus.

Hematopoietic stem cells of W/Wv mice failed to produce macroscopically visible hematopoietic spleen colonies in irradiated recipient mice. Infection of W/Wv mice of the spleen focus-forming virus-susceptible genotype Fv-2ss (DBA/2) or Fv-2rs (BD2F1) with Rauscher leukemia virus (RLV) restored the spleen colony-forming capacity of the stem cells. The resulting spleen colonies had normal size and cellularity; the frequency of and ratio between granulocyte-macrophage and erythroid progenitor cells were also normal, without excessive production of erythroid cells. The frequency of spleen colony-forming units (CFU-S) appeared to be strongly reduced in W/Wv mice. The seeding fraction of RLV-infected W/Wv stem cells in the recipient spleens did not differ from that of uninfected or RLV-infected +/+ stem cells. At equivalent numbers of CFU-S, spleen suspensions of RLV-infected W/Wv mice were equally effective as +/+ control suspensions in protecting irradiated mice from death due to bone marrow failure. Thus the number of CFU-S observed appeared to be predictive for the number of W/Wv cells required for effective radioprotection. In irradiated W/Wv mice that received transplants of RLV-infected W/Wv cells, circulating erythrocyte numbers approached those of control mice; the erythrocytes were of normal size, in contrast to the macrocytic red cells of untreated W/Wv mice. The reduced frequency of CFU-S in RLV-infected W/Wv mice can be readily explained by a reduced self-replicating capacity, attributable to the W/Wv genes, which was not reconstituted by infection with RLV. The data indicate a direct involvement of pluripotent stem cells upon infection with RLV.

Anemia, Macrocytic↗
PubMed1984

Transplantation of non-purified autologous bone marrow in patients with AML in first remission.

Four patients with acute myeloid leukemia (AML) were treated with high-dose cyclophosphamide and total body irradiation followed by reinfusion of a portion of their own bone marrow collected during remission. This procedure was applied when the patients were in complete remission. They did not receive further maintenance chemotherapy after grafting. The use of bone marrow for grafting that had been pre-exposed to high-dose chemotherapy for remission induction did not preclude good hematologic regeneration. All patients showed stable remissions that lasted for 64+, 21, 40+, and 19+ months, respectively. Death in the second patient was due to a medullary relapse of the leukemia. Autologous bone marrow transplantation in patients with AML in remission may permit lasting remissions, even when applied without additional chemotherapy and attempts to purify the marrow of neoplastic cells.

Adolescent↗
PubMed1984

Detection of murine bone marrow granulocyte/macrophage progenitor cells (GM-CFU) in serum-free cultures stimulated with purified M-CSF or GM-CSF.

Colony formation by mouse granulocyte/macrophage progenitors (GM-CFU) responding to purified colony-stimulating factors (CSF) in serum-free cultures is described. Analysis of the lipid requirements for colony growth stimulated by purified macrophage CSF (M-CSF) demonstrated that cholesterol is essential. Linoleic acid further promoted colony growth only if cholesterol was present, but phospholipid was inhibitory. More colonies were obtained in serum-free cultures, than in serum-supplemented controls. This difference could not be attributed to a change in the range of sensitivity to M-CSF. Stimulation of GM-CFU with granulocyte/macrophage CSF (GM-CSF) required further supplementation with hydrocortisone for optimal expression of colony-forming capacity in serum-free cultures. Hydrocortisone slightly inhibited colony growth stimulated with M-CSF. Under these culture conditions, the number of GM-CFU responding to GM-CSF was twice that obtained with M-CSF.

Animals↗
PubMed1981

Analysis of the cell cycle of late erythroid progenitor cells by sedimentation at unit gravity.

Approximately 70% of the late erythroid progenitor cells (E-CFU) which are present in normal bone marrow are killed by exposure to high specific activity tritiated thymidine (3H-TdR) in vitro or to hydroxyurea in vivo, indicating that a high proportion of these cells synthesize DNA. Their cell cycle was further analyzed by sedimentation at unit gravity. The modal sedimentation rate of 7.4 mm/h appeared to correspond to the fraction that is killed by 3H-TdR. Cells sedimenting at 6.0 and 9.5 mm/h were not susceptible to kill by 3H-TdR and correspond to cells in, respectively, G1 and G2/M. Based on a buoyant density of 1.077 g/cm3, the modal diameter of cells in G1 was calculated to be 8.3 micron, in S 9.2 micron and in G2/M 10.5 micron. The E-CFU population appeared to be composed of about 18% G1 cells, about 70% S phase cells and about 12% G2/M cells. The surviving fraction of E-CFU 2 h after intraperitoneal administration of 1 g/kg hydroxyurea was identified as being mainly a synchronous population in early S phase. The data are best explained by a short duration of G1 and G2/M phases.

Animals↗
PubMed1980

Erythropoietin-independent regeneration of erythroid progenitor cells following multiple injections of hydroxyurea.

It wa shown previously that colony formation in vitro by early erythroid progenitor cells (BFUe) requires sequential stimulation with a specific glycoprotein termed BFA and erythropoietin (EP). The action exerted by BFA was characterized as induction of proliferation in BFUe resulting after several cell divisions in EP-responsive progeny. The present study is directed at detection of EP-independent regulation of erythroid progenitor cells in vivo. Haemopoietic regeneration was induced by multiple administrations of hydroxyurea (HU). The femoral regeneration patterns of haemopoietic stem cells (CFUs), granulocyte/macrophage progenitor cells (CFUgm) and erythroid progenitor cells (BFUe, day 3 BFUe and CFUe) were studied in hypertransfused mice in comparison to nontransfused controls. The results show that (1) the phase of exponential regeneration of none of the cell populations studied is affected by hypertransfusion; (2) each of these cell populations exhibit a distinct regeneration pattern, indicating that they behave as separate functional entities; and (3) the three erythroid cell populations are suppressed by hypertransfusion in the post-exponential phase of regeneration in contrast to CFUs and CFUgm. The results support a two-regulator model of erythropoiesis.

Animals↗
PubMed1979

Structural identity of the pluripotential hemopoietic stem cell.

A review is presented of the experiments that resulted in the identification of a specific morphologic entity representing the pluripotential hemopoietic stem cell (HSC) in mouse bone marrow. This entity was subsequently discovered in concentrated HSC preparations from bone marrow of rats, monkeys, and humans. In the mouse, a set of physical parameters (of the HSC) has been collected which agree with its morphologic description. It was also shown that these physical properties, and a number of cell surface properties, do not enable a distinction between HSC and its immediate descendants, the G/M CFU 1 and the E-BFU. The factors that stimulate proliferation of these three cell types have been isolated from human leukocyte conditioned medium and mouse spleen conditioned medium and were partly purified and characterized. The information at present indicates that the three cell types respond to closely related, if not identical, factors. Direct counts of HSC in electron microscopic preparations of density gradient fractions of different enrichment have been compared with HSC values computed from spleen colony counts and f factors for rat and mouse marrow. A high degree of correlation was found between the two types of observations. The slopes of the regression lines for mouse marrow fractions, for concentrates of normal rat marrow, and for concentrates of cycling rat marrow were the same, namely, 0.5. The deviation of this value from the expected value of 1.0 is probably not due to the use of erroneous f values. It is proposed that the observed discrepancy may be due to heterogeneity of spleen colony forming cells, in that a proportion of them may not be pluripotential.

Animals↗
PubMed1979

Induction of erythropoietin responsiveness in vitro by a distinct population of bone marrow cells.

Bone marrow contains a small population of primitive erythroid progenitor cells which can be detected by their capacity to form large numbers of erythroid progeny in viscous cultures containing erythropoietin (EP). These cells have been termed erythroid 'burst-forming units' (BFUe). The present study demonstrates that expression of the erythroid differentiation potential of BFUe requires the presence of an activity additional to EP. This activity has been designated as BFA (burst feeder activity). It is shown that the number of BFUe detected and their apparent sensitivity to EP are directly related to the BFA concentration of the cultures. BFA was found to be associated with a population of bone marrow cells of high buoyant density and small volume, which are sensitive to irradiation. The radiation dose-effect curve provided strong evidence that bone marrow BFA is independent of cell proliferation; this was supported by showing that BFA is unaffected by in vivo treatment with hydroxyurea. The findings are compatible with a two-step regulation model for erythroid differentiation in which BFA-induced progeny of BFUe acquire sensitivity to EP.

Animals↗
PubMed1979

Particle-induced erythropoietin-independent effects of erythroid precursor cells in murine bone marrow.

A possible regulatory action of phagocytic cells on erythropoiesis was investigated by infusion of inert polystyrene latex particles (LAT). LAT appeared to induce changes in the femoral content of erythroid progenitor cells. These changes were most pronounced in primitive erythroid progenitor cells (BFUe) and appeared to be gradually damped in more differentiated populations (CFUe and erythroblasts). LAT did not influence granulocyte/macrophage progenitor cells (CFUc). The effects of LAT could not be attributed to changes in the systemic erythropoietin (EP) concentration. Administration of dexamethason nullified the effect of low doses of LAT, suggesting that phagocytosis of the particles is essential to the observed effects. Erythroid burst formation was previously found to be dependent on a bone marrow associated activity, termed BFA (burst feeder activity). BFA acts as an in vitro inducer of EP-responsiveness in BFUe. In this study it was found that LAT-induced changes in femoral erythroid progenitor cell content were characteristically preceded by corresponding changes in BFA. It was concluded that BFA-associated cells probably play a role in vivo in the early differentiation of erythroid progenitor cells. The present data are interpreted as direct in vivo evidence supporting a two-step regulatory model operating in erythropoiesis and provide evidence that phagocytic cells are a component of the erythroid haemopoietic inductive micro-environment.

Animals↗