Enrichment of Early Fetal-Liver Hemopoietic Stem Cells of the Rat Using Monoclonal Antibodies Against the Transferrin Receptor, Thy-1, and MRC-Oχ82

Abstract

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-Oχ26, used for rosetting), Cdw90 (anti- Thy-1, MRC-Oχ7), and the newly described MRC-Oχ82 (reacting with myeloid 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 (Oχ82 + or Oχ43- ), and macrophage (Oχ43+) 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 Oχ26(CD71)-negative, Oχ7(CDw90)-positive, Oχ82-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-Oχ44), MRC-Oχ39, MRC-Oχ59, and 144.2.15. These would provide a strategy for their enrichment by depletion.

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