Microscopy of Living Bone Marrow In Situ. II. Influence of the Microenvironment on Hemopoiesis

Abstract The present study examined in vivo the dynamic changes of hypocellular bone marrow during increased hemopoietic activity induced by phlebotomy or administration of erythropoietin. During increased hemopoietic activity, large venules and venous sinusoids located within the marrow spaces were replaced by dilated or polygonal networks of sinusoids supplied by small arterioles or capillaries that coursed from surrounding spicules of bone into the marrow spaces. An increased cellularity (hemopoietic and fat cells) was associated with this change in vascularity. The results suggest the presence of two functional parts of the microvascular system in bone marrow. The first is associated with hemopoietic areas of the marrow and is erythropoietin-sensitive; the second, with regeneration and resorption of bone. The presence of cancellous bone and the morphology of the microvascular system may determine the extent of hemopoietic activity of bone marrow. During increased hemopoietic activity, the intimate relationship between the microvascular system, bone spicules, and the developing cellular compartment suggests local factors resident in bone are transported to these proliferating cells to exert a hemopoietic effect.

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Cell proliferation assay – method optimisation for in vivo labeling of DNA in the rat forestomach

Acta veterinaria ◽

10.1515/acve-2017-0001 ◽

2017 ◽

Vol 67 (1) ◽

pp. 1-10

Author(s):

Gordana Joksić ◽

Mileva Mićić ◽

Jelena Filipović ◽

Dunja Drakulić ◽

Miloš Stanojlović ◽

...

Keyword(s):

Cell Proliferation ◽

Immunohistochemical Analysis ◽

Optimal Dose ◽

Cell Labeling ◽

Assay Method ◽

Proliferating Cells ◽

Adult Rats ◽

In Vivo Labeling

AbstractThe study of cell proliferation is a useful tool in the fields of toxicology, pathophysiology and pharmacology. Cell proliferation and its degree can be evaluated using 5-bromo-2′-deoxyuridine which is incorporated into the newly synthesized DNA. The aim of this study was the optimization of subcutaneous application of 5-bromo-2′-deoxyuridine implantation for continuous and persistent marking of proliferating cells in the rat forestomach. 3-tert-Butyl-4-hydroxyanisole was used as the agent that ensures cell proliferation. In order to determine the optimal dose for proliferating cells labeling, 5-bromo-2′-deoxyuridine doses of 50 mg, 100 mg, 200 mg or 350 mg were implemented 2 days prior to sacrifice by flat-faced cylindrical matrices. Immunohistochemical analysis using 5-bromo-2′-deoxyuridine in situ detection kit was performed for the detection of 5-bromo-2′-deoxyuridine labeled cells. The results showed that for adult rats, the optimum 5-bromo-2′-deoxyuridine dose is 200 mg per animal for subcutaneous application. The here described manner of 5-bromo-2′-deoxyuridine in vivo labeling provides a simple, efficient, and reliable method for cell labeling, and at the same minimizes stress to animals.

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Microscopy of Living Bone Marrow In Situ

Blood ◽

10.1182/blood.v38.1.87.87 ◽

1971 ◽

Vol 38 (1) ◽

pp. 87-95 ◽

Cited By ~ 18

Author(s):

ROBERT S. MCCUSKEY ◽

SAMUEL G. MCCLUGAGE ◽

WALDO J. YOUNKER

Keyword(s):

Bone Marrow ◽

Microscopic Examination ◽

Hemopoietic Cells ◽

Highly Active ◽

Preliminary Examination ◽

Yellow Marrow ◽

Living Bone

Abstract A chamber has been designed to permit chronic microscopic examination of living bone marrow in situ. The amount of metal adjacent to the tissue within the gap of the chamber appears to be critical if normal hemopoiesis is to occur, since excessive metal resulted in the regeneration of a hypocellular, gelatinous marrow. When the amount of metal was reduced, relatively normal marrow regenerated into the chamber. Most frequently this histologically resembled yellow marrow but on occasion highly active marrow was observed. Preliminary examination of the microvascular system confirmed previous reports that sinusoids generally were arranged in interconnecting polygonal networks surrounding individual or clusters of fat and hemopoietic cells. The sinusoids were lined by a definite endothelium. No "intersinusoidal capillaries" were observed.

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The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation

Blood ◽

10.1182/blood-2007-07-099648 ◽

2008 ◽

Vol 111 (1) ◽

pp. 42-49 ◽

Cited By ~ 144

Author(s):

Antje M. Wengner ◽

Simon C. Pitchford ◽

Rebecca C. Furze ◽

Sara M. Rankin

Keyword(s):

Bone Marrow ◽

Intraperitoneal Injection ◽

Fold Increase ◽

In Situ Perfusion ◽

Acute Peritonitis ◽

Femoral Bone Marrow ◽

Prior Administration

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1α–mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 × 106 and 5.4 × 106 neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 × 106 neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.

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Evidence for Increased Bone Marrow Lymphoplasmocytoid Cells and SDF1 Secretion in imatinib Treated CML. Relationship with Clinical/hemathological Response

Blood ◽

10.1182/blood.v112.11.4256.4256 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4256-4256

Author(s):

Ivana Pierri ◽

Silvia Catellani ◽

Francesca Olcese ◽

Raffaella Grasso ◽

Nicoletta Colombo ◽

...

Keyword(s):

Bone Marrow ◽

B Lymphocytes ◽

Kinase Inhibitors ◽

Myelogenous Leukemia ◽

Imatinib Treatment ◽

First Line ◽

Polymerase Chain ◽

Chemotactic Effect

Abstract Tyrosine kinase inhibitors, such as imatinib mesylate (Gleevec, Novartis, formerly known as STI571) are confirmed to be the first line treatment of Chronic Myelogenous Leukemia (CML) and of a rare form of gastroenteric stromal cancer. It has been reported that in the latter case, the response to the drug in vivo is mainly due to immunocompetent cells, able to produce cytokines with antineoplastic activity. In this study, peripheral blood and bone marrow of 20 CML patients were studied, prior and during treatment with imatinib, to assess morphologic, phenotypic, cytogenetic and biomolecular patterns. Plasma from BM and PB was also tested to evaluate (by ELISPOT and real time polymerase chain reaction) cytokines able to induce B lymphocytes differentiation, and/or proliferation, such as interleukin (IL)-4, IL-6 (ligand for CD126), IL-3, IL-10 or IL-21 together with chemokines MCP-1, SDF-1, IP-10 and IL-8. In 14 out of 20 CML patients a significant increase in the percentage of BM lymphoplasmocytoid cells was observed on imatinib treatment with >10% (range 8–12%) of CD20+CD126+cells. Among this population, two third of cells coexpressed IgM and one third was IgD+, moreover a lower fraction of IgM+CD126+CD20− (3–4%) or IgD+CD126+CD20− (2–3%) cells was found too. In all these patients SDF1 increased in the BM plasma after imatinib (from 10–80pg/ml to 150–450pg/ml) and its receptor CXCR4 was up-regulated on CD20+CD126+cells. In 4 patients the amount of IP-10 in BM plasma and the expression of its receptor CXCR3 were also increased. No significant modification in transcription and secretion of IL-3, IL-4, IL-6, IL-10, IL-21, IL-8 or MCP1 were observed. The lasting 6 patients had<5% of CD20 +CD126+ lymphocytes (range2–4%), 2/3 coexpressing IgM and 1/3 coexpressing IgD. Every patients with increased number of CD126+ B lymphocytes achieved hemathologic remission, 7 of them complete cytogenetic and biomolecular remission. On the other hand, among the patients with low or undetectable CD20+CD126+cells, 2 obtained only hemathological remission. In the 2 relapsed patients, BM CD20+CD126+ lymphocytes decreased from 11% and 8% to 7 and 5%, respectively, with undetectable IgM+ CD126+CD20− or IgD+ CD126+CD20− cells. The increased production of SDF-1, following imatinib administration, might increase BM lymphoplasmocitoid cells, thanks to the double proliferative/chemotactic effect of the cytokine on B cells, with redistribution and in situ differentiation of CD20+ CD126+ lymphocytes. These findings shed some light on the possibility that, even in CML, immunological events may play a role in disease control;moreover they could be useful in monitoring disease outcome.

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Eotaxin Induces a Rapid Release of Eosinophils and Their Progenitors From the Bone Marrow

Blood ◽

10.1182/blood.v91.7.2240.2240_2240_2248 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2240-2248 ◽

Cited By ~ 4

Author(s):

Roger T. Palframan ◽

Paul D. Collins ◽

Timothy J. Williams ◽

Sara M. Rankin

Keyword(s):

Bone Marrow ◽

Guinea Pig ◽

Allergic Inflammation ◽

Interleukin 5 ◽

In Situ Perfusion ◽

Dose Dependent ◽

Femoral Bone Marrow

The CC-chemokine eotaxin is a potent eosinophil chemoattractant that stimulates recruitment of eosinophils from the blood to sites of allergic inflammation. Mobilization from the bone marrow is an important early step in eosinophil trafficking during the allergic inflammatory response. In this paper we examine the potential of eotaxin to mobilize eosinophils and their progenitors from bone marrow. Eotaxin stimulated selective, dose-dependent chemotaxis of guinea pig bone marrow eosinophils in vitro. Intravenous injection of eotaxin (1 nmol/kg) into guinea pigs in vivo stimulated a rapid blood eosinophilia (from 3.9 ± 1.2 to 28 ± 9.9 × 104eosinophils/mL at 30 minutes) and a corresponding decrease in the number of eosinophils retained in the femoral marrow (from 9.0 ± 0.8 to 4.8 ± 0.8 × 106 eosinophils per femur). To show a direct release of eosinophils from the bone marrow an in situ perfusion system of the guinea pig femoral bone marrow was developed. Infusion of eotaxin into the arterial supply of the perfused femoral marrow stimulated a rapid and selective release of eosinophils into the draining vein. In addition, eotaxin stimulated the release of colony-forming progenitor cells. The cytokine interleukin-5 was chemokinetic for bone marrow eosinophils and exhibited a marked synergism with eotaxin with respect to mobilization of mature eosinophils from the femoral marrow. Thus, eotaxin may be involved in both the mobilization of eosinophils and their progenitors from the bone marrow into the blood and in their subsequent recruitment into sites of allergic inflammation.

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Eotaxin Induces a Rapid Release of Eosinophils and Their Progenitors From the Bone Marrow

Blood ◽

10.1182/blood.v91.7.2240 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2240-2248 ◽

Cited By ~ 197

Author(s):

Roger T. Palframan ◽

Paul D. Collins ◽

Timothy J. Williams ◽

Sara M. Rankin

Keyword(s):

Bone Marrow ◽

Guinea Pig ◽

Allergic Inflammation ◽

Interleukin 5 ◽

In Situ Perfusion ◽

Dose Dependent ◽

Femoral Bone Marrow

Abstract The CC-chemokine eotaxin is a potent eosinophil chemoattractant that stimulates recruitment of eosinophils from the blood to sites of allergic inflammation. Mobilization from the bone marrow is an important early step in eosinophil trafficking during the allergic inflammatory response. In this paper we examine the potential of eotaxin to mobilize eosinophils and their progenitors from bone marrow. Eotaxin stimulated selective, dose-dependent chemotaxis of guinea pig bone marrow eosinophils in vitro. Intravenous injection of eotaxin (1 nmol/kg) into guinea pigs in vivo stimulated a rapid blood eosinophilia (from 3.9 ± 1.2 to 28 ± 9.9 × 104eosinophils/mL at 30 minutes) and a corresponding decrease in the number of eosinophils retained in the femoral marrow (from 9.0 ± 0.8 to 4.8 ± 0.8 × 106 eosinophils per femur). To show a direct release of eosinophils from the bone marrow an in situ perfusion system of the guinea pig femoral bone marrow was developed. Infusion of eotaxin into the arterial supply of the perfused femoral marrow stimulated a rapid and selective release of eosinophils into the draining vein. In addition, eotaxin stimulated the release of colony-forming progenitor cells. The cytokine interleukin-5 was chemokinetic for bone marrow eosinophils and exhibited a marked synergism with eotaxin with respect to mobilization of mature eosinophils from the femoral marrow. Thus, eotaxin may be involved in both the mobilization of eosinophils and their progenitors from the bone marrow into the blood and in their subsequent recruitment into sites of allergic inflammation.

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Studies of the Hemopoietic Microenvironment. I. Changes in the Microvascular System and Stroma During Erythropoietic Regeneration and Suppression in the Spleens of CF1 Mice

Blood ◽

10.1182/blood.v39.5.697.697 ◽

1972 ◽

Vol 39 (5) ◽

pp. 697-712 ◽

Cited By ~ 45

Author(s):

Robert S. McCuskey ◽

Howard A. Meineke ◽

Samuel F. Townsend

Keyword(s):

Bone Marrow ◽

Blood Flow ◽

Linear Velocity ◽

Proliferating Cells ◽

Hemopoietic Microenvironment ◽

Murine Spleen ◽

Flow Through ◽

Tissue Compartments ◽

Red Pulp

Abstract Specific alterations in the microvascular and connective tissue compartments of the hemopoietic microenvironment have been examined during erythropoietic regeneration and suppression in the murine spleen and bone marrow using in vivo microscopic and histochemical methods. The results have confirmed the concept of specific hemopoietic microenvironments and have demonstrated specific alterations in the microenvironment during erythropoietic stimulation and repression. Elevated erythropoiesis in the splenic red pulp is accompanied by an elevation in blood flow through the microvascular system. Both the linear velocity of flow and the number of sinusoids with blood flow in them increased significantly. In contrast, erythropoietic repression was accompanied by a decreased linear velocity of blood flow, as well as a marked increase in the amount of blood being stored in the splenic sinusoids. This also was the picture when diffuse granulopoiesis was present in the red pulp, or when granuloid or undifferentiated colonies were present. The chemical composition of the stroma in the spleen and bone marrow also varied during states of hemopoietic activity and, in addition, there were differences in the composition of the stroma between these two organs. In both organs, foci of early proliferating cells were enveloped by a coating of sulfated acid mucopolysaccharide. This coat persisted on cells in later stages of granulopoiesis but not on cells in the later stages of erythropoiesis. The latter were enveloped with a coating of neutral mucopolysaccharide. A tentative hypothesis to explain the mechanisms involved in producing these changes is discussed.

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Ectopic Human Mesenchymal Stem Cell-Coated Scaffolds Create An In Vivo Leukemia Niche in NOD/SCID Mice.

Blood ◽

10.1182/blood.v114.22.559.559 ◽

2009 ◽

Vol 114 (22) ◽

pp. 559-559

Author(s):

Sarah Rivkah Vaiselbuh ◽

Morris Edelman ◽

Jeffrey Michael Lipton ◽

Johnson M. Liu

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Stem Cell Niche ◽

Scid Mice ◽

Cxcr4 Antagonist ◽

Cell Niche

Abstract Abstract 559 Introduction: Different cellular components of the normal hematopoietic niche have been identified. However, the niche for malignant hematopoiesis remains to be elucidated. Recent work of other groups has suggested that hematopoietic stem cells (HSC) within the bone marrow anchor themselves in place by attaching to osteoblasts and/or vascular sinusoid endothelial cells. We have recently identified mesenchymal stem cells (MSC) as niche-maker cells and found a crucial role of the SDF-1/CXCR4 axis in this process. Stromal Derived Factor-1 (SDF-1/CXCL12) regulates stem cell trafficking and the cell cycle via its receptor CXCR4. Methods: Polyurethane scaffolds, coated in vitro with human bone marrow MSC, were implanted subcutaneously in non-irradiated NOD/SCID mice. CD34+ HSC or primary AML cells (from a leukapheresis product) were injected either in situ or retro-orbitally in the mice and analyzed for engraftment. The mice were treated twice per week with in situ injections of SDF-1, AMD3100 (a CXCR4 antagonist) or PBS (control). After 2 to 4 weeks, the scaffolds were processed and evaluated for cell survival in the mesenchymal niche by immunohistochemistry. Results: We created in vitro MSC-coated scaffolds that retained inoculated AML cells in the presence of SDF-1, while AML cells seeded on empty scaffolds were not retained. In vivo in NOD/SCID mice, the MSC-coated scaffolds, in the presence of SDF-1 enabled homing of both in situ injected normal CD34+ HSC and retroorbital- or in situ injected primary human AML cells. The scaffolds were vascularized and showed osteoclasts and adipocytes present, suggestive of an ectopic human bone marrow microenvironment in the murine host. Finally, the SDF-1-treated scaffolds showed proliferation of the MSC stromal layer with multiple adherent AML cells, while in the AMD3100-treated scaffolds the stromal lining was thin and disrupted at several points, leaving AML cells free floating in proximity. The PBS-treated control-scaffold showed a thin single cell MSC stromal layer without disruption, with few AML cells attached. Conclusion: The preliminary data of this functional ectopic human microenvironment in NOD/SCID mice suggest that AMD3100 (a CXCR4 antagonist) can disrupt the stem cell niche by modulation of the mesenchymal stromal. Further studies are needed to define the role of mesenchymal stem cells in maintaining the hematopoietic/leukemic stem cell niche in vivo. In Vivo Leukemia Stem Cell Niche: (A) Empty polyurethane scaffold. (B)Vascularization in SQ implanted MSC-coated scaffold (s) niche in NOD/SCID mice. (C) DAB Peroxidase (brown) human CD45 positive nests of AML cells (arrows) 1 week after direct in situ AML injection. (D) Human CD45 positive myeloid cells adhere to MSC in vivo (arrows). Disclosures: No relevant conflicts of interest to declare.

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CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization

Blood ◽

10.1182/blood-2010-08-304287 ◽

2011 ◽

Vol 117 (19) ◽

pp. 5067-5077 ◽

Cited By ~ 251

Author(s):

Ariane Tormin ◽

Ou Li ◽

Jan Claas Brune ◽

Stuart Walsh ◽

Birgit Schütz ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem ◽

Close Proximity ◽

Marrow Mesenchymal Stem Cells ◽

Similar Phenotype ◽

Marrow Stroma ◽

Cd146 Expression

Abstract Nonhematopoietic bone marrow mesenchymal stem cells (BM-MSCs) are of central importance for bone marrow stroma and the hematopoietic environment. However, the exact phenotype and anatomical distribution of specified MSC populations in the marrow are unknown. We characterized the phenotype of primary human BM-MSCs and found that all assayable colony-forming units-fibroblast (CFU-Fs) were highly and exclusively enriched not only in the lin−/CD271+/CD45−/CD146+ stem-cell fraction, but also in lin−/CD271+/CD45−/CD146−/low cells. Both populations, regardless of CD146 expression, shared a similar phenotype and genotype, gave rise to typical cultured stromal cells, and formed bone and hematopoietic stroma in vivo. Interestingly, CD146 was up-regulated in normoxia and down-regulated in hypoxia. This was correlated with in situ localization differences, with CD146 coexpressing reticular cells located in perivascular regions, whereas bone-lining MSCs expressed CD271 alone. In both regions, CD34+ hematopoietic stem/progenitor cells were located in close proximity to MSCs. These novel findings show that the expression of CD146 differentiates between perivascular versus endosteal localization of non-hematopoietic BM-MSC populations, which may be useful for the study of the hematopoietic environment.

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