Generation of dendritic cells from human bone marrow mononuclear cells: Advantages for clinical application in comparison to peripheral blood monocyte derived cells

Abstract Colonies of cells with distinctive dendritic appearance were observed in methylcellulose cultures of human bone marrow and peripheral blood mononuclear cells (PBMC). Such cells appeared alone in colonies of less than 50 cells, together with macrophages in mixed colonies and also within clusters of T lymphocytes at high culture cell numbers. The morphologic resemblance to lymphoid dendritic cells was confirmed by electron microscopy and the cells were distinguished from macrophages by immunoenzymatic and immunogold labeling with monoclonal antibodies (MoAbs). Like macrophages they were HLA-DR+ and CD4+. However, they lacked nonspecific esterase and the macrophage cytoplasmic marker Y1/82A. Most strikingly, cells were strongly HLA-DQ+ and expressed CD1a (T6), which is characteristic of skin Langerhans cells. Their functional similarity to lymphoid dendritic cells was demonstrated by their ability to stimulate allogeneic mixed leukocyte reactions. Dendritic cell colony numbers were estimated in both bone marrow and peripheral blood of controls and in leukemia and lymphoma patients before and after chemotherapy. Colony numbers were low in control blood and in patients before treatment (less than 1.0 to 3.7/10(5) cells). However, during hematopoietic recovery the mean value increased to 37.5/10(5) cells and this increase correlated closely with the observed increase in circulating colony forming unit-granulocyte macrophage (CFU- GM) in individual patients. Autoradiographic studies demonstrated mitotic activity within CD1a+ colonies and a linear relationship between cultured cells and both pure and mixed colonies was consistent with their derivation from a single precursor. These data indicate that a novel hematopoietic progenitor of dendritic/Langerhans cells (DL-CFU) may now be identified in a clonal assay system and suggest a probable common progenitor for these cells and macrophages.

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Identification of hematopoietic progenitors of macrophages and dendritic Langerhans cells (DL-CFU) in human bone marrow and peripheral blood

Blood ◽

10.1182/blood.v76.6.1139.bloodjournal7661139 ◽

1990 ◽

Vol 76 (6) ◽

pp. 1139-1149 ◽

Cited By ~ 1

Author(s):

CD Reid ◽

PR Fryer ◽

C Clifford ◽

A Kirk ◽

J Tikerpae ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Peripheral Blood ◽

Langerhans Cells ◽

Mononuclear Cells ◽

Cultured Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Nonspecific Esterase ◽

Hla Dq

Colonies of cells with distinctive dendritic appearance were observed in methylcellulose cultures of human bone marrow and peripheral blood mononuclear cells (PBMC). Such cells appeared alone in colonies of less than 50 cells, together with macrophages in mixed colonies and also within clusters of T lymphocytes at high culture cell numbers. The morphologic resemblance to lymphoid dendritic cells was confirmed by electron microscopy and the cells were distinguished from macrophages by immunoenzymatic and immunogold labeling with monoclonal antibodies (MoAbs). Like macrophages they were HLA-DR+ and CD4+. However, they lacked nonspecific esterase and the macrophage cytoplasmic marker Y1/82A. Most strikingly, cells were strongly HLA-DQ+ and expressed CD1a (T6), which is characteristic of skin Langerhans cells. Their functional similarity to lymphoid dendritic cells was demonstrated by their ability to stimulate allogeneic mixed leukocyte reactions. Dendritic cell colony numbers were estimated in both bone marrow and peripheral blood of controls and in leukemia and lymphoma patients before and after chemotherapy. Colony numbers were low in control blood and in patients before treatment (less than 1.0 to 3.7/10(5) cells). However, during hematopoietic recovery the mean value increased to 37.5/10(5) cells and this increase correlated closely with the observed increase in circulating colony forming unit-granulocyte macrophage (CFU- GM) in individual patients. Autoradiographic studies demonstrated mitotic activity within CD1a+ colonies and a linear relationship between cultured cells and both pure and mixed colonies was consistent with their derivation from a single precursor. These data indicate that a novel hematopoietic progenitor of dendritic/Langerhans cells (DL-CFU) may now be identified in a clonal assay system and suggest a probable common progenitor for these cells and macrophages.

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Arachidonic Acid and Freshly Isolated Human Bone Marrow Mononuclear Cells

Mediators of Inflammation ◽

10.1080/09629359990694 ◽

1999 ◽

Vol 8 (1) ◽

pp. 31-35 ◽

Cited By ~ 3

Author(s):

Y. Denizot ◽

V. Desplat ◽

C. Dulery ◽

F. Trimoreau ◽

V. Praloran

Keyword(s):

Bone Marrow ◽

Arachidonic Acid ◽

Fatty Acid ◽

Mononuclear Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Bone Marrow Mononuclear Cells ◽

Gm Csf ◽

Chromatography Analysis ◽

Bone Marrow Plasma

Arachidonic acid (AA), a fatty acid found in the human bone marrow plasma, is the precursor of eicosanoids that modulate bone marrow haematopoiesis. To further our understanding of the role of AA in the bone marrow physiology, we have assessed its incorporation in human bone marrow mononuclear cells. Gas chromatography analysis indicates the presence of AA in their fatty acid composition. In bone marrow mononuclear cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids, a result not observed with blood mononuclear cells. Prelabelling-chase experiments indicate a trafficking of labelled AA from phosphatidylcholine to phosphatidylethanolamine. Stimulation of prelabelled bone marrow mononuclear cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the release of a part of the incorporated labelled AA. Finally, exogenous AA (up to 1 μM) has no significant effect on cell growth. In conclusion, human bone marrow mononuclear cells participate to the control of marrow AA concentrations by incorporating AA into phospholipids and triglycerides. In turn, bone marrow mononuclear cells can release AA in response to the potent haematopoietic growth factor GM-CSF.

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Cryopreserved human bone marrow mononuclear cells as a source of mesenchymal stromal cells: application in osteoporosis research

Cytotherapy ◽

10.1080/14653240802192644 ◽

2008 ◽

Vol 10 (5) ◽

pp. 460-468 ◽

Cited By ~ 33

Author(s):

A. Casado-Díaz ◽

R. Santiago-Mora ◽

R. Jiménez ◽

J. Caballero-Villarraso ◽

C. Herrera ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Mononuclear Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Bone Marrow Mononuclear Cells ◽

Osteoporosis Research

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HUMAN BONE MARROW MONONUCLEAR CELLS AS EFFECTORS AND TARGETS OF NATURAL KILLING

NK Cells and Other Natural Effector Cells ◽

10.1016/b978-0-12-341360-4.50160-8 ◽

1982 ◽

pp. 1085-1090 ◽

Cited By ~ 1

Author(s):

Sudhir Gupta ◽

Gabriel Fernandes

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Bone Marrow Mononuclear Cells ◽

Natural Killing

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Characterisation of Side Population (SP) Cells Obtained from Different Hematopoietic Progenitor/Stem Cell Compartments in Human Bone Marrow and Peripheral Blood.

Blood ◽

10.1182/blood.v108.11.4170.4170 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4170-4170

Author(s):

Dag Josefsen ◽

Lise Forfang ◽

Marianne Dyrhaug ◽

Gunnar Kvalheim

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Population ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Hematopoietic Stem ◽

Cell Compartments ◽

Cd34 Cells

Abstract Side population (SP) cells are characterised by their ability to exclude Hoechst 33342 dye from the cells. Using this method, it has been demonstrated that cells within the SP+ fraction of mononuclear cells from both murine and human hematopoietic systems are enriched for primitive hematopoietic stem- and progenitor cells. Moreover, most of the SP+ cells did not express CD34, indicating the presence of a CD34 negative hematopoietic stem cell population. To explore this further, we have examined SP+ cells obtained from different cell compartments in human bone marrow and peripheral blood. Human bone marrow (BM) was obtained from healthy volunteer donors by iliac crest aspiration after informed consent. Mononuclear cells (MNC) were obtained by Ficoll grade centrifugation. CD34+ cells were then isolated from MNC. Highly enriched CD34+ cells were isolated from PBPC obtained from patients with Hodgkin lymphoma. To identify the SP+ cells, the cells were stained with Hoechst 33342 dye. Using flowcytometric techniques (FACStar+, FACSDiva, Becton Dickinson, San Jose, CA) we were able to visualize the dye efflux in SP+ cells. SP+ cells were functionally confirmed using Verapamil. Phenotypical characterisation of the different cell populations using flow cytometric methods was performed. The level of SP+ cells in BM-MNC was 1,3% (mean, n=3) In line with previous findings, we observed that SP+ cells obtained from BM-MNC lack expression of several lineage committed markers, including CD15 and CD19. Most of the cells were CD34− (mean=2,2%), which was lower than in the main population (MP; mean=5%). The level of CD133 expression was low and similar in both populations. Furthermore we found a higher fraction of CD3+ T-cells in the SP fraction than in the MP fraction (mean: 69% vs 51%). To further investigate the SP+CD34+ cell fraction, we examined CD34+ cells isolated from both human bone marrow and peripheral blood. The percentage of SP+CD34+ cells varied from 0,4 up to 18% of the total CD34+ cell population obtained from PBPC (n= 16), whereas the level of SP+CD34+ cells obtained from bone marrow was 5% of the total CD34+ cell population (n=3). Expression of lineage committed markers, including CD10, CD15 and CD19 was less then 10% of the whole CD34+ cell population obtained from PBPC, whereas we found a higher level of expression of these markers in CD34+ cells isolated from bone marrow. However, when we examined the SP+CD34+ cells from either PBPC or bone marrow, we observed that the phenotypic profile of these cells were similar with almost no expression of lineage markers. The frequency of LTC-IC was markedly increased in SP+MNC, in line with previous findings. In addition we also observed a marked increase in LTC-IC in SP+CD34+ cells compared to SP-CD34+ cells in both BM and PB (BM: 7-fold increase; PB: 3–4 fold). In conclusion, SP cells are present in different hematopoietic progenitor cell populations, including BM-MNC, BM-CD34+ cells and PB-CD34+ cells. In SP+CD34+ cell fractions from both BM and PB we observed an increased expression of stem cell markers like CD90 and CD133, whereas in SP+MNC we found low levels of CD34, CD90 and CD133 expression. However, the LTC-IC frequency was markedly higher in all SP+fractions compared to MP fractions, suggesting that sorting of SP+ cells from different hematopoietic stem- and progenitor cell compartments identify immature hematopoietic cells.

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Proteomic Characteristics ofex vivo-Enriched Adult Human Bone Marrow Mononuclear Cells in Continuous Perfusion Cultures

Journal of Proteome Research ◽

10.1021/pr801064u ◽

2009 ◽

Vol 8 (4) ◽

pp. 2079-2089 ◽

Cited By ~ 9

Author(s):

Agasanur K. Prahalad ◽

Janet M. Hock

Keyword(s):

Bone Marrow ◽

Mononuclear Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Bone Marrow Mononuclear Cells ◽

Adult Human ◽

Perfusion Cultures ◽

Continuous Perfusion

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Human Bone Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine Production by Peripheral Blood Monocytes Subpopulations and Myeloid Dendritic Cells

Stem Cells International ◽

10.1155/2015/819084 ◽

2015 ◽

Vol 2015 ◽

pp. 1-15 ◽

Cited By ~ 16

Author(s):

Paula Laranjeira ◽

Joana Gomes ◽

Susana Pedreiro ◽

Monia Pedrosa ◽

Antonio Martinho ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Protein Expression ◽

Peripheral Blood ◽

Immune Cell ◽

Human Bone ◽

Human Bone Marrow ◽

Mrna Levels ◽

Myeloid Dendritic Cells ◽

Experimental Conditions

The immunosuppressive properties of mesenchymal stromal/stem cells (MSC) rendered them an attractive therapeutic approach for immune disorders and an increasing body of evidence demonstrated their clinical value. However, the influence of MSC on the function of specific immune cell populations, namely, monocyte subpopulations, is not well elucidated. Here, we investigated the influence of human bone marrow MSC on the cytokine and chemokine expression by peripheral blood classical, intermediate and nonclassical monocytes, and myeloid dendritic cells (mDC), stimulated with lipopolysaccharide plus interferon (IFN)γ. We found that MSC effectively inhibit tumor necrosis factor- (TNF-)αand macrophage inflammatory protein- (MIP-) 1βprotein expression in monocytes and mDC, without suppressing CCR7 and CD83 protein expression. Interestingly, mDC exhibited the highest degree of inhibition, for both TNF-αand MIP-1β, whereas the reduction of TNF-αexpression was less marked for nonclassical monocytes. Similarly, MSC decreased mRNA levels of interleukin- (IL-) 1βand IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and nonclassical monocytes, and IL-1βand CXCL10 in mDC. MSC do not impair the expression of maturation markers in monocytes and mDC under our experimental conditions; nevertheless, they hamper the proinflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses.

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The Induction of Dendritic Cells with IFN-alpha and GM-CSF from Bone Marrow Mononuclear Cells from Patients with Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v106.11.4873.4873 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4873-4873

Author(s):

Shuier Zhen ◽

Jie Jin ◽

Xiangmin Tong

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

T Cell ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Bone Marrow Mononuclear Cells ◽

Gm Csf

Abstract Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease arising from the clonal expansion of a stem cell with the typical Philadelphia (Ph) chromosome cytogenetic abnormality. IFN-a has been proven to be effective for patients in the chronic phase of myelogenous leukemia (CML), yet the mechanisms of the antitumor action of these cytokines are still a matter of debate. Dendritc cells (DCs) are potent antigen-presenting cells that prime effective T-cell response aginst tumour antigens. Recent studies have shown that IFN-a can exert a variety of effects on dendritic cells (DCs), which may play an important role in the induction of an antitumor immunity. Human DCs can be generated in vitro from peripheral blood(PB) monocytes or from CD34+ haematopoietic precursor cells in culture medium containing human granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4 and some other cytokines. Previous studies have shown a new effective protocol for the generation of human DCs from unseparated BM aspirate cells with excellent functional capacity of antigen uptake and of stimulating naive and memory T cell responses superior to that of DCs from peripheral blood(PB) monocytes. We, therefore, explored whether treatment with IFN-a may influence the CML bone marrow mononuclear cells(BMMNCs) derived DCs in vitro. Treatment BMMNCs of 12 patients with CML in chronic phase with IFN-a+rhGM-CSF(IFN-a-DC) generated DCs with more mature phenotype properties expressing higher of CD80,CD86,HLA-DR,CD83 compared to the CML- BMMNCs treated with rhGM-CSF+IL-4(IL-4-DC). And in parallel with phenotypes, IFN-a-DC also showed more effective than IL-4-DC in eliciting an allogeneic mixed lymphocyte reaction by MTT assay. FISH confirmed the DCs of both groups were leukemic origin. These findings demonstrate that IFN-a promotes the differentiation/maturation of DCs derived from BMMNCs of patients with CML in vitro, these studies also broaden the clinical scope of IFN-a as a promising agent in the immunotherapy of CML.

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