scholarly journals Induction of human granulocyte differentiation in vitro by ubiquitin and thymopoietin

Blood ◽  
1977 ◽  
Vol 50 (2) ◽  
pp. 275-288 ◽  
Author(s):  
WA Kagan ◽  
GJ O'Neill ◽  
GS Incefy ◽  
G Goldstein ◽  
RA Good
Keyword(s):  
Bone Marrow Cells ◽  
Functional Differentiation ◽  
Velocity Sedimentation ◽  
Differentiation Markers ◽  
Band Form ◽  
Immature Granulocyte ◽  
Differentiation Marker ◽  
Cell Fractions ◽  
Human And Mouse

Abstract Human bone marrow cells were separated according to density by centrifugation on Ficoll-Hypaque gradients and then according to size by velocity sedimentation. This procedure resulted in fractions enriched for immature granulocytes, mature granulocytes, and lymphocytes. Cells in these fractions were analyzed for their expression of certain surface and functional differentiation markers and for their ability to respond to thymopoietin and ubiquitin with the expression of additional differentiation markers. A higher percentage of band form and segmented granulocytes than of more immature granulocytes expressed complement receptors on their surfaces. Thymopoietin and ubiquitin induced a significant percentage of the cells in the immature granulocyte fraction to express this marker. These data suggested that the complement receptor may be viewed as a differentiation marker on human granulocytes, the expression of which can be induced in vitro by thymopoietin and ubiquitin. Furthermore, fractions containing predominantly band form granulocytes were induced by ubiquitin (but not thymopoietin) to develop the capacity to respond to chemotactic agents, and cell fractions containing predominantly myelocytes and metamyelocytes were induced by thymopoietin and ubiquitin to develop the capacity to phagocytose latex particles. These findings indicated that thymopoietin and ubiquitin, two agents known to induce a number of stages of human and mouse lymphocyte differentiation, are also capable of inducing some stages of human granulocyte differentiation in vitro.

scholarly journals Induction of human granulocyte differentiation in vitro by ubiquitin and thymopoietin

Blood ◽  
1977 ◽  
Vol 50 (2) ◽  
pp. 275-288
Author(s):  
WA Kagan ◽  
GJ O'Neill ◽  
GS Incefy ◽  
G Goldstein ◽  
RA Good
Keyword(s):  
Bone Marrow Cells ◽  
Functional Differentiation ◽  
Velocity Sedimentation ◽  
Differentiation Markers ◽  
Band Form ◽  
Immature Granulocyte ◽  
Differentiation Marker ◽  
Cell Fractions ◽  
Human And Mouse

Human bone marrow cells were separated according to density by centrifugation on Ficoll-Hypaque gradients and then according to size by velocity sedimentation. This procedure resulted in fractions enriched for immature granulocytes, mature granulocytes, and lymphocytes. Cells in these fractions were analyzed for their expression of certain surface and functional differentiation markers and for their ability to respond to thymopoietin and ubiquitin with the expression of additional differentiation markers. A higher percentage of band form and segmented granulocytes than of more immature granulocytes expressed complement receptors on their surfaces. Thymopoietin and ubiquitin induced a significant percentage of the cells in the immature granulocyte fraction to express this marker. These data suggested that the complement receptor may be viewed as a differentiation marker on human granulocytes, the expression of which can be induced in vitro by thymopoietin and ubiquitin. Furthermore, fractions containing predominantly band form granulocytes were induced by ubiquitin (but not thymopoietin) to develop the capacity to respond to chemotactic agents, and cell fractions containing predominantly myelocytes and metamyelocytes were induced by thymopoietin and ubiquitin to develop the capacity to phagocytose latex particles. These findings indicated that thymopoietin and ubiquitin, two agents known to induce a number of stages of human and mouse lymphocyte differentiation, are also capable of inducing some stages of human granulocyte differentiation in vitro.

scholarly journals GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO

1974 ◽  
Vol 140 (6) ◽  
pp. 1511-1521 ◽  
Author(s):  
H. Robson MacDonald ◽  
Jean-Charles Cerottini ◽  
K. Theodor Brunner
Keyword(s):  
T Lymphocytes ◽  
Physical Properties ◽  
Cell Lineage ◽  
Kinetic Studies ◽  
Large Cell ◽  
Velocity Sedimentation ◽  
Cell Fractionation ◽  
Anamnestic Response ◽  
Cell Fractions

Separation of cells by velocity sedimentation at unit gravity was utilized to investigate the physical properties of cytotoxic thymus-derived lymphocytes (CTL) generated in long-term mixed leukocyte cultures (MLC). In kinetic studies, CTL were found almost exclusively in the large cell fractions at the peak of the response on day 4, whereas the majority of CTL in day 14 MLC had the sedimentation properties of small lymphocytes. Reculture until day 14 of cells fractionated on the basis of size on day 4 indicated that the small CTL were derived exclusively from cells which had been large on day 4. Re-exposure of day 14 MLC cells to the original stimulating alloantigens resulted in significant cell proliferation and rapid regeneration of CTL activity. Cell fractionation experiments demonstrated that the cells in the day 14 MLC population which responded to the secondary allogeneic stimulus were small T lymphocytes, and that these cells rapidly developed into large, highly cytotoxic CTL following stimulation. Moreover, by restimulating on day 14 fractions which were selected on the basis of size on day 4, it was found that the responding small lymphocytes were themselves the progeny of cells which were large at the peak of the response. Since CTL and CTL progenitors showed concomitant changes in physical properties with time, the possibility exists that they belong to the same cell lineage, and hence that CTL can differentiate into cells which are no longer cytotoxic, but capable of mounting an anamnestic response.

Involvement of suppressors of cytokine signaling in toll-like receptor–mediated block of dendritic cell differentiation

Blood ◽  
2006 ◽  
Vol 108 (13) ◽  
pp. 4102-4108 ◽  
Author(s):  
Holger Bartz ◽  
Nicole M. Avalos ◽  
Andrea Baetz ◽  
Klaus Heeg ◽  
Alexander H. Dalpke
Keyword(s):  
Bone Marrow Cells ◽  
Functional Differentiation ◽  
Precursor Cells ◽  
Cytokine Signaling ◽  
Inhibitory Effects ◽  
Gm Csf ◽  
Dendritic Cell Differentiation ◽  
Maturation Stages ◽  
Macrophage Colony

Abstract Dendritic cells (DCs) are important sentinels within innate immunity, monitoring the presence of infectious microorganisms. They operate in 2 different maturation stages, with transition from immature to mature DCs being induced by activation of toll-like receptors (TLRs). However, TLRs are also expressed on precursor cells of DCs. Here we analyzed the effects of TLR stimulation during the process of granulocyte-macrophage–colony-stimulating factor (GM-CSF)–mediated in vitro generation of immature DCs from precursor cells. We show that TLR triggering deviated phenotypic and functional differentiation from CD14+ monocytes to CD1a+ DCs. Similar results were obtained when differentiation of murine myeloid DCs from bone marrow cells was analyzed. The inhibitory effects were independent of soluble factors. TLR stimulation in DC precursor cells induced proteins of the suppressor of cytokine signaling family (SOCS), which correlated with loss of sensitivity to GM-CSF. Overexpression of SOCS-1 abolished GM-CSF signal transduction. Moreover, forced SOCS-1 expression in DC precursors mimicked the inhibitory effects on DC generation observed for TLR stimulation. The results indicate that TLR stimulation during the period of DC generation interferes with and deviates DC differentiation and that these effects are mediated particularly by SOCS-1.

scholarly journals Productive infection by B19 parvovirus of human erythroid bone marrow cells in vitro

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 384-391 ◽  
Author(s):  
K Ozawa ◽  
G Kurtzman ◽  
N Young
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Productive Infection ◽  
Similar Proportion ◽  
Erythroid Cells ◽  
B19 Parvovirus ◽  
Transient Aplastic Crisis ◽  
Cell Fractions ◽  
Marrow Cells

Abstract B19 parvovirus, the cause of fifth disease and transient aplastic crisis, has been successfully propagated in suspension cultures of human erythroid bone marrow cells obtained from patients with sickle cell disease and stimulated by erythropoietin. B19 inoculation in vitro resulted in a marked decline in identifiable erythroid cells over seven to nine days of incubation. Characteristic giant early erythroid cells were seen on Wright's-Giemsa stain of infected cultures. By in situ hybridization, 30% to 40% of erythroblasts were infected at 48 hours; a similar proportion of cells showed B19 capsid protein by immunofluorescence. B19 DNA was present in erythroblasts but not in the leukocyte fraction of bone marrow. B19 replication, as determined by Southern analysis, and B19 encapsidation, as determined by sensitivity of isolated cell fractions to DNase I, were restricted to the nuclei. B19 DNA was detectable in the nuclei from infected cultures beginning at 18 hours and in the supernatant at 32 hours; B19 genome copy number was estimated at about 25,000 to 30,000/infected cell at 48 hours. Recovery of virus depended on the multiplicity of infection (moi); at low moi, approximately 200x input virus was recovered from total cultures and 50x from the culture supernatants. Virus released into the supernatant was as infectious or more infectious than virus obtained from sera of infected patients. Human erythroid bone marrow culture represents a safe in vitro system for the elucidation of the cellular and molecular biology of the pathogenic B19 parvovirus.

scholarly journals Separation of haemopoietic cells for biochemical investigation. Preparation of erythroid and myeloid cells from human and laboratory-animal bone marrow and the separation of erythroblasts according to their state of maturation

1981 ◽  
Vol 194 (3) ◽  
pp. 789-796 ◽  
Author(s):  
F L Harrison ◽  
T M Beswick ◽  
C J Chesterton
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Velocity Sedimentation ◽  
Laboratory Animals ◽  
Adhesive Molecule ◽  
Blast Cells ◽  
Unit Gravity Sedimentation ◽  
Haemopoietic Cells ◽  
Marrow Cells

The separation of haemopoietic bone-marrow cells by centrifugation through discontinuous density gradients of Percoll is described. This method was used to prepare fractions enriched in erythroblasts, myeloid blast cells or reticulocytes from bone marrow of anaemic and non-anaemic rabbits, from the marrow of other anaemic laboratory animals and from human samples. It is a simple, rapid, reproducible and inexpensive technique that can be readily adapted to suit individual requirements. Secondly, a convenient method is presented for the separation of large quantities of bone-marrow cells into fractions enriched in erythroblasts at different stages of maturation, by velocity sedimentation through a linear gradient of 1-2% sucrose at unit gravity. In vitro, erythroblasts adhere together strongly via a mechanism almost certainly involving a beta-galactoside-specific surface lectin termed erythroid developmental agglutinin. Since the efficiency of cell-separation techniques depends heavily on the maintenance of a single cell suspension in which each unit can move independently, the presence of an adhesive molecule at the cell surface is of considerable significance. The effect of washing the marrow with a lactose-containing medium, which has been shown to remove the agglutinin, was therefore investigated in relation to both methods. The separation on Percoll gradients is considerably enhanced by this treatment. In addition, the unit-gravity sedimentation gradient can be loaded with 5-10 times more cells after lactose extraction in comparison with intact marrow. Although enrichment is less, a useful fractionation according to maturation is still obtained.

scholarly journals Enrichment of spinal cord cell cultures with motoneurons

1978 ◽  
Vol 77 (1) ◽  
pp. 83-98 ◽  
Author(s):  
DK Berg ◽  
GD Fischbach
Keyword(s):  
Spinal Cord ◽  
Cell Cultures ◽  
Cholinergic Neurons ◽  
Velocity Sedimentation ◽  
Spinal Cord Neurons ◽  
Initial Suspension ◽  
Spinal Cord Cell ◽  
Muscle Cultures ◽  
Cell Fractions

Spinal cord cell cultures contain several types of neurons. Two methods are described for enriching such cultures with motoneurons (defined here simply as cholinergic cells that are capable of innervating muscle). In the first method, 7-day embryonic chick spinal cord neurons were separated according to size by 1 g velocity sedimentation. It is assumed that cholinergic motoneurons are among the largest cells present at this stage. The spinal cords were dissociated vigorously so that 95-98% of the cells in the initial suspension were isolated from one another. Cells in leading fractions (large cell fractions: LCFs) contain about seven times as much choline acetyltransferase (CAT) activity per unit cytoplasm as do cells in trailing fractions (small cell fractions: SCFs). Muscle cultures seeded with LCFs develop 10-70 times as much CAT as cultures seeded with SCFs and six times as much CAT as cultures seeded with control (unfractionated) spinal cord cells. More than 20% of the large neurons in LCF-muscle cultures innervate nearby myotubes. In the second method, neurons were gently dissociated from 4-day embryonic spinal cords and maintained in vitro. This approach is based on earlier observations that cholinergic neurons are among the first cells to withdraw form the mitotic cycle in the developing chick embryo (Hamburger, V. 1948. J. Comp. Neurol. 88:221-283; and Levi-Montalcini, R. 1950. J. Morphol. 86:253-283). 4-Day spinal cord-muscle cultures develop three times as much CAT as do 7-day spinal cord-muscle plates, prepared in the same (gentle) manner. More than 50% of the relatively large 4-day neurons innervate nearby myotubes. Thus, both methods are useful first steps toward the complete isolation of motoneurons. Both methods should facilitate study of the development of cholinergic neurons and of nerve-muscle synapse formation.

scholarly journals Culture of isolated bovine megakaryocytes on reconstituted basement membrane matrix leads to proplatelet process formation

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 912-924 ◽  
Author(s):  
KS Topp ◽  
F Tablin ◽  
J Levin
Keyword(s):  
Basement Membrane ◽  
Bone Marrow Cells ◽  
Human Platelets ◽  
Velocity Sedimentation ◽  
Thin Filaments ◽  
Membrane Matrix ◽  
Process Formation ◽  
Vitro Model

Abstract We have enriched for bovine megakaryocytes and identified a culture system that may provide an in vitro model for platelet formation. Mature megakaryocytes with an unusually high ploidy distribution were obtained after differential centrifugation and velocity sedimentation of bone marrow cells through gradients of bovine serum albumin (BSA). The cell membranes of isolated megakaryocytes and megakaryocytes in vivo stained with antisera to human platelets and human platelet membrane GPIIIa. The microenvironment of bovine megakaryocytes in vivo was investigated using antibodies to types I and IV collagen and laminin. In an attempt to duplicate the microenvironment in vitro, bovine megakaryocytes were cultured on a reconstituted basement membrane matrix (Matrigel). The cells adhered to the gel, extended radial lamellipodia, and occasionally formed lengthy pseudopodia. Ultrastructural examination of these cells showed widening and coalescence of the megakaryocyte demarcation membranes (DMS), and inclusion of platelet granules, thin filaments, and microtubules in the processes. Very few DMS vesicles were present distally in the processes. The culture of megakaryocytes on a reconstituted basement membrane may closely model the in vivo megakaryocyte microenvironment and allow the study of thrombocytopoiesis in vitro.

scholarly journals Productive infection by B19 parvovirus of human erythroid bone marrow cells in vitro

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 384-391 ◽  
Author(s):  
K Ozawa ◽  
G Kurtzman ◽  
N Young
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Productive Infection ◽  
Similar Proportion ◽  
Erythroid Cells ◽  
B19 Parvovirus ◽  
Transient Aplastic Crisis ◽  
Cell Fractions ◽  
Marrow Cells

B19 parvovirus, the cause of fifth disease and transient aplastic crisis, has been successfully propagated in suspension cultures of human erythroid bone marrow cells obtained from patients with sickle cell disease and stimulated by erythropoietin. B19 inoculation in vitro resulted in a marked decline in identifiable erythroid cells over seven to nine days of incubation. Characteristic giant early erythroid cells were seen on Wright's-Giemsa stain of infected cultures. By in situ hybridization, 30% to 40% of erythroblasts were infected at 48 hours; a similar proportion of cells showed B19 capsid protein by immunofluorescence. B19 DNA was present in erythroblasts but not in the leukocyte fraction of bone marrow. B19 replication, as determined by Southern analysis, and B19 encapsidation, as determined by sensitivity of isolated cell fractions to DNase I, were restricted to the nuclei. B19 DNA was detectable in the nuclei from infected cultures beginning at 18 hours and in the supernatant at 32 hours; B19 genome copy number was estimated at about 25,000 to 30,000/infected cell at 48 hours. Recovery of virus depended on the multiplicity of infection (moi); at low moi, approximately 200x input virus was recovered from total cultures and 50x from the culture supernatants. Virus released into the supernatant was as infectious or more infectious than virus obtained from sera of infected patients. Human erythroid bone marrow culture represents a safe in vitro system for the elucidation of the cellular and molecular biology of the pathogenic B19 parvovirus.

In Childhood ALL, Both Blasts with a CD20−/Low and a CD20High Immunophenotype Have the Ability to Transfer the Leukemia Onto Immune-Deficient NOD/Scid Y−/− Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1348-1348
Author(s):  
Kerrie Wilson ◽  
Klaus Rehe ◽  
Simon Bomken ◽  
Marian Case ◽  
Leonard Shultz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Stem Cell Marker ◽  
Differentiation Markers ◽  
Childhood All ◽  
Flow Sorting ◽  
Differentiation Marker ◽  
Transcription Factor Expression

Abstract There is an ongoing controversy as to whether cancer is always maintained by a rare population of highly specialized cancer stem cells or whether cancer-propagating cells may be more abundant in some cancer types. We have previously shown that in a heterogeneous group of childhood ALL different blast populations, regardless of their expression of the progenitor/stem cell marker CD34 or the lymphoid differentiation antigen CD19, contain leukemia-initiating activity (Cancer Cell 2008, 14(1), 47–58). By profiling B cell transcription factor expression, these different populations appeared to mirror stages of normal B cell development. Here we extend our experiments to another lymphoid differentiation marker, CD20, to provide further evidence that ALL blasts at different stages of maturation possess the ability to re-initiate the leukemia. Patient Transplant Mice Population Cell dose Engrafted L736 Secondary 9 CD20High 10.000–100.000 6 11 CD20−/Low 10 Tertiary 4 CD20High 10.000 4 4 CD20−/Low 1 L754 Primary 4 CD20High 100.000 2 4 CD20−/Low 3 Secondary 11 CD20High 5.000-100.000 9 11 CD20−/Low 7 A67 Secondary 6 CD20High 9.000-20.000 6 6 CD20−/Low 6 Unsorted bone marrow cells from 3 different ALL patients (L736, L754 and A67) were transplanted into 12 primary mice. Bone marrow was harvested from leukemic mice and flow sorted candidate populations (CD19+CD20−/Low and CD19+CD20High) were re-transplanted into 52 secondary and 8 tertiary mice. As expected from our previous experiments both CD19+CD20−/Low and CD19+CD20High cells were able to re-establish the disease in unconditioned NOD/scid y−/− mice (see table). Leukemic engraftment ranged from 0.5 to 73% as determined by flow cytometry on bone marrow aspirates. Both populations re-established the complete phenotype of the original leukemia including CD20−/Low and CD20High blasts. These results were confirmed by directly sorting primary ALL blasts from L754 without prior passage in the mice. Cell purity after flow sorting was high (81–99%) and low numbers of cells engrafted (5000 for both CD19+CD20−/Low and CD19+CD20High). The three patients reflected different ALL subtypes (L736 intermediate risk ALL: high WBC/MRD low risk, L754:high hyperdiploid/MRD high risk; and A67: high risk ALL/t(9;22)). In conclusion, these results confirm our previous observation that ALL blasts irrespective of the expression of lymphoid differentiation markers are able to engraft immune-deficient mice. Therefore, leukemia-propagating cells in childhood ALL may be more abundant than previously thought.

scholarly journals Culture of isolated bovine megakaryocytes on reconstituted basement membrane matrix leads to proplatelet process formation

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 912-924 ◽  
Author(s):  
KS Topp ◽  
F Tablin ◽  
J Levin
Keyword(s):  
Basement Membrane ◽  
Bone Marrow Cells ◽  
Human Platelets ◽  
Velocity Sedimentation ◽  
Thin Filaments ◽  
Membrane Matrix ◽  
Process Formation ◽  
Vitro Model

We have enriched for bovine megakaryocytes and identified a culture system that may provide an in vitro model for platelet formation. Mature megakaryocytes with an unusually high ploidy distribution were obtained after differential centrifugation and velocity sedimentation of bone marrow cells through gradients of bovine serum albumin (BSA). The cell membranes of isolated megakaryocytes and megakaryocytes in vivo stained with antisera to human platelets and human platelet membrane GPIIIa. The microenvironment of bovine megakaryocytes in vivo was investigated using antibodies to types I and IV collagen and laminin. In an attempt to duplicate the microenvironment in vitro, bovine megakaryocytes were cultured on a reconstituted basement membrane matrix (Matrigel). The cells adhered to the gel, extended radial lamellipodia, and occasionally formed lengthy pseudopodia. Ultrastructural examination of these cells showed widening and coalescence of the megakaryocyte demarcation membranes (DMS), and inclusion of platelet granules, thin filaments, and microtubules in the processes. Very few DMS vesicles were present distally in the processes. The culture of megakaryocytes on a reconstituted basement membrane may closely model the in vivo megakaryocyte microenvironment and allow the study of thrombocytopoiesis in vitro.

Sign in / Sign up

Export Citation Format

Share Document