MULTIPARAMETER ANALYSIS OF TRANSPLANTABLE HEMATOPOIETIC STEM-CELLS .1. THE SEPARATION AND ENRICHMENT OF STEM-CELLS HOMING TO MARROW AND SPLEEN ON THE BASIS OF RHODAMINE-123 FLUORESCENCE

Abstract

A multiparameter cell separative procedure is described that enables normal transplantable hemopoietic stem cells that preferentially home to the marrow of lethally irradiated mice to be enriched and separated from the majority of spleen colony-forming cells that are assayed 13 days after transplantation (CFU-S13). First, bone marrow cells are centrifuged in a discontinuous bovine serum albumin gradient. Low-density cells (1.060-1.068 g/cm3) are harvested and labeled with the supravital cationic fluorochrome rhodamine 123 (Rh123). Labeled cells are analyzed using a fluorescence-activated cell sorter, and cells are sorted on the basis of relative Rh123 fluorescence within a predetermined forward versus 90.degree. red light scatter window that has been optimized for the recovery and enrichment of cells with marrow repopulating ability (MRA). Cells with MRA were characterized by relatively low Rh123 fluorescence and could be separated from a fraction that fluoresced more intensely and contained the majority of CFU-S13 but low MRA. The ratios of low-fluorescent:high-fluorescent MRA and CFU-S13 were 20:1 and 0.5:1, respectively, and the resultant ratio of MRA:CFU-S13 was 556:1 in the low-fluorescent fraction, and 48:1 in the high-fluorescent fraction. Cells with platelet repopulating ability cofractionate with MRA whereas cells with erythroid repopulating ability remain associated with CFU-S13. High-proliferative-potential macrophage colony-forming cells that form colonies in agar in the presence of the combined stimulus of pregnant mouse uterus extract plus human-spleen-conditioned medium was present in both fractions. Between 20% and 30% of transplantable hemopoietic stem cells has been removed in approximately 1% of total nucleated cells from the starting preparation, and enrichments of 25- to 30-fold have been achieved permitting hemopoietic reconstitution of lethally irradiated host mice with as few as 400 donor cells.