C-kit positive porcine bone marrow progenitor cells identified and enriched using recombinant stem cell factor.
Porcine haematological studies have been hampered by the lack of monoclonal antibodies against porcine CD34 or CD117 expressed on haematological progenitors. The present report describes the enumeration, phenotyping and isolation of porcine haematopoietic progenitor cells expressing stem cell factor (SCF, c-kit ligand) receptor (c-kit, CD117). Recombinant porcine (rp) SCF and granulocyte-macrophage colony-stimulating factor (GM-CSF) were expressed in the mammalian HEK293 cell-based expression system. Both were biologically active and induced the proliferation of the human erythroleukemic cell line TF-1, as well as of porcine bone marrow haematopoietic cells (BMHC), in a concentration-dependent manner. The effect of rpSCF on BMHC proliferation was synergistic with rpGM-CSF. Furthermore, rpSCF had a synergistic effect on the generation of BMHC-derived dendritic cells (DC) induced by GM-CSF and TNF-alpha. RpSCF was expressed with a 6-histidine epitope, permitting both its purification and immunological detection. Binding studies with BMHC demonstrated ligation of SCF to 4-11% of BMHC. These cells represented the SWC3(low/-)SWC8- BMHC subset, with characteristics of immature proliferative progenitor BMHC. In contrast, no expression was noted on the SWC3+SWC8- monocytic, the SWC3+SWC8+ granulocytic or the SWC3-SWC8+ B cell lineage cells. Using magnetic or fluorescence-activated cell sorting, SCF-ligating BMHC were enriched for pluripotent progenitor cells. In this manner, porcine haematological studies can be pursued in a detailed manner not before possible.
Summerfield
Artur
A
Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, CH-3147 Mittelhäusern, Switzerland. Artur.Summerfield@ivi.admin.ch
Horn
Michael P
MP
Lozano
Gabriela
G
Carrasco
Carlos P
CP
Atze
Kristine
K
McCullough
Ken
K
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000145:classification / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D019089:Stem Cell Factor / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D034421:Sus scrofa
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019009:Proto-Oncogene Proteins c-kit