Enrichment of early fetal-liver hemopoietic stem cells of the rat using monoclonal antibodies against the transferrin receptor, Thy-1, and MRC-OX82.
Fetal livers from inbred rat fetuses at 14 days' gestation were dispersed into a single-cell suspension by physical disruption and collagenase digestion. Pluripotent stem cells were characterized and partially purified by a combination of monoclonal antibodies. These included CD71 (anti-transferrin receptor, MRC-OX26, used for rosetting), Cdw90 (anti-Thy-1, MRC-OX7), and the newly described MRC-OX82 (reacting with myeloiid cells in peritoneal exudate), employed in FACS sorting. Enrichment was monitored by long-term reconstitution of lethally irradiated congenic rats genetically distinguishable from the donor by an allelomorphic variant of the CD45 cell-surface antigen. At intervals from 3 months to 1 year, lymph-node cells and peritoneal exudate cells were biopsied for analysis by two-color flow cytometry--one color to determine donor origin, the other to identify Th cell (CD4+), Tc cell (CD8+), B cell (sIg+ or CD45RC+), neutrophil (OX82+ or OX43-), and macrophage (OX43+) compartments. The degree of chimaerism was taken as the read out of stem-cell activity. No significant differentials between lymph-node and peritoneal exudate chimaerisms were detected in any of the recipients; therefore, the enrichment procedure revealed only pluripotent cells, not stem cells of restricted potency. All recovered stem-cell activity was in the OX26(CD71)-negative, OX7(CDw90)-positive, OX82-positive fraction. In the optimum case, an enrichment of very roughly 200-fold in cell-for-cell activity was obtained. Rat bone-marrow colony-forming units in the spleen (CFUs-12) were found to lack the surface antigens recognized by the monoclonal antibodies CD53 (MRC-OX44), MRC-OX39, MRC-OX59, and 144.2.15. These would provide a strategy for their enrichment by depletion.
Crook
K
K
Dunn School of Pathology, University of Oxford, England.
Hunt
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SV
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D008099:Liver / Q000166:cytology* / Q000196:embryology*; D008198:Lymph Nodes / Q000276:immunology; D010537:Peritoneum / Q000276:immunology; D051381:Rats; D011919:Rats, Inbred Strains; D011990:Receptors, Transferrin / Q000276:immunology*; D012397:Rosette Formation; D018800:Thy-1 Antigens / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D011990:Receptors, Transferrin; D018800:Thy-1 Antigens