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A simplified method for enrichment of mouse hematopoietic stem cells.

A simplified method for enriching mouse hematopoietic stem cells using standard two-color fluorescence-activated cell sorting (FACS) has been developed. By eliminating one fluorescence parameter from a previously described four-parameter (three-color) method, FACS enrichment of mouse hematopoietic stem cells to a purity within twofold of that accomplished by the more complex method could be achieved using a single-laser, two-color FACS instrument. The method involves positive selection of mouse bone marrow cells expressing the Ly-6A.2 molecule (previously termed stem cell antigen-1, or Sca-1) and negative selection for expression of a number of cell surface markers characteristic of differentiated cells of hematolymphoid lineages (Lin-). Cell populations selected by this method contain 480 +/- 100 day-13 splenic colony-forming units (CFUs-13) per 10(4) cells, whereas the day-8 splenic colony-forming unit (CFUs-8) content is about tenfold lower. The frequency of thymic colony-forming units (CFUt) is about one in ten cells. Long-term hematopoietic repopulation of irradiated animals was observed following the transfer of 60-100 cells. However, as few as 20 cells could mediate radioprotection of lethally irradiated mice. An analysis of the cell surface phenotype of isolated Ly-6A.2+Lin- cells showed that 30%-50% expressed low levels of the Thy-1 antigen and that the CFUs-13 activity was predominantly associated with the Thy-1lo cells. The Ly-6A.2+Lin- cells expressed intermediate levels of phagocyte glycoprotein-1 (Pgp-1), low levels of heat-stable antigen (HSA), and high levels of class I major histocompatibility antigens (H2 K/D), leukocyte common antigen (Ly-5), and carbohydrate binding sites for the lectin wheat-germ agglutinin (WGA). By these criteria, Ly-6A.2+Lin- cells are phenotypically similar to mouse hematopoietic stem cells isolated by other methods.