Enrichment of early fetal-liver hemopoietic stem cells of the rat using monoclonal antibodies against the transferrin receptor, Thy-1, and MRC-OX82

Dev Immunol. 1996;4(4):235-46. doi: 10.1155/1995/85036.

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-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Antigens, CD / immunology*
  • Cell Separation / methods*
  • Cells, Cultured
  • Flow Cytometry
  • Hematopoietic Stem Cells / immunology*
  • Liver / cytology*
  • Liver / embryology*
  • Lymph Nodes / immunology
  • Peritoneum / immunology
  • Rats
  • Rats, Inbred Strains
  • Receptors, Transferrin / immunology*
  • Rosette Formation
  • Thy-1 Antigens / immunology*
  • Thy-1 Antigens / isolation & purification

Substances

  • Antibodies, Monoclonal
  • Antigens, CD
  • Receptors, Transferrin
  • Thy-1 Antigens