scholarly journals The Development of Human Megakaryocytes: III. Development of Mature Megakaryocytes From Highly Purified Committed Progenitors in Synthetic Culture Media and Inhibition of Thrombopoietin-Induced Polyploidization by Interleukin-3

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 426-434 ◽  
Author(s):  
Alexandr Dolzhanskiy ◽  
Ross S. Basch ◽  
Simon Karpatkin
Keyword(s):  
Bone Marrow ◽  
Cell Sorting ◽  
Stem Cell Factor ◽  
Magnetic Beads ◽  
Culture Media ◽  
Serum Free ◽  
Interleukin 3 ◽  
Colony Forming Units ◽  
Affinity Techniques ◽  
Synthetic Media

Abstract Megakaryocyte (MK) progenitors, CD34+CD41+ cells, were isolated from human bone marrow with a purity greater than 98% and a viability of 95%, using affinity techniques with magnetic beads followed by fluorescence-activated cell sorting. These cells were incubated in synthetic media containing the cytokines thrombopoietin (TPO), interleukin-3 (IL-3), stem cell factor (SCF ), and IL-6, obviating the confounding effects of serum growth factors or cytokine secretions of non-MK cells on MK maturation. MK number, MK colony-forming units (CFU-MK), and MK ploidy and phenotype were examined during 7 days in culture. TPO in serum-free cultures without any other exogenously added cytokine supported MK growth and maturation. SCF synergized with TPO to augment MK production and maturation and could partially replace it under some conditions. Both TPO and IL-3 alone increased MK number (12- and 5-fold, respectively) and CFU-MK (∼15-fold each). SCF alone had no effect on MK proliferation in the absence of TPO, but increased both MK number and CFU-MK by 1.5- to 2.0-fold in the presence of TPO. When combined with IL-3, SCF increased both MK number and CFU-MK by 15- to 20-fold in the absence of TPO. In the presence of TPO, the combination of IL-3 and SCF produced only modest increases (1.5- to 2.0-fold) in both MK number and CFU-MK. The proportion of polyploid MK increased greater than fivefold in the presence of TPO. SCF had little effect on MK ploidy in the presence of TPO, but enhanced ploidy twofold to threefold in the absence of TPO. IL-3 alone never increased the level of polyploidization. Rather, it consistently inhibited TPO- and SCF-induced polyploidization of MK. This inhibition was observed in cultures with or without SCF or IL-6. Although IL-3 also supported the proliferation of CD41+ cells and CFU-MK production, the cells that developed under the influence of IL-3 were phenotypically unusual (CD41dim, CD42dim) and of relatively low ploidy. Mature MK were not produced. When added with TPO, IL-3 suppressed polyploidization. Therefore, TPO stimulates MK growth and maturation, whereas IL-3 stimulates growth without maturation and may serve to conserve the immature MK compartment.

scholarly journals Effects of interleukin-3 and erythropoietin on in vivo erythropoiesis and F-cell formation in primates

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1571-1576 ◽  
Author(s):  
T Umemura ◽  
A al-Khatti ◽  
RE Donahue ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Cell Formation ◽  
Erythroid Progenitors ◽  
Hemoglobin F ◽  
Interleukin 3 ◽  
Colony Forming Units ◽  
Early Increase ◽  
Bone Marrow Cultures

Abstract To test the in vivo cooperativity between interleukin-3 (IL-3) and erythropoietin (Epo) in stimulating erythropoiesis and hemoglobin F (HbF) production in primates, we administered recombinant human IL-3 and recombinant human Epo to baboons and macaques. The effect of these treatments was assessed by serial bone marrow cultures and by measuring HbF production in the progeny of bone marrow progenitors and in peripheral-blood reticulocytes. Administration of IL-3 alone to hematologically normal or anemic baboons produced an early increase in erythroid colony-forming units (CFUe) and erythroid clusters (e- clusters) with an increase in reticulocyte counts and a late increment in the relative frequency of erythroid burst-forming units (BFUe). In parallel to the increase in peripheral-blood reticulocytes, IL-3 increased the frequency of F reticulocytes in the normal and anemic animals. When administration of IL-3 was followed by administration of Epo, expansion in all classes of erythroid progenitors and increase in reticulocytes occurred, beyond the levels observed when the animals were treated with Epo alone. The combination of IL-3 and Epo, however, did not increase consistently the rate of F reticulocytes beyond the level induced by Epo alone. These results suggest that IL-3 enhances the effect of Epo on erythropoiesis, but the combination of the two growth factors does not lead to a preferential and significant enhancement of HbF production.

scholarly journals Effects of stem cell factor (kit-ligand) and interleukin-3 on the growth and serine proteinase expression of rat bone-marrow-derived or serosal mast cells

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 72-83 ◽  
Author(s):  
DM Haig ◽  
JF Huntley ◽  
A MacKellar ◽  
GF Newlands ◽  
L Inglis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mast Cells ◽  
Stem Cell Factor ◽  
Synergistic Effects ◽  
Serine Proteinase ◽  
In Vitro Growth ◽  
Interleukin 3 ◽  
Kit Ligand

Abstract The effects of rat stem-cell factor (SCF) and interleukin-3 (IL-3), alone or in combination, on the in vitro growth and serine proteinase expression of rat serosal/connective-tissue mast cells (CTMC) or bone marrow-derived mast cells (BMMC) were examined. Rat SCF stimulated the growth of both CTMC and BMMC. IL-3 stimulated BMMC growth to a lesser extent than did SCF, whereas CTMC numbers did not increase in IL-3. However, SCF and IL-3 had synergistic effects on the growth of both BMMC and CTMC. SCF favoured the maintenance of rat mast cell proteinase- I (RMCP-I) in CTMC, but did not induce detectable production of RMCP-I in BMMC. In contrast, when IL-3 or lymph node-conditioned medium (LNCM) was added to SCF, a subpopulation of CTMC expressed and stored the soluble proteinase RMCP-II. In BMMC, the RMCP-II content of cells maintained in SCF was significantly less than that of cells maintained in IL-3 or LNCM. RMCP-II also appeared in the supernatants of BMMC, especially when BMMC numbers were increasing rapidly in SCF with or without IL-3 or LNCM. Thus, SCF and IL-3 can regulate the growth of rat BMMC and CTMC, as well as influence their production and release of proteinases.

scholarly journals Surface antigenic determinants on human pluripotent and unipotent hematopoietic progenitor cells

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 1006-1010 ◽  
Author(s):  
MP Bodger ◽  
CA Izaguirre ◽  
HA Blacklock ◽  
AV Hoffbrand
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Sorting ◽  
Progenitor Cell ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Colony Forming Units

RFB-1 is a monoclonal antibody previously shown to react with granulocyte-monocyte progenitors (CFU-GM) and immature lymphoid cells in human bone marrow. RFB-HLA-DR is a monoclonal antibody that reacts with HLA-DR (la-like) antigens. The present study shows that the bone marrow subset reactive with both RFB-1 and RFB-HLA-DR contains all the cells that give rise to mixed hematopoietic colonies (derived from CFU- GEMM; a pluripotent human progenitor cell) as well as to megakaryocytic (megakaryocyte-CFU-derived) and erythropoietic (derived from erythroid burst-forming units, BFU-E) colonies, as shown by fluorescence- activated cell sorting and complement-mediated cytotoxicity. These results indicate that CFU-GEMM, BFU-E, and megakaryocyte-CFU express RFB-1 and la-like antigens. RFB-1 antigen is also expressed on erythroid colony-forming units (CFU-E). RFB-1 and RFB-HLA-DR are useful reagents in the study of hematopoietic stem cells.

scholarly journals Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

2013 ◽  
Vol 33 (suppl 1) ◽  
pp. 20-24 ◽  
Author(s):  
Gesiane Ribeiro ◽  
Cristina O. Massoco ◽  
José Corrêa de Lacerda Neto
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
Adipose Tissue ◽  
Culture Media ◽  
Stromal Vascular Fraction ◽  
Bone Marrow Aspiration ◽  
Cell Culture Media ◽  
Colony Forming Units ◽  
Fetal Bovine ◽  
Equine Bone

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

Synergism of interferon-gamma and stem cell factor on the development of murine hematopoietic progenitors in serum-free culture

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1435-1441 ◽  
Author(s):  
M Shiohara ◽  
K Koike ◽  
T Nakahata
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Interferon Gamma ◽  
Stem Cell Factor ◽  
Bone Marrow Cells ◽  
Colony Growth ◽  
Hematopoietic Progenitors ◽  
Ifn Gamma ◽  
Serum Free ◽  
Marrow Cells

Abstract We examined the effects of interferon-gamma (IFN-gamma) on the growth of murine hematopoietic progenitors supported by interleukin-3 (IL-3) or stem cell factor (SCF) in a serum-free culture system. IFN-gamma inhibited IL-3-dependent granulocyte-macrophage colony growth by normal bone marrow cells, but increased the number of pure and mixed megakaryocyte colonies by post-5-fluorouracil bone marrow cells. The addition of IFN-gamma to the culture containing SCF resulted in a synergistic action on the development of primitive hematopoietic progenitors as well as on the development of mature populations. Primitive progenitors responding to SCF + IFN-gamma were suggested to be supported by SCF in the early stage of development and require IFN- gamma for subsequent growth. Replating experiments of blast cell colonies and comparison of total colony growth among SCF + IFN-gamma, SCF + IL-3, and SCF + IFN-gamma + IL-3 suggest that multipotential progenitors supported by SCF + IFN-gamma are a part of those reactive to SCF + IL-3. These findings suggest that IFN-gamma has bifunctional activity on murine hematopoiesis.

scholarly journals Surface antigenic determinants on human pluripotent and unipotent hematopoietic progenitor cells

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 1006-1010 ◽  
Author(s):  
MP Bodger ◽  
CA Izaguirre ◽  
HA Blacklock ◽  
AV Hoffbrand
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Sorting ◽  
Progenitor Cell ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Colony Forming Units

Abstract RFB-1 is a monoclonal antibody previously shown to react with granulocyte-monocyte progenitors (CFU-GM) and immature lymphoid cells in human bone marrow. RFB-HLA-DR is a monoclonal antibody that reacts with HLA-DR (la-like) antigens. The present study shows that the bone marrow subset reactive with both RFB-1 and RFB-HLA-DR contains all the cells that give rise to mixed hematopoietic colonies (derived from CFU- GEMM; a pluripotent human progenitor cell) as well as to megakaryocytic (megakaryocyte-CFU-derived) and erythropoietic (derived from erythroid burst-forming units, BFU-E) colonies, as shown by fluorescence- activated cell sorting and complement-mediated cytotoxicity. These results indicate that CFU-GEMM, BFU-E, and megakaryocyte-CFU express RFB-1 and la-like antigens. RFB-1 antigen is also expressed on erythroid colony-forming units (CFU-E). RFB-1 and RFB-HLA-DR are useful reagents in the study of hematopoietic stem cells.

scholarly journals Macrophage colony formation supported by purified CSF-1 and/or interleukin 3 in serum-free culture: evidence for hierarchical difference in macrophage colony-forming cells

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 859-864 ◽  
Author(s):  
K Koike ◽  
ER Stanley ◽  
JN Ihle ◽  
M Ogawa
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Culture System ◽  
Bone Marrow Cells ◽  
Serum Free ◽  
Interleukin 3 ◽  
Two Factors ◽  
Macrophage Colony ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Using a serum-free culture system, we examined murine macrophage colony formation from bone marrow cells cultured in the presence of purified CSF-1, interleukin 3 (IL 3) or a combination of the two factors. CSF-1 supported macrophage and neutrophil-macrophage colony formation, whereas IL-3 supported the formation of various types of single lineage and multilineage colonies. CSF-1 supported more macrophage colonies from bone marrow cells of normal mice than IL 3, whereas in cultures of bone marrow cells of 5-fluorouracil-treated mice, IL 3 supported more macrophage colonies. A combination of CSF-1 and IL 3 resulted in granulocyte-macrophage (GM) colony formation that was equal to or greater than the sum of GM colony formation supported by the factors individually. The combination of CSF-1 and IL 3 resulted in significant increases in the size of both macrophage and neutrophil-macrophage colonies. Similar increases in colony size were observed when CSF-1 was added to cultures five days after incubation of marrow cells with IL 3. These data support the concept that some of the macrophage colony- forming cells that respond to IL 3 are more primitive than those that are sensitive to CSF-1.

scholarly journals Interleukin 3 perfusion in W/Wv mice allows the development of macroscopic hematopoietic spleen colonies and restores cutaneous mast cell number.

1990 ◽  
Vol 172 (1) ◽  
pp. 403-406 ◽  
Author(s):  
C Ody ◽  
V Kindler ◽  
P Vassalli
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Bone Marrow Cells ◽  
Cell Number ◽  
Interleukin 3 ◽  
Normal Number ◽  
Colony Forming Units ◽  
Mast Cell Number ◽  
Cutaneous Mast Cell ◽  
Marrow Cells

The genetically anemic W/Wv mice are characterized by the inability of their bone marrow cells to form macroscopic pluripotent hematopoietic colonies in the spleen of irradiated recipients upon transfer (colony-forming units). Furthermore, they almost totally lack mast cells, notably in the skin. In the present study, we have tested the effect of recombinant murine interleukin 3 (rmIL-3) on W/Wv mice hematopoiesis. Transfer of W/Wv bone marrow cells into lethally irradiated recipients perfused with rmIL-3 is followed by the appearance of macroscopic spleen colonies. Moreover, perfusion of rmIL-3 in W/Wv mice: (a) restores almost normal total numbers of hematopoietic precursors (colony-forming cells), but without modification of anemia; and (b) leads to the appearance of a normal number of mastocytes in the skin.

Mechanisms of Erythroid Precursor Expansion in Response to Erythropoietin and Stem Cell Factor: Requirements for Cooperative Signal Transduction.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2182-2182 ◽  
Author(s):  
Murat O. Arcasoy ◽  
Xiaohong Jiang
Keyword(s):  
Signal Transduction ◽  
Bone Marrow ◽  
Stem Cell ◽  
Red Blood Cells ◽  
Tyrosine Phosphorylation ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Stem Cell Factor ◽  
Erythroid Precursor ◽  
Serum Free

Abstract The adult bone marrow produces 2x1011 red blood cells daily to maintain the oxygen carrying capacity of peripheral blood. Certain pathologic conditions such as blood loss or hemolysis that result in increased oxygen demands lead to a physiologic bone marrow response characterized by increased rate of erythropoiesis and expansion of bone marrow proerythroblasts, the precursor cells that differentiate into mature red blood cells. We studied the mechanisms by which erythropoietin (EPO) and stem cell factor (SCF) regulate the expansion of primary human proerythroblasts. Using liquid cultures of peripheral blood CD34+ cells isolated from healthy volunteers, we generated uniform populations of transferrin receptor (CD71)+ human proerythroblasts. We established a serum-free culture model to study the effect of EPO and SCF on the survival and proliferative capacity of the cells. Primary proerythroblasts failed to survive in the presence of EPO or SCF alone, but exhibited marked synergistic proliferation in response to EPO plus SCF, exhibiting one log expansion in 5 days under serum-free conditions. Characterization of EPO receptor (EPOR) and SCF receptor (KIT)-mediated signal transduction in proerythroblasts revealed a requirement for EPOR, but not KIT, signaling for tyrosine phosphorylation of STAT5 (Tyr694), a downstream target for the cytoplasmic tyrosine kinase JAK2. MAP kinases ERK 1/2 were phosphorylated (Thr202/Tyr204) in response to either EPO or SCF alone, with phosphorylation of ERK1/2 induced predominantly by SCF. We found increased phosphorylation of ERK 1/2 when proerythroblasts were treated with both EPO and SCF. Phosphorylation of protein kinase B/Akt (Ser473), a signaling molecule downstream of phosphatidylinositol 3-kinase (PI3K), was observed following SCF treatment. Treatment with kinase inhibitors targeting JAK, PI3K and MAP kinase kinase (MEK1) during EPO and SCF stimulation revealed that JAK inhibitor AG490 attenuated STAT5 tyrosine phosphorylation, MEK inhibitor PD98059 abolished ERK 1/2 phosphorylation and the PI3K inhibitor LY294002 inhibited Akt phosphorylation. To determine the contribution of specific signaling pathways to synergistic proliferation of proerythroblasts in response to cooperative effects of EPO and SCF, we performed proliferation assays with increasing concentrations of each inhibitor (5 and 50 μM) or DMSO vehicle. We found significant, dose-dependent inhibition of proerythroblast proliferation in response to all three JAK, PI3K or MEK inhibitors (figure 1, *P<0.001 by ANOVA, n=6). We conclude that 1)- EPO or SCF alone fail to support proerythroblast survival, 2)- The cooperative and synergistic effects of EPO and SCF are required for the expansion of primary erythroid precursors, 3)- EPOR but not KIT signaling mediates tyrosine phosphorylation of STAT5 in proerythroblasts, 4)- Phosphorylation of Akt and ERK1/2 in proerythroblasts is mediated primarily by SCF, 5)- EPO and SCF together lead to increased phosphorylation of ERK 1/2, and 6)- The cooperative effect of EPO and SCF that mediates synergistic erythroid precursor expansion requires activation of multiple signaling pathways, including the JAK-STAT, PI3K and MAP kinase pathways. Figure Figure

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