ENRICHMENT OF EARLY FETAL-LIVER HEMATOPOIETIC 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 disper sed into a single-cell suspension by physical disruption and collagena se digestion. Pluripotent stem cells were characterized and partially purified by a combination of monoclonal antibodies. These included CD7 1 (anti-transferrin receptor, MRC-OX26, used for resetting), Cdw90 (an ti-Thy-1, MRC-OX7), and the newly described MRC-OX82 (reacting with my eloid cells in peritoneal exudate), employed in FAGS sorting. Enrichme nt was monitored by long-term reconstitution of lethally irradiated co ngenic rats genetically distinguishable from the donor by an allelomor phic variant of the CD45 cell-surface antigen. At intervals from 3 mon ths to 1 year, lymph-node cells and peritoneal exudate cells were biop sied for analysis by two-color flow cytometry-one color to determine d onor origin, the other to identify Th cell (CD4+), Tc cell (CD8+), B c ell (sIg+ or CD45RC+), neutrophil (OX82+ or OX43-), and macrophage (OX 43+) 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 recipi ents; therefore, the enrichment procedure revealed only pluripotent ce lls, not stem cells of restricted potency. All recovered stem-cell act ivity was in the OX26(CD71)-negative, OX7(CDw90)-positive OX82-positiv e fraction. In the optimum case, an enrichment of very roughly 200-fol d in cell-for-cell activity was obtained. Rat bone-marrow colony-formi ng units in the spleen (CFUs-12) were found to lack the surface antige ns recognized by the monoclonal antibodies CD53 (MRC-OX44), MRC-OX39, MRC-OX59, and 144.2.15. These would provide a strategy for their enric hment by depletion.