scholarly journals Some observations of the hematopoietic status in vivo and in vitro on mice of genotype S1/S1d

Blood ◽  
1976 ◽  
Vol 48 (4) ◽  
pp. 601-608
Author(s):  
FD Wilson ◽  
L O'Grady
Keyword(s):  
Bone Marrow ◽  
Colony Growth ◽  
Hematopoietic Stem ◽  
Quantitative Studies ◽  
Bone Marrow Cultures ◽  
Marrow Stroma ◽  
Constant Cell ◽  
Hematopoietic Stroma

Studies on the mechanism of anemia in mice of genotype S1/S1d have implicated the hematopoietic stroma (the hematopoietic inductive microenvironment, HIM) rather than hematopoietic stem cells as the site of the defect. Using methylcellulose-supported bone marrow culture systems, we have observed, in addition to classical hematopoietic colonies, the formation of surface associated fibroblastic plaques that could stimulate hematopoietic colony growth. These plaques were hypothesized to be derived from bone marrow stroma precursors. In view of the reported stromal-based defect in S1/S1d mice, studies were initiated, using our culture system, to determine if abnormalities exist in the plaque-forming potentials of these mice. Relative to controls, bone marrow derived from S1/S1d mice exhibited a significant decrease in hematopoietic colonly-forming units in culture, but no differences were apparent in the absolute numbers of fibroblastic plaque-forming units or in the ability of such plaques once derived to stimulate hematopoietic colony growth when overlain with fresh normal bone marrow preparations. Quantitative studies on the bone marrow of the S1/S1d mice revealed a marked reduction in total nucleated cells per femur. The importance of evaluating the results of bone marrow cultures in an absolute (i.e., number of units per femur) rather than a relative (i.e., number of units forming in a constant cell inoculum) term was underlined by these studies.

scholarly journals Some observations of the hematopoietic status in vivo and in vitro on mice of genotype S1/S1d

Blood ◽  
1976 ◽  
Vol 48 (4) ◽  
pp. 601-608 ◽  
Author(s):  
FD Wilson ◽  
L O'Grady
Keyword(s):  
Bone Marrow ◽  
Colony Growth ◽  
Hematopoietic Stem ◽  
Quantitative Studies ◽  
Bone Marrow Cultures ◽  
Marrow Stroma ◽  
Constant Cell ◽  
Hematopoietic Stroma

Abstract Studies on the mechanism of anemia in mice of genotype S1/S1d have implicated the hematopoietic stroma (the hematopoietic inductive microenvironment, HIM) rather than hematopoietic stem cells as the site of the defect. Using methylcellulose-supported bone marrow culture systems, we have observed, in addition to classical hematopoietic colonies, the formation of surface associated fibroblastic plaques that could stimulate hematopoietic colony growth. These plaques were hypothesized to be derived from bone marrow stroma precursors. In view of the reported stromal-based defect in S1/S1d mice, studies were initiated, using our culture system, to determine if abnormalities exist in the plaque-forming potentials of these mice. Relative to controls, bone marrow derived from S1/S1d mice exhibited a significant decrease in hematopoietic colonly-forming units in culture, but no differences were apparent in the absolute numbers of fibroblastic plaque-forming units or in the ability of such plaques once derived to stimulate hematopoietic colony growth when overlain with fresh normal bone marrow preparations. Quantitative studies on the bone marrow of the S1/S1d mice revealed a marked reduction in total nucleated cells per femur. The importance of evaluating the results of bone marrow cultures in an absolute (i.e., number of units per femur) rather than a relative (i.e., number of units forming in a constant cell inoculum) term was underlined by these studies.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

Thrombopoietin-Induced Stimulation of Megakaryocyte- Enriched Bone Marrow Cultures

1979 ◽  
Author(s):  
K. L. Kellar ◽  
B. L. Evatt ◽  
C. R. McGrath ◽  
R. B. Ramsey
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Bone Marrow Cells ◽  
Rabbit Plasma ◽  
Bone Marrow Cultures ◽  
Plasma Fractions ◽  
Marrow Cultures ◽  
Stimulation Of

Liquid cultures of bone marrow cells enriched for megakaryocytes were assayed for incorporation of 3H-thymidine (3H-TdR) into acid-precipitable cell digests to determine the effect of thrombopoietin on DNA synthesis. As previously described, thrombopoietin was prepared by ammonium sulfate fractionation of pooled plasma obtained from thrombocytopenic rabbits. A control fraction was prepared from normal rabbit plasma. The thrombopoietic activity of these fractions was determined in vivo with normal rabbits as assay animals and the rate of incorporation of 75Se-selenomethionine into newly formed platelets as an index of thrombopoietic activity of the infused material. Guinea pig megakaryocytes were purified using bovine serum albumin gradients. Bone marrow cultures containing 1.5-3.0x104 cells and 31%-71% megakaryocytes were incubated 18 h in modified Dulbecco’s MEM containing 10% of the concentrated plasma fractions from either thrombocytopenic or normal rabbits. In other control cultures, 0.9% NaCl was substituted for the plasma fractions. 3H-TdR incorporation was measured after cells were incubated for 3 h with 1 μCi/ml. The protein fraction containing thrombopoietin-stimulating activity caused a 25%-31% increase in 3H-TdR incorporation over that in cultures which were incubated with the similar fraction from normal plasma and a 29% increase over the activity in control cultures to which 0.9% NaCl had been added. These data suggest that thrombopoietin stimulates DNA synthesis in megakaryocytes and that this tecnique may be useful in assaying thrombopoietin in vitro.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

scholarly journals Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

2021 ◽  
Author(s):  
Zixian Liu ◽  
Jinhong Wang ◽  
Miner Xie ◽  
Peng Wu ◽  
Yao Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Pcr Analysis ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Colony Assay ◽  
Megakaryocyte Progenitors ◽  
Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

scholarly journals The Kinetics of the Normal, the Megaloblastic and the Sideroachrestic Erythropoiesis Estimated by Colchicine Blocking In Vitro

Blood ◽  
1971 ◽  
Vol 37 (2) ◽  
pp. 204-210 ◽  
Author(s):  
I. T. M. BOLL ◽  
H.-P. KOENIGS
Keyword(s):  
Bone Marrow ◽  
Maturation Stage ◽  
Ineffective Erythropoiesis ◽  
Cell Doubling Time ◽  
Bone Marrow Cultures ◽  
Delayed Maturation ◽  
Kinetics Of ◽  
Very High

Abstract By adding colchicine to bone marrow cultures we developed further parameters for kinetics in normal, megaloblastic and sideroachrestic bone marrow. The increased regeneration in megalopoiesis is demonstrated by an increased mitotic index, an increased stathmokinetic index, a shortened cell doubling time and the prolongation of the divisable pool to the oxyphile erythroblasts which only mature in the normal state. To get ineffective erythropoiesis, the maturation in vivo must have been delayed by an increased number of generations up to the formation of megalocytes. From the stathmokinetic test in vitro, the maturation in megalopoiesis is accelerated as a result of the inhibition of α-2 α-divisions. In normal erythropoiesis stopping mitoses by colchicine probably causes a delayed maturation because the next maturation stage cannot be reached without the regular n-2n-division. In sideroachrestic anemia, the maturation behaves normally but the stathmokinetic test is very high. We conclude that the maturation and mode of division in sideroachrestic anemia is nearly normal.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3216-3226 ◽  
Author(s):  
Aisha V. Sauer ◽  
Emanuela Mrak ◽  
Raisa Jofra Hernandez ◽  
Elena Zacchi ◽  
Francesco Cavani ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Severe Combined Immunodeficiency ◽  
Intrinsic Defect ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Bone Phenotype

Abstract Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4680-4686 ◽  
Author(s):  
Kent W. Christopherson ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mouse Bone Marrow ◽  
Peptidase Activity ◽  
Hematopoietic Stem ◽  
Migratory Response ◽  
Stromal Cell Derived Factor ◽  
Marrow Cells

AbstractCXC ligand 12 (CXCL12; also known as stromal cell–derived factor 1α/SDF-1α) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin— hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit—lin— cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin— or Sca-1+c-kit—lin— mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin— mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit—lin— cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)–induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.

scholarly journals CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy

Blood ◽  
2009 ◽  
Vol 113 (18) ◽  
pp. 4341-4351 ◽  
Author(s):  
Abdel Kareem Azab ◽  
Judith M. Runnels ◽  
Costas Pitsillides ◽  
Anne-Sophie Moreau ◽  
Feda Azab ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Therapeutic Agents ◽  
Cytotoxic Agents ◽  
Parp Cleavage ◽  
Hematopoietic Stem ◽  
Akt Phosphorylation ◽  
Enhanced Sensitivity

Abstract The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) provides a protective environment and resistance to therapeutic agents. We hypothesized that disruption of the interaction of MM cells with their BM milieu would lead to their sensitization to therapeutic agents such as bortezomib, melphalan, doxorubicin, and dexamethasone. We report that the CXCR4 inhibitor AMD3100 induces disruption of the interaction of MM cells with the BM reflected by mobilization of MM cells into the circulation in vivo, with kinetics that differed from that of hematopoietic stem cells. AMD3100 enhanced sensitivity of MM cell to multiple therapeutic agents in vitro by disrupting adhesion of MM cells to bone marrow stromal cells (BMSCs). Moreover, AMD3100 increased mobilization of MM cells to the circulation in vivo, increased the ratio of apoptotic circulating MM cells, and enhanced the tumor reduction induced by bortezomib. Mechanistically, AMD3100 significantly inhibited Akt phosphorylation and enhanced poly(ADP-ribose) polymerase (PARP) cleavage as a result of bortezomib, in the presence of BMSCs in coculture. These experiments provide a proof of concept for the use of agents that disrupt interaction with the microenvironment for enhancement of efficacy of cytotoxic agents in cancer therapy.

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