Direct morphological and functional examination of murine pluripotent hemopoietic stem cells

Pluripotent hemopoietic stem cells (PHSC) were isolated from adult mouse bone marrow by a combination of equilibrium density centrifugation and light-activated cell sorting for WGA-positive and H-2k antigen-positive cells. The sorted cells gave rise to 2 spleen colonies per 100 injected cells at 8 days and 6.6 colonies per 100 cells at 12 days after transplantation into lethally irradiated syngeneic recipients. The average enrichment factor for day 12 CFU-S (colony forming unit-spleen) equalled 135 (range, 90-230; n = 15). Enrichment for the cell type that provides radioprotection was equal to 180 +/- 70, indicating that PHSC and CFU-S are identical. Evidence is provided that the spleen seeding efficiency (the f-factor) of these cells was 0.10 and, therefore, that the average purity of the sorted PHSC was 65% (range in 15 experiments, 35-110%). The sorted cells were all in the G0 or G1 phase of the cycle. They appeared to be undifferentiated blasts by morphological criteria, the majority of the cells being similar in structure to the PHSC previously identified in less purified concentrates. Electron microscopy revealed that the sorted cells consisted primarily of two cell types, possibly representing G0 and G1 cells. The FACS was used to deposit single selected cells into individual microwells of Terasaki trays. Thirty percent of the sorted cells could be induced to form progeny in vitro. This procedure facilitates direct examination of the first events of hemopoietic regulation.