Expression and function of c-kit in hemopoietic progenitor cells.

The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c-kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.

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Thrombopoietin Is Synthesized by Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v90.9.3444 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3444-3455 ◽

Cited By ~ 72

Author(s):

Anastasia Guerriero ◽

Lydia Worford ◽

H. Kent Holland ◽

Gui-Rong Guo ◽

Kevin Sheehan ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Stromal Cells ◽

Cell Sorting ◽

Bone Marrow Cells ◽

Adult Bone Marrow ◽

Hla Dr ◽

Adult Bone ◽

Marrow Cells

Abstract We have previously characterized stromal progenitor cells contained in fetal bone marrow by fluorescence-activated cell sorting (FACS) using the differential expression of CD34, CD38, and HLA-DR, and found that a small number were contained within the CD34+ cell fraction. In the present study, the frequency of stromal progenitors in both the CD34+ and CD34− subpopulations from samples of fetal and adult bone marrow was approximately one in 5,000 of the mononuclear cell fraction. Using multiparameter single-cell sorting, one in 20 fetal bone marrow cells with the CD34+, CD38−, HLA-DR−, CDw90+ phenotype were clonogenic stromal progenitors, whereas greater than one in five single cells with the CD34−, CD38−, HLA-DR−, CDw90+ phenotype formed stromal cultures. We found that cultures initiated by hematopoietic and stromal progenitors contained within the CD34+ fraction of bone marrow cells formed mixed hematopoietic/stromal cell cultures that maintained the viability of the hematopoietic progenitor cells for 3 weeks in the absence of added hematopoietic cytokines. We characterized some of the hematopoietic cytokines synthesized by stromal cultures derived from either CD34+ or CD34− bone marrow cells using reverse transcriptase–polymerase chain reaction (RT-PCR) amplification of interleukin-3 (IL-3), stem cell factor (SCF), CD34, Flt3/Flk2 ligand (FL), and thrombopoietin (TPO) mRNA sequences. We found ubiquitous expression of TPO mRNA in greater than 90% of stromal cultures initiated by either CD34+ or CD34− cells, and variable expression of SCF, FL, and CD34 mRNA. In particular, SCF and CD34 mRNA were detected only in stromal cultures initiated by CD34+ bone marrow cells, although the differences between CD34+ and CD34− stromal cells were not statistically significant. IL-3 mRNA was not found in any stromal cultures. An enzyme-linked immunosorbent assay (ELISA) of soluble SCF and TPO present in culture supernatants demonstrated that biologically significant amounts of protein were secreted by some cultured stromal cells: eight of 16 samples of conditioned media from stromal cultures initiated by fetal and adult bone marrow contained more than 32 pg/mL SCF (in the linear range of the ELISA), with a median value of 32 pg/mL (range, 9 to 230), while 13 of 24 samples of conditioned media had more than 16 pg/mL TPO (in the linear range of the ELISA), with a median of 37 pg/mL (range, 16 to 106). Our data indicate that stromal cultures initiated by single bone marrow cells can make FL, SCF, and TPO. Local production of early-acting cytokines and TPO by stromal cells may be relevant to the regulation of hematopoietic stem cell self-renewal and megakaryocytopoiesis in the bone marrow microenvironment.

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Antibody stimulation of hemopoietic progenitor cells

Blood ◽

10.1182/blood.v65.4.869.869 ◽

1985 ◽

Vol 65 (4) ◽

pp. 869-876 ◽

Cited By ~ 4

Author(s):

RM Crapper ◽

JW Schrader

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Exchange Chromatography ◽

Staphylococcal Protein A ◽

Igg Antibodies ◽

Hemopoietic Progenitor ◽

Hemopoietic Progenitor Cells ◽

Stimulation Of

Abstract Antisera were raised by immunizing rabbits with cloned lines of murine hemopoietic progenitor cells (P cells) that depended on the presence of a specific hemopoietic growth factor, persisting cell-stimulating factor (PSF), for their growth and survival. The unabsorbed antiserum was inhibitory, but after absorption with murine spleen cells and the mastocytoma, P815, significant stimulation of both P cell growth and thymidine incorporation was evident. IgG antibodies isolated from the antiserum by staphylococcal protein A chromatography or further purified by diethylaminoethyl anion exchange chromatography, ammonium sulphate precipitation, and gel filtration using Sephacryl S-300 were responsible for the stimulation. The absorbed antiserum promoted the survival of normal murine bone marrow cells in liquid culture over a four-day period, and the inclusion of IgG antibodies in agar cultures of normal bone marrow promoted the in vitro survival, over a 48-hour period, of cells capable of subsequently generating, in the presence of a source of PSF, colonies of neutrophils, macrophages, and megakaryocytes. It is postulated that the antibodies act by stimulating the PSF receptor on both the factor-dependent cell lines and normal myeloid progenitor cells.

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Enrichment of pluripotent hemopoietic progenitor cells from human bone marrow

Blood ◽

10.1182/blood.v64.4.774.774 ◽

1984 ◽

Vol 64 (4) ◽

pp. 774-779 ◽

Cited By ~ 12

Author(s):

MP Bodger ◽

IM Hann ◽

RF Maclean ◽

ME Beard

Keyword(s):

Bone Marrow ◽

T Cells ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Fraction ◽

Light Scatter ◽

Hemopoietic Progenitor ◽

Hemopoietic Progenitor Cells

Abstract Pluripotent hemopoietic progenitor cells (CFU-GEMM, cells forming mixed hemopoietic colonies in methylcellulose) from human bone marrow were enriched 90-fold by positive selection on the fluorescence-activated cell sorter using monoclonal antibody RFB-1. Bone marrow cells were separated by cell size, using log 90 degrees light scatter, and the cell fraction containing CFU-GEMM was further separated by relative fluorescence intensity for the RFB-1 antigen. Further enrichment, up to 150-fold, was achieved by depleting bone marrow of T cells and mature myeloid cells prior to RFB-1 selection. These procedures yield a cell fraction containing 51% blast cells, 2% promyelocytes, and 47% undifferentiated (lymphocyte-like) mononuclear cells, although only 1% of the cells formed a mixed colony. CFU-GEMM are strongly positive for the RFB-1 antigen, whereas morphologically identifiable erythroblasts, myeloblasts, and promyelocytes are weakly RFB-1+. This suggests that the relative concentration of the RFB-1 antigen on bone marrow cells is inversely related to their maturity. The greatly increased recovery of CFU-GEMM after the separation of bone marrow by log 90 degrees light scatter and the removal of T cells and mature myeloid cells suggested that accessory cells that normally regulate the cloning efficiency of CFU-GEMM were removed.

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Antibody stimulation of hemopoietic progenitor cells

Blood ◽

10.1182/blood.v65.4.869.bloodjournal654869 ◽

1985 ◽

Vol 65 (4) ◽

pp. 869-876

Author(s):

RM Crapper ◽

JW Schrader

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Exchange Chromatography ◽

Staphylococcal Protein A ◽

Igg Antibodies ◽

Hemopoietic Progenitor ◽

Hemopoietic Progenitor Cells ◽

Stimulation Of

Antisera were raised by immunizing rabbits with cloned lines of murine hemopoietic progenitor cells (P cells) that depended on the presence of a specific hemopoietic growth factor, persisting cell-stimulating factor (PSF), for their growth and survival. The unabsorbed antiserum was inhibitory, but after absorption with murine spleen cells and the mastocytoma, P815, significant stimulation of both P cell growth and thymidine incorporation was evident. IgG antibodies isolated from the antiserum by staphylococcal protein A chromatography or further purified by diethylaminoethyl anion exchange chromatography, ammonium sulphate precipitation, and gel filtration using Sephacryl S-300 were responsible for the stimulation. The absorbed antiserum promoted the survival of normal murine bone marrow cells in liquid culture over a four-day period, and the inclusion of IgG antibodies in agar cultures of normal bone marrow promoted the in vitro survival, over a 48-hour period, of cells capable of subsequently generating, in the presence of a source of PSF, colonies of neutrophils, macrophages, and megakaryocytes. It is postulated that the antibodies act by stimulating the PSF receptor on both the factor-dependent cell lines and normal myeloid progenitor cells.

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Enrichment of pluripotent hemopoietic progenitor cells from human bone marrow

Blood ◽

10.1182/blood.v64.4.774.bloodjournal644774 ◽

1984 ◽

Vol 64 (4) ◽

pp. 774-779

Author(s):

MP Bodger ◽

IM Hann ◽

RF Maclean ◽

ME Beard

Keyword(s):

Bone Marrow ◽

T Cells ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Fraction ◽

Light Scatter ◽

Hemopoietic Progenitor ◽

Hemopoietic Progenitor Cells

Pluripotent hemopoietic progenitor cells (CFU-GEMM, cells forming mixed hemopoietic colonies in methylcellulose) from human bone marrow were enriched 90-fold by positive selection on the fluorescence-activated cell sorter using monoclonal antibody RFB-1. Bone marrow cells were separated by cell size, using log 90 degrees light scatter, and the cell fraction containing CFU-GEMM was further separated by relative fluorescence intensity for the RFB-1 antigen. Further enrichment, up to 150-fold, was achieved by depleting bone marrow of T cells and mature myeloid cells prior to RFB-1 selection. These procedures yield a cell fraction containing 51% blast cells, 2% promyelocytes, and 47% undifferentiated (lymphocyte-like) mononuclear cells, although only 1% of the cells formed a mixed colony. CFU-GEMM are strongly positive for the RFB-1 antigen, whereas morphologically identifiable erythroblasts, myeloblasts, and promyelocytes are weakly RFB-1+. This suggests that the relative concentration of the RFB-1 antigen on bone marrow cells is inversely related to their maturity. The greatly increased recovery of CFU-GEMM after the separation of bone marrow by log 90 degrees light scatter and the removal of T cells and mature myeloid cells suggested that accessory cells that normally regulate the cloning efficiency of CFU-GEMM were removed.

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The γ-globin gene promoter progressively demethylates as the hematopoietic stem progenitor cells differentiate along the erythroid lineage in baboon fetal liver and adult bone marrow

Experimental Hematology ◽

10.1016/j.exphem.2006.09.001 ◽

2007 ◽

Vol 35 (1) ◽

pp. 48-55 ◽

Cited By ~ 9

Author(s):

Mahipal Singh ◽

Donald Lavelle ◽

Kestis Vaitkus ◽

Nadim Mahmud ◽

Maria Hankewych ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Fetal Liver ◽

Globin Gene ◽

Gene Promoter ◽

Hematopoietic Stem ◽

Adult Bone Marrow ◽

Adult Bone

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Allergy and the Bone Marrow: Transmigration Pathways of Hemopoietic Progenitor Cells from the Bone Marrow

Allergy Frontiers: Classification and Pathomechanisms ◽

10.1007/978-4-431-88315-9_25 ◽

2009 ◽

pp. 421-432 ◽

Cited By ~ 1

Author(s):

Roma Sehmi ◽

Jennifer V. Thomson ◽

Adriana Catalli

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Hemopoietic Progenitor ◽

Hemopoietic Progenitor Cells

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The Characterization, Molecular Cloning, and Expression of a Novel Hematopoietic Cell Antigen From CD34+ Human Bone Marrow Cells

Blood ◽

10.1182/blood.v89.8.2706 ◽

1997 ◽

Vol 89 (8) ◽

pp. 2706-2716 ◽

Cited By ~ 49

Author(s):

Nobuko Uchida ◽

Zhi Yang ◽

Jesse Combs ◽

Olivier Pourquié ◽

Megan Nguyen ◽

...

Keyword(s):

Bone Marrow ◽

Molecular Cloning ◽

Bone Marrow Cells ◽

Hematopoietic Cell ◽

Cell Antigen ◽

Adult Bone Marrow ◽

Cd34 Cells ◽

Adult Bone ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123lo, Thy-1+, and CD38−/lo CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.

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