scholarly journals Identification and characterization of osteoclast progenitors by clonal analysis of hematopoietic cells

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1710-1716 ◽  
Author(s):  
MY Lee ◽  
JL Lottsfeldt ◽  
KL Fevold
Keyword(s):  
Bone Marrow ◽  
Tumor Cell ◽  
Mononuclear Cells ◽  
Fetal Liver ◽  
Tumor Cell Line ◽  
Adherent Cell ◽  
Mouse Bone Marrow ◽  
Hematopoietic Progenitors ◽  
Adult Mice

Abstract We have identified a distinct population of colony-forming cells that give rise to mononuclear cells expressing an enzyme marker and other features of the osteoclast in bone marrow cultures stimulated by conditioned medium of a murine tumor cell line. These colony-forming cells were defined as osteoclast colony-forming units (CFU-O). The tumor cell-derived activity was recently isolated and was named osteoclast colony-stimulating factor (O-CSF). To understand the development of osteoclast progenitors and to clarify the relationship of osteoclast progenitors to other hematopoietic progenitors, we examined CFU-O in hematopoietic tissues obtained from normal adult mice, mouse fetuses, and mice with 5-fluorouracil (5FU) treatment. CFU- O were present in the adult mouse bone marrow, adherent cell-depleted marrow, in the spleen, and in the day 14 fetal liver, with an incidence similar to other hematopoietic progenitors. The culture period required for the development of CFU-O-derived colonies in vitro and the manner in which CFU-O responded to 5FU suggested that CFU-O belonged to a relatively primitive progenitor population; they are clearly more immature than macrophage progenitors that respond to macrophage-CSF, but more mature than multilineage progenitors that respond to stem cell factor. Our studies have defined and characterized an osteoclast progenitor and distinguished it from other hematopoietic progenitors for the first time.

scholarly journals Identification and characterization of osteoclast progenitors by clonal analysis of hematopoietic cells

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1710-1716 ◽  
Author(s):  
MY Lee ◽  
JL Lottsfeldt ◽  
KL Fevold
Keyword(s):  
Bone Marrow ◽  
Tumor Cell ◽  
Mononuclear Cells ◽  
Fetal Liver ◽  
Tumor Cell Line ◽  
Adherent Cell ◽  
Mouse Bone Marrow ◽  
Hematopoietic Progenitors ◽  
Adult Mice

We have identified a distinct population of colony-forming cells that give rise to mononuclear cells expressing an enzyme marker and other features of the osteoclast in bone marrow cultures stimulated by conditioned medium of a murine tumor cell line. These colony-forming cells were defined as osteoclast colony-forming units (CFU-O). The tumor cell-derived activity was recently isolated and was named osteoclast colony-stimulating factor (O-CSF). To understand the development of osteoclast progenitors and to clarify the relationship of osteoclast progenitors to other hematopoietic progenitors, we examined CFU-O in hematopoietic tissues obtained from normal adult mice, mouse fetuses, and mice with 5-fluorouracil (5FU) treatment. CFU- O were present in the adult mouse bone marrow, adherent cell-depleted marrow, in the spleen, and in the day 14 fetal liver, with an incidence similar to other hematopoietic progenitors. The culture period required for the development of CFU-O-derived colonies in vitro and the manner in which CFU-O responded to 5FU suggested that CFU-O belonged to a relatively primitive progenitor population; they are clearly more immature than macrophage progenitors that respond to macrophage-CSF, but more mature than multilineage progenitors that respond to stem cell factor. Our studies have defined and characterized an osteoclast progenitor and distinguished it from other hematopoietic progenitors for the first time.

Lateral Gene Transfer Contributing to Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1235-1235
Author(s):  
Joo Hyun Lee ◽  
Cynthia R. Giver ◽  
Sravanti Rangaraju ◽  
Edmund K Waller
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Tumor Cell ◽  
Cell Line ◽  
Tumor Cell Line ◽  
Hematopoietic Stem ◽  
Precursor Frequency ◽  
Marrow Cells

Abstract The uncontrolled proliferation of genetically mutated cells is the commonly understood mechanism for cancer growth and invasion, with accumulation of new mutations in daughter cells leading to clonal diversity of cancer derived from a single founding event. The genetic alterations are passed to new generations by cell division and vertical gene transfer. Viral transmission of oncogenes represents a known mechanism of lateral gene transfer in cancer initiation. Some experimental systems have also suggested that circulating DNA or micro-vesicles may contribute to lateral oncogene transfer in tumorigenesis. We hypothesized that interactions between leukemic cells and adjacent normal hematopoietic stem or progenitor cells may provide an alternative mechanism for the accumulation of mutated genes and the multiplicity of distinct clones in leukemia. To test this hypothesis, we performed experiments to determine whether tumorigenic properties could be transferred from a tumor cell line to normal mouse bone marrow cells using both in vivo and in vitro and systems. B6-GFP+ mice were injected i.v. with 200,000 C1498-Luc cells (a B6-derived NKT-cell-like mouse tumor cell line expressing luciferase and DSRed). Bioluminescent imaging was used to monitor the progression of tumor cell growth in recipients. At 1 month after tumor-cell inoculation, marrow from these mice was harvested and FACS-sorted for GFP+ cells (to eliminate C1498 cells), and then cultured on irradiated stromal cell layers in 96-well plates in a limiting dilution analysis for Poisson analysis of GFP+ clonogenic precursor frequency on day 9. On day 10, cells were harvested from culture and GFP+ cells resorted onto fresh stromal layers for second and third determinations of GFP+ clonogenic precursor frequency on days 15 and 18. As shown in Figure 1, the frequency of clonogenic precursors increased with each successive determination for marrow from C1498-injected mice, while control cultures from non-injected mice showed no increase in precursor frequency, suggesting that exposure to C1498 cells conferred a growth advantage to the marrow cells in the tumor-cell injected mice. Similar results were obtained using an in vitro system of co-culture using C1498 cells and GFP+ bone marrow cells, followed by serial rounds of GFP+ sorting and Poisson analysis, showing increases in clonogenic frequency over 5 successive sorts and re-cultures over a 2-month period, while control cultures showed decreased clonogenic frequencies over the course of the experiment. To confirm these observations in vivo, B6-GFP mice were injected with C1498-Luc and marrow was harvested after a month and sorted for GFP+ cells. The sorted marrow was transplanted into 11Gy-irradiated (FVB x B6albino)F1 recipients (5 x 106 cells per recipient, n=5). Control recipients were irradiated and transplanted with GFP+ marrow from non-injected donors. All recipients developed full hematopoietic engraftment with GFP+ cells. At 6 months post-transplant, a tumor was observed near the left shoulder of one of the recipients of C1498-exposed GFP+ marrow. Figure 2 shows IVIS GFP imaging of this mouse with the GFP+ tumor along with control animals. The tumor was not positive for luciferase expression. The mouse was sacrificed and the tumor excised and a portion was dissociated for flow cytometric analysis and culturing (with other segments reserved for subsequent histological and genetic analysis). Both GFP+ and non-GFP cells were found in the dissociated tumor cell suspension. The GFP+ cells were hematopoietic in origin (CD45+) and exhibited a mixed phenotype containing markers expressed on C1498 (DX5+) and myeloid lineage cells (CD11b+) as well as Sca-1, a stem cell marker. Cultures of the GFP+ tumor yielded a population of GFP+ mononuclear cells. These data are consistent with a model in which growth-promoting or transforming genes from cancer cells become incorporated within a healthy hematopoietic stem or progenitor cell, which contributes to the genetic diversity of the cancer through the initiation a new transformed clone. Genetic analysis with deep sequencing will compare the DNA sequences between the parental C1498 cell line, sorted populations of clonogenic GFP+ cells obtained from the in vitro and in vivo experiments, and the GFP+ tumor cells to confirm the transformation of healthy bone marrow hematopoietic stem cells with genetic sequences derived from the C1498 cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Chronic Myelogenous Leukemia Cells Contribute to the Stromal Myofibroblasts in Leukemic NOD/SCID MouseIn Vivo

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Ryosuke Shirasaki ◽  
Haruko Tashiro ◽  
Yoko Oka ◽  
Takuji Matsuo ◽  
Tadashi Yamamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myelogenous Leukemia ◽  
Mononuclear Cells ◽  
Severe Combined Immunodeficiency ◽  
Myelogenous Leukemia ◽  
Mouse Bone Marrow ◽  
Murine Bone Marrow ◽  
Endothelial Growth Factor

We recently reported that chronic myelogenous leukemia (CML) cells converted into myofibroblasts to create a microenvironment for proliferation of CML cellsin vitro. To analyze a biological contribution of CML-derived myofibroblastsin vivo, we observed the characters of leukemic nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse. Bone marrow nonadherent mononuclear cells as well as human CD45-positive cells obtained from CML patients were injected to the irradiated NOD/SCID mice. When the chimericBCR-ABLtranscript was demonstrated in blood, human CML cells were detected in NOD/SCID murine bone marrow. And CML-derived myofibroblasts composed with the bone marrow-stroma, which produced significant amounts of human vascular endothelial growth factor A. When the parental CML cells were cultured with myofibroblasts separated from CML cell-engrafted NOD/SCID murine bone marrow, CML cells proliferated significantly. These observations indicate that CML cells make an adequate microenvironment for their own proliferationin vivo.

scholarly journals Mouse fetal liver cells lack functional amphotropic retroviral receptors

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 433-439 ◽  
Author(s):  
C Richardson ◽  
M Ward ◽  
S Podda ◽  
A Bank
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Retroviral Vectors ◽  
Liver Cells ◽  
Mouse Bone Marrow ◽  
Adult Mice ◽  
Fetal Liver Cells ◽  
Marrow Cells

Abstract We have been transducing mouse hematopoietic cells with the human MDR1 (MDR) gene in retroviral vectors to determine the optimal conditions for retroviral gene transfer as a model system for potential human gene therapy. In these studies, we have demonstrated transduction and expression of the human MDR gene using ecotropic and amphotropic MDR- retroviral producer lines. To obtain more mouse hematopoietic cells for detailed study, mouse fetal liver cells (FLC) have been used for MDR transduction and expression, and to reconstitute the ablated marrows of live adult mice. FLC contain hematopoietic cells that have a reconstituting capacity comparable to that of adult mouse bone marrow cells. However, to our surprise, FLC can only be transduced with ecotropic retrovirus and not with amphotropic virus. This restriction of transduction of FLC cannot be overcome by higher titer virus. The resistance to amphotropic transduction by FLC may be part of a changing developmental program that results in a different antigen repertoire on FLC as compared with adult bone marrow cells.

scholarly journals Effect of Campath-1H antibody on human hematopoietic progenitors in vitro

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 807-812
Author(s):  
MH Gilleece ◽  
TM Dexter
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Fluorescein Isothiocyanate ◽  
Lymphoid Cells ◽  
Human Complement ◽  
Hematopoietic Progenitors ◽  
Pilot Studies ◽  
Significant Difference ◽  
And Control

The humanized antibody CAMPATH-1H has been shown in pilot studies to be beneficial in the treatment of lymphoid malignancy and other lymphoproliferative diseases. The antigen recognized by this antibody is not confined to lymphoid cells, and work with rat antibodies of similar specificity has not eliminated the possibility of damage to human hematopoietic progenitors, particularly those capable of repopulating bone marrow and sustaining hematopoiesis. This study aimed to discover if hematopoietic progenitor cells were affected by treatment with CAMPATH-1H, with or without human complement. Bone marrow mononuclear cells from healthy volunteers were treated with saturating concentrations of CAMPATH-1H, human complement, or CAMPATH- 1H plus human complement. The CD34-positive fraction of the mononuclear cells was treated similarly. Residual progenitor activity was measured in the colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte assay and compared with untreated controls. There was no significant difference (at the 5% level) between treated and control cells. Mononuclear cells were divided into CAMPATH-1H-positive and CAMPATH-1H-negative fractions by fluorescein isothiocyanate-CAMPATH-1H labeling and fluorescence-activated cell sorter separation. Hematopoietic progenitors were predominantly found in the CAMPATH-1H- negative fraction. Furthermore, mononuclear cells treated with CAMPATH- 1H and complement were equivalent to controls in experiments that investigated the capacity of these cells to form hematopoietic foci in long-term cultures.

scholarly journals Mouse fetal liver cells lack functional amphotropic retroviral receptors

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 433-439
Author(s):  
C Richardson ◽  
M Ward ◽  
S Podda ◽  
A Bank
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Retroviral Vectors ◽  
Liver Cells ◽  
Mouse Bone Marrow ◽  
Adult Mice ◽  
Fetal Liver Cells ◽  
Marrow Cells

We have been transducing mouse hematopoietic cells with the human MDR1 (MDR) gene in retroviral vectors to determine the optimal conditions for retroviral gene transfer as a model system for potential human gene therapy. In these studies, we have demonstrated transduction and expression of the human MDR gene using ecotropic and amphotropic MDR- retroviral producer lines. To obtain more mouse hematopoietic cells for detailed study, mouse fetal liver cells (FLC) have been used for MDR transduction and expression, and to reconstitute the ablated marrows of live adult mice. FLC contain hematopoietic cells that have a reconstituting capacity comparable to that of adult mouse bone marrow cells. However, to our surprise, FLC can only be transduced with ecotropic retrovirus and not with amphotropic virus. This restriction of transduction of FLC cannot be overcome by higher titer virus. The resistance to amphotropic transduction by FLC may be part of a changing developmental program that results in a different antigen repertoire on FLC as compared with adult bone marrow cells.

scholarly journals Effect of Campath-1H antibody on human hematopoietic progenitors in vitro

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 807-812 ◽  
Author(s):  
MH Gilleece ◽  
TM Dexter
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Fluorescein Isothiocyanate ◽  
Lymphoid Cells ◽  
Human Complement ◽  
Hematopoietic Progenitors ◽  
Pilot Studies ◽  
Significant Difference ◽  
And Control

Abstract The humanized antibody CAMPATH-1H has been shown in pilot studies to be beneficial in the treatment of lymphoid malignancy and other lymphoproliferative diseases. The antigen recognized by this antibody is not confined to lymphoid cells, and work with rat antibodies of similar specificity has not eliminated the possibility of damage to human hematopoietic progenitors, particularly those capable of repopulating bone marrow and sustaining hematopoiesis. This study aimed to discover if hematopoietic progenitor cells were affected by treatment with CAMPATH-1H, with or without human complement. Bone marrow mononuclear cells from healthy volunteers were treated with saturating concentrations of CAMPATH-1H, human complement, or CAMPATH- 1H plus human complement. The CD34-positive fraction of the mononuclear cells was treated similarly. Residual progenitor activity was measured in the colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte assay and compared with untreated controls. There was no significant difference (at the 5% level) between treated and control cells. Mononuclear cells were divided into CAMPATH-1H-positive and CAMPATH-1H-negative fractions by fluorescein isothiocyanate-CAMPATH-1H labeling and fluorescence-activated cell sorter separation. Hematopoietic progenitors were predominantly found in the CAMPATH-1H- negative fraction. Furthermore, mononuclear cells treated with CAMPATH- 1H and complement were equivalent to controls in experiments that investigated the capacity of these cells to form hematopoietic foci in long-term cultures.

Expression of MUC-1 Epitopes on Normal Bone Marrow: Implications for the Detection of Micrometastatic Tumor Cells

1999 ◽  
Vol 17 (5) ◽  
pp. 1535-1535 ◽  
Author(s):  
Wolfram Brugger ◽  
Hans-Jörg Bühring ◽  
Frank Grünebach ◽  
Wichard Vogel ◽  
Sepp Kaul ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cell ◽  
Western Blotting ◽  
Mononuclear Cells ◽  
Cell Detection ◽  
Tumor Cell Detection ◽  
Peripheral Blood Progenitor Cells ◽  
Normal Human ◽  
Positive Results

PURPOSE: The expression of the carcinoma-associated mucin MUC-1 is thought to be restricted to epithelial cells and is used for micrometastatic tumor cell detection in patients with solid tumors, including those with breast cancer. Little is known, however, about the expression of MUC-1 epitopes in normal hematopoietic cells. MATERIALS AND METHODS: MUC-1 expression was analyzed by flow cytometry and immunocytology on bone marrow (BM) mononuclear cells and purified CD34+ cells from healthy volunteers, using different anti-MUC-1–specific monoclonal antibodies. In addition, Western blotting of MUC-1 proteins was performed. RESULTS: Surprisingly, 2% to 10% of normal human BM mononuclear cells expressed MUC-1, as defined by the anti–MUC-1 antibodies BM-2 (2E11), BM-7, 12H12, MAM-6, and HMFG-1. In contrast, two antibodies recognizing the BM-8 and the HMFG-2 epitopes of MUC-1 were not detected. MUC-1+ cells from normal BM consisted primarily of erythroblasts and normoblasts. In agreement with this, normal CD34+ cells cultured in vitro to differentiate into the erythroid lineage showed a strong MUC-1 expression on day 7 proerythroblasts. Western blotting of these cells confirmed that the reactive species is the known high molecular weight MUC-1 protein. CONCLUSION: Our data demonstrate that some MUC-1 epitopes are expressed on normal BM cells and particularly on cells of the erythroid lineage. Hence the application of anti–MUC-1 antibodies for disseminated tumor cell detection in BM or peripheral blood progenitor cells may provide false-positive results, and only carefully evaluated anti–MUC-1 antibodies (eg, HMFG-2) might be selected. Furthermore, MUC-1–targeted immunotherapy in cancer patients might be hampered by the suppression of erythropoiesis.

scholarly journals Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 55-62 ◽  
Author(s):  
PJ Simmons ◽  
B Torok-Storb
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Stromal Cell ◽  
Mononuclear Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Adherent Cell ◽  
Glycophorin A

Murine IgM monoclonal antibody STRO-1 identifies a cell surface antigen expressed by stromal elements in human bone marrow (BM). STRO-1 binds to approximately 10% of BM mononuclear cells, greater than 95% of which are nucleated erythroid precursors, but does not react with committed progenitor cells (colony-forming unit granulocyte-macrophage [CFU-GM], erythroid bursts [BFU-E], and mixed colonies [CFU-Mix]). Fibroblast colony-forming cells (CFU-F) are present exclusively in the STRO-1+ population. Dual-color cell sorting using STRO-1 in combination with antibody to glycophorin A yields a population approximately 100-fold enriched in CFU-F in the STRO-1+/glycophorin A+ population. When plated under long-term BM culture (LTBMC) conditions, STRO-1+ cells generate adherent cell layers containing multiple stromal cell types, including adipocytes, smooth muscle cells, and fibroblastic elements. STRO-1+ cells isolated from LTBMC at later times retain the capacity to generate adherent layers with a cellular composition identical to that of the parent cultures. The STRO-1-selected adherent layers are able to support the generation of clonogenic cells and mature hematopoietic cells from a population of CD34+ cells highly enriched in so-called long-term culture-initiating cells. We conclude that antibody STRO-1 binds to BM stromal elements with the capacity to transfer the hematopoietic microenvironment in vitro.

Efficient Expression of a Kit/GFP Transgene in Hematopoietic Stem Cells of Mouse Fetal Liver and Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4159-4159
Author(s):  
Francesco Cerisoli ◽  
Letizia Cassinelli ◽  
Giuseppe Lamorte ◽  
Stefania Citterio ◽  
Maria Cristina Magli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Fetal Liver ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Gfp Fluorescence ◽  
Adult Bone

Abstract The hierarchy of transcription factors and signalling molecules involved in hematopoietic development has been dissected through transgenic and knock-out experiments, leading to the identification of several important genes. Less well known are the networks of transcription factors which regulate the activities of the main genes identified. Kit, encoding the membrane receptor of Stem Cell Factor (SCF), is a critical molecule for Hematopoietic Stem Cells (HSC) and some early progenitors, in which it is expressed. In a previous work (Cairns et al., Blood102, 3954;2003), we used mouse lines expressing transgenic Green Fluorescent Protein (GFP) under the control of Kit regulatory elements to investigate Kit regulation in different cell systems such as the hematopoietic and germ cell lineages. We generated a mouse Kit transgene capable of efficiently driving GFP expression both in PGC and in hematopoietic progenitors, such as CFU-Mix and BFU-Es. In the present work, we evaluated the functional efficiency of the same transgene also in HSC residing in the Fetal Liver (FL) and adult Bone Marrow (BM). To test if the construct is expressed in HSC, we transplanted FL or BM cells, fractionated on the basis of Kit expression and the level of GFP fluorescence, into irradiated non-transgenic mice. At the same time, the proportion of hematopoietic progenitors in the various fractions was assessed by in vitro colony assays. Following long term hematological reconstitution, the contribution of transplanted GFP cells was evaluated by the proportion of fluorescent mixed colonies in colture as well as by the proportion of fluorescent bone marrow cells, as assessed by FACS analysis. Long term reconstitution was confirmed by secondary transplants. Results show that the repopulating cells derived from fetal liver and adult bone marrow reside in a fraction of Kit+ cells with intermediate GFP fluorescence level, whereas CFU-Mix and BFU-E are in the highly GFP fluorescent fraction. Furthermore, flow cytometry of fetal liver shows that the intermediate fluorescence fraction is highly enriched in Kit+, Sca1+, CD11b+ cells (the expected HSC immunophenotype), whereas the high fluorescence fraction contains mainly Kit+, Sca1−, CD11b− cells. Similarly, the HSC-enriched tip of the Side Population (SP) of adult bone marrow is highly enriched in Kit+, Sca1+ cells of intermediate GFP fluorescence, whereas the upper part of the SP is enriched in Kit+, Sca1− cells of high GFP fluorescence. Our results indicate that the transgene (and possibly the endogenous Kit gene as well) might be transcribed at relatively low levels in HSC versus other progenitors. Noteworthy, the same transgene is also highly expressed in PGC and in Cardiac Stem Cells (CSC) (Messina et al., Circ. Res. 95,911;2004) and in blastocyst inner mass grown in vitro, indicating that the most 5′ part of the intron (4kb), added to the otherwise inactive promoter might include sites regulating Kit expression in multiple stem cell types.

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