scholarly journals Measurement of ploidy distribution in megakaryocyte colonies obtained from culture: with studies of the effects of thrombocytopenia

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 287-297 ◽  
Author(s):  
J Levin ◽  
FC Levin ◽  
DG Penington ◽  
D Metcalf
Keyword(s):  
Bone Marrow ◽  
Ploidy Level ◽  
Dna Content ◽  
Soft Agar ◽  
Distribution Characteristic ◽  
Cell Type ◽  
Big Cell ◽  
Platelet Depletion ◽  
Number Of Cells

Abstract Microdensitometric measurement of the DNA content of individual megakaryocytes was performed using megakaryocyte colonies obtained following culture, in soft agar, of hematopoietic cells from C57BL/6J mice. Two types of colonies were detected. After 7 days of culture, the big cell type contained 16 /+- 2.3 acetylcholinesterase (AChE) positive cells/colony, with a mean ploidy level of 16.8 /+- 0.8/cell and the ploidy distribution characteristic of recognizable megakaryocytes in bone marrow. The heterogeneous type contained 44 /+- 9.6 cells/colony (some of which were AChE negative), with a mean ploidy level of 6.8 /+- 0.7/cell. The ploidy distribution of heterogeneous colonies differed markedly from big cell colonies, with preponderance of 2N and 4N cells. Colony-forming cells, obtained 4–5 days after induction of acute thrombocytopenia, gave big cell colonies with a marked increase in DNA content. Mean ploidy level increased to 21.5 /%- 1.8/cell; the frequency of 32N cells increased from 17% to 30% and 64N cells from 0% to 6%. This is the pattern of change observed in bone marrow, in vivo, 24 to 48 hr after induction of acute thrombocytopenia. The number of cells/colony did not increase. In contrast, acute thrombocytopenia did not alter the ploidy of heterogeneous colonies. The different responses to the stimulus of acute thrombocytopenia suggest that there are at least two types of Meg-CFC. The delayed appearance of altered Meg-CFC that produced big cell colonies indicates that the pool of stem cells, from which committed megakaryocyte precursors are derived, may respond indirectly to the stimulus of platelet depletion.

scholarly journals Measurement of ploidy distribution in megakaryocyte colonies obtained from culture: with studies of the effects of thrombocytopenia

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 287-297 ◽  
Author(s):  
J Levin ◽  
FC Levin ◽  
DG Penington ◽  
D Metcalf
Keyword(s):  
Bone Marrow ◽  
Ploidy Level ◽  
Dna Content ◽  
Soft Agar ◽  
Distribution Characteristic ◽  
Cell Type ◽  
Big Cell ◽  
Platelet Depletion ◽  
Number Of Cells

Microdensitometric measurement of the DNA content of individual megakaryocytes was performed using megakaryocyte colonies obtained following culture, in soft agar, of hematopoietic cells from C57BL/6J mice. Two types of colonies were detected. After 7 days of culture, the big cell type contained 16 /+- 2.3 acetylcholinesterase (AChE) positive cells/colony, with a mean ploidy level of 16.8 /+- 0.8/cell and the ploidy distribution characteristic of recognizable megakaryocytes in bone marrow. The heterogeneous type contained 44 /+- 9.6 cells/colony (some of which were AChE negative), with a mean ploidy level of 6.8 /+- 0.7/cell. The ploidy distribution of heterogeneous colonies differed markedly from big cell colonies, with preponderance of 2N and 4N cells. Colony-forming cells, obtained 4–5 days after induction of acute thrombocytopenia, gave big cell colonies with a marked increase in DNA content. Mean ploidy level increased to 21.5 /%- 1.8/cell; the frequency of 32N cells increased from 17% to 30% and 64N cells from 0% to 6%. This is the pattern of change observed in bone marrow, in vivo, 24 to 48 hr after induction of acute thrombocytopenia. The number of cells/colony did not increase. In contrast, acute thrombocytopenia did not alter the ploidy of heterogeneous colonies. The different responses to the stimulus of acute thrombocytopenia suggest that there are at least two types of Meg-CFC. The delayed appearance of altered Meg-CFC that produced big cell colonies indicates that the pool of stem cells, from which committed megakaryocyte precursors are derived, may respond indirectly to the stimulus of platelet depletion.

scholarly journals Leukokinetic Studies. VII. Morphology of the Bone Marrow and Blood of Dogs Given Vinblastine Sulfate

Blood ◽  
1964 ◽  
Vol 23 (1) ◽  
pp. 53-67 ◽  
Author(s):  
DANE R. BOGGS ◽  
JOHN W. ATHENS ◽  
OTTO P. HAAB ◽  
PASQUALE A. CANCILLA ◽  
SPENCER O. RAAB ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mortality Rate ◽  
Single Injection ◽  
Gastrointestinal Toxicity ◽  
Blood Neutrophil ◽  
Cell Type ◽  
Large Numbers ◽  
Blood Neutrophils ◽  
Vinblastine Sulfate ◽  
Number Of Cells

Abstract The effect of a single injection of vinblastine sulfate was studied in 50 mongrel dogs. Nine of 34 dogs given 0.2 mg./Kg. of VLB died with gastrointestinal toxicity and the mortality rate increased as the dosage of VLB was increased. The morphologic pattern of leukocyte suppression and recovery in the bone marrow and blood was studied in detail in surviving animals. The cells of the bone marrow were markedly affected by VLB. Within 4 hours there was an increase in the number of cells in metaphase and, by day 1, virtually all proliferating leukocytes and erythrocytes had disappeared. An orderly repopulation of the bone marrow followed. The neutrophils, eosinophils, lymphocytes and monocytes of the blood were all markedly altered in concentration after VLB. Each type of cell first decreased to abnormally small numbers and then increased to abnormally large numbers in the blood. The curve of disappearance from and reappearance in the blood differed for each cell type. The changes in blood neutrophil number and morphology were correlated with changes in the blood neutrophil precursor cells of the marrow. The following conclusions were reached concerning the neutrophils and the assumptions implicit to these conclusions were detailed. 1. In the dog, the marrow contains enough post-mitotic granulocytes to replace those lost from the blood for at least 3 to 4 days. 2. The release of mature neutrophils from the bone marrow is a function of the rate at which blood neutrophils are lost and proceeds normally even when the marrow granulocyte reserve is partially depleted.

scholarly journals Ultrastructural analysis of murine megakaryocyte maturation in vitro: comparison of big-cell and heterogeneous megakaryocyte colonies

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1509-1518
Author(s):  
PE Stenberg ◽  
J Levin
Keyword(s):  
Bone Marrow ◽  
Soft Agar ◽  
Small Cells ◽  
Specific Marker ◽  
Mouse Bone Marrow ◽  
Structural Level ◽  
Agar Culture ◽  
Big Cell ◽  
Mature Bone

Two morphologically distinct types of murine megakaryocyte (MK) colonies are present after three to seven days in soft agar culture: (a) “big-cell” colonies composed of ten to 30 large, mature-appearing megakaryocytes and (b) “heterogeneous” colonies consisting of approximately 100 or more cells at various stages of differentiation. Cytochemical and immunocytochemical techniques were used to study MK maturation in colonies as well as normal mouse bone marrow. Acetylcholinesterase (AChE), a specific marker for murine platelets and MK, was found in the perinuclear cisterna, endoplasmic reticulum, and occasionally, Golgi cisternae of MK in three-day big-cell colonies and immature bone marrow MK. MK in seven-day big-cell colonies and mature bone marrow MK showed additional reaction sites in the demarcation membrane system and occasional granules. In seven-day heterogeneous colonies, small cells resembled immature bone marrow MK with respect to AChE localization, whereas large cells corresponded to mature bone marrow MK. With immunogold procedures at the ultrastructural level, polyclonal antibodies against human platelet membrane glycoprotein IIIa and antimouse platelet antiserum labeled bone marrow MK and all MK from colonies grown in soft agar cultures for three to seven days. Granulocytes and macrophages in both bone marrow and soft agar cultures were negative for AChE and these immunocytochemical markers. These data indicate that the pattern of expression of AChE during maturation of MK is similar in vivo and in vitro and demonstrate, when using this marker at the fine-structural level, that a greater range of MK maturational stages is present in heterogeneous colonies than is observed in MK in big-cell colonies. Furthermore, we have confirmed that small cells in heterogeneous colonies are MK and that these colonies are composed solely of MK and their precursors.

A Novel Real-Time In Vivo Homing Model of Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 242-242
Author(s):  
Judith M. Runnels ◽  
Costas Pitsillides ◽  
Joel A. Spencer ◽  
Joji Fujisaki ◽  
Hai Ngo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Count ◽  
Real Time ◽  
Intact Mouse ◽  
Cell Homing ◽  
New Model ◽  
Cell Counts ◽  
Number Of Cells

Abstract Background: Multiple myeloma (MM) is characterized by widespread involvement of the bone marrow (BM) at diagnosis, implying a continuous (re) circulation of the MM cells in the peripheral blood and (re) entrance into the BM. The normal process of B cell homing is regulated by cytokines and receptors such as SDF-1, CXCR4, VLA4, LFA-1, VCAM-1 and ICAM-1. In order to better understand the role of homing in MM, we developed an in vivo model which allows the continuous real-time imaging of MM cells as they home and adhere to the BM, as well as quantifying the numbers of cells in the circulation. Methods: MM.1S (2 ×106/ml) were fluorescently labeled by incubation with the dialkylcarbocyanine membrane dye “DiD” (Molecular Probes) 1uM dye for 15 min at 37°C. Cells were i.v injected in Balb/c mice. Appropriate arterioles in the ear pinnae of the mice were chosen for obtaining measurements, and the fluorescence signal on the MM cells was excited as the labeled cells passed through a slit of light (from a 632 nm He:Ne laser) focused across the vessel. Cell counts were obtained every 5 min from the time of injection. MM cell homing to bone marrow vasculature of the skull was analyzed using fluorescence confocal microscopy. A small incision was made in the scalp so as to expose the underlying dorsal skull surface. The mouse was placed on a warmed microscope stage. Imaging duration was 1–3 hours per session. DiD was excited with a 635 nm diode laser. High-resolution images with cellular details were obtained through the intact mouse skull at depths of up to 250 um from the surface of the skull. Images from several depths were obtained and z-stacking was performed to merge the images. Quantitative evaluation was made by dividing the bone marrow into pre-determined quadrants (areas 1 to 4) and counting numbers of fluorescent cells per field. To demonstrate that that this new model identifies changes in homing of MM cells, we used the CXCR4 inhibitor AMD3100 and anti-VLA-4 antibody to inhibit homing to the BM. MM cells were pre-incubated with AMD3100 (50 uM overnight, Sigma, MO) or anti-VLA-4 antibody (1hr incubation with 10 ug/ml, BD Pharmingen, CA) or control PBS under the same conditions. In vivo flow cytometry and confocal imaging were then performed on control and AMD3100 treated mice. Results: The number of cells in the control group decreased dramatically (86% decrease) after 1 hour indicating homing, whereas there was only a 47% reduction in the cells at 1 hour in the AMD3100 treated cohort, (p=0.002). Similarly, we demonstrated that the number of cells present in the perivascular bone marrow niches of the skull was significantly higher in the control mice as compared to the AMD3100-treated group at 1 hour after injection. The mean cell count in the AMD3100 treated mice decreased to 38% as compared to controls, p=0.01 Likewise, the use of anti-VLA-4 antibody demonstrated significant retention of MM cells in the circulation 1 hr after injection (4% reduction in circulating cell count vs 82% for control). Conclusion: We describe a new model that detects in vivo real-time homing of MM cells from the peripheral circulation into BM niches, which can be used to study the trafficking of MM cells into and out of the BM, as well as the effect of novel agents on this dynamic process.

scholarly journals In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 312-316 ◽  
Author(s):  
DM Rennick ◽  
L Thompson-Snipes ◽  
RL Coffman ◽  
BW Seymour ◽  
JD Jackson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Soft Agar ◽  
Nippostrongylus Brasiliensis ◽  
Antibody Treatment ◽  
Interleukin 5 ◽  
In Vivo Experiments ◽  
Regulatory Molecule ◽  
Time Of Infection ◽  
Eosinophil Colonies

Abstract Bone marrow of mice parasitized with Nippostrongylus brasiliensis showed increased numbers of eosinophils as early as 4 days after infection. By day 7, their bone marrow also contained elevated numbers of progenitors that form small eosinophil colonies (20 to 50 cells) in soft agar cultures supplemented with interleukin-5 (IL-5). However, when mice were infused with anti-IL-5 antibodies at the time of infection, the number of recognizable eosinophils present in bone marrow remained low and eventually dropped below normal levels. The antibody treatment also prevented increased generation of IL-5- responsive precursors capable of differentiating into mature eosinophils in liquid culture and inhibited the generation of progenitor cells capable of forming small eosinophil colonies or clusters in soft agar cultures. The results of these in vivo experiments directly show that IL-5 is an essential regulatory molecule required for the bone marrow-dependent phase of a parasite-induced eosinophilia.

scholarly journals Genetic analysis reveals cell type-specific regulation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase SHP1.

1996 ◽  
Vol 184 (3) ◽  
pp. 1111-1126 ◽  
Author(s):  
U Lorenz ◽  
A D Bergemann ◽  
H N Steinberg ◽  
J G Flanagan ◽  
X Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Genetic Analysis ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Receptor Protein ◽  
Cell Type ◽  
Protein Tyrosine ◽  
Cell Type Specific

Receptor protein tyrosine kinases (RTKs) transmit downstream signals via interactions with secondary signaling molecules containing SH2 domains. Although many SH2-phosphotyrosyl interactions have been defined in vitro, little is known about the physiological significance of specific RTK/SH2 interactions in vivo. Also, little is known about the mechanisms by which specific RTKs interact with and/or are regulated by specific protein tyrosine phosphatases (PTPs). To address such issue, we carried out a genetic analysis of the previously reported biochemical interaction between the RTK c-Kit, encoded at the W locus, and the SH2-containing non-transmembrane PTP SHP1, encoded at the motheaten (me) locus (1). Mice carrying a kinase-defective allele of c-Kit (Wv/+) were crossed with me/+ mice, which carry one effectively null allele of SHP1, and then backcrossed to generate all possible allelic combinations. Our results indicate strong intergenic complementation between these loci in hematopoietic progenitor cells. Compared to progenitors purified from normal mice, bone marrow progenitor cells (lin-) from me/me mice markedly hyper-proliferated in response to Kit ligand (KL). stimulation. Superimposition of the me/me genotype increased the number of one marrow-derived CFU-E from Wv/+ mice. Conversely, the presence of one or two copies of Wv decreased the number of macrophages and granulocytes in me/me lung, skin, peripheral blood and bone marrow, thereby decreasing the severity of the me/me phenotype. The decrease in dermal mast cells in Wv/Wv mice was rescued to levels found in Wv/+mice by superimposition of the me/me genotype. Surprisingly, however, the presence or absence of SHP1 had no effect on the proliferative response of bone marrow-derived cultured mast cells to KL or IL3 ex vivo. Nevertheless, the immediate-early response to KL stimulation, as measured by KL-induced tyrosyl phosphorylation, was substantially increased in mast cells from Wv/+:me/me compared to Wv/ +:+/+ mice, strongly suggesting that SHP1 directly dephosphorylates and regulates c-Kit. Taken together, our results establish that SHP1 negatively regulates signaling from c-Kit in vivo, but in a cell type-specific manner.

scholarly journals Ultrastructural analysis of murine megakaryocyte maturation in vitro: comparison of big-cell and heterogeneous megakaryocyte colonies

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1509-1518 ◽  
Author(s):  
PE Stenberg ◽  
J Levin
Keyword(s):  
Bone Marrow ◽  
Soft Agar ◽  
Small Cells ◽  
Specific Marker ◽  
Mouse Bone Marrow ◽  
Structural Level ◽  
Agar Culture ◽  
Big Cell ◽  
Mature Bone

Abstract Two morphologically distinct types of murine megakaryocyte (MK) colonies are present after three to seven days in soft agar culture: (a) “big-cell” colonies composed of ten to 30 large, mature-appearing megakaryocytes and (b) “heterogeneous” colonies consisting of approximately 100 or more cells at various stages of differentiation. Cytochemical and immunocytochemical techniques were used to study MK maturation in colonies as well as normal mouse bone marrow. Acetylcholinesterase (AChE), a specific marker for murine platelets and MK, was found in the perinuclear cisterna, endoplasmic reticulum, and occasionally, Golgi cisternae of MK in three-day big-cell colonies and immature bone marrow MK. MK in seven-day big-cell colonies and mature bone marrow MK showed additional reaction sites in the demarcation membrane system and occasional granules. In seven-day heterogeneous colonies, small cells resembled immature bone marrow MK with respect to AChE localization, whereas large cells corresponded to mature bone marrow MK. With immunogold procedures at the ultrastructural level, polyclonal antibodies against human platelet membrane glycoprotein IIIa and antimouse platelet antiserum labeled bone marrow MK and all MK from colonies grown in soft agar cultures for three to seven days. Granulocytes and macrophages in both bone marrow and soft agar cultures were negative for AChE and these immunocytochemical markers. These data indicate that the pattern of expression of AChE during maturation of MK is similar in vivo and in vitro and demonstrate, when using this marker at the fine-structural level, that a greater range of MK maturational stages is present in heterogeneous colonies than is observed in MK in big-cell colonies. Furthermore, we have confirmed that small cells in heterogeneous colonies are MK and that these colonies are composed solely of MK and their precursors.

scholarly journals In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 312-316 ◽  
Author(s):  
DM Rennick ◽  
L Thompson-Snipes ◽  
RL Coffman ◽  
BW Seymour ◽  
JD Jackson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Soft Agar ◽  
Nippostrongylus Brasiliensis ◽  
Antibody Treatment ◽  
Interleukin 5 ◽  
In Vivo Experiments ◽  
Regulatory Molecule ◽  
Time Of Infection ◽  
Eosinophil Colonies

Bone marrow of mice parasitized with Nippostrongylus brasiliensis showed increased numbers of eosinophils as early as 4 days after infection. By day 7, their bone marrow also contained elevated numbers of progenitors that form small eosinophil colonies (20 to 50 cells) in soft agar cultures supplemented with interleukin-5 (IL-5). However, when mice were infused with anti-IL-5 antibodies at the time of infection, the number of recognizable eosinophils present in bone marrow remained low and eventually dropped below normal levels. The antibody treatment also prevented increased generation of IL-5- responsive precursors capable of differentiating into mature eosinophils in liquid culture and inhibited the generation of progenitor cells capable of forming small eosinophil colonies or clusters in soft agar cultures. The results of these in vivo experiments directly show that IL-5 is an essential regulatory molecule required for the bone marrow-dependent phase of a parasite-induced eosinophilia.

scholarly journals Computerized measurement of the DNA content, areas, and autoradiographic grains of the same nuclei: demonstration that lightly (3H)thymidine-labeled bone marrow cells are predominantly in G0/G1 and G2.

1984 ◽  
Vol 32 (11) ◽  
pp. 1197-1203 ◽  
Author(s):  
D C Allison ◽  
J Meyne ◽  
P F Ridolpho ◽  
K Bose ◽  
M Chakerian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Content ◽  
Nuclear Dna ◽  
Bone Marrow Cells ◽  
Nuclear Dna Content ◽  
Mouse Bone Marrow ◽  
Computer File ◽  
Cellular Dna ◽  
Marrow Cells

A computerized "flying spot" microdensitometer and scanning stage have been combined to measure the cellular DNA content, nuclear areas, and autoradiographic grain areas of the same cells. The slide positions of the Feulgen-stained, (3H)thymidine-labeled cells are mapped with the computerized stage, and nuclear DNA content and areas are then determined by integral absorbance measurements at 588 nm. Following autoradiograph preparation, the cells are relocated and the areas of the autoradiographic grains over each nucleus are measured at a light wavelength (625 nm) and an optical density setting (greater than 0.10) that do not detect the Feulgen stain. The microcomputer calculates the portion of each nucleus covered with autoradiographic grains (grain area proportion, GAP), and it links the GAP value to the DNA content of each nucleus in the computer file for subsequent sorting and analysis. By using this system in a study of mouse bone marrow cells labeled in vivo with (3H)thymidine, we found that all S-phase cells were clearly labeled after 8 or more days of autoradiographic exposure. Prolonged exposures (up to 64 days) led to detection of lightly labeled cells (0.1 less than GAP less than 0.8) with G1/G0 and G2 DNA content.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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