LONG-TERM REPOPULATION OF IRRADIATED MICE WITH LIMITING NUMBERS OF PURIFIED HEMATOPOIETIC STEM-CELLS - IN-VIVO EXPANSION OF STEM-CELL PHENOTYPE BUT NOT FUNCTION

Hematopoietic stem cells were isolated from normal adult mouse bone ma rrow based on surface antigen expression (Thy-1.1(low)Lin(neg)Ly-6A/E( +)) and further selected for low retention of rhodamine 123. This popu lation of cells (Rh-123(low)) could mediate radioprotection and long-t erm (:greater than 12 months) repopulation after transplantation of as few as 25 cells. Transfer of five genetically marked Rh-123(low) cell s in the presence of 10(5) normal bone marrow cells resulted in recons titution of peripheral blood by greater than 10% donor cells in 64% (3 0 of 47) of recipient mice. Of 46 animals surviving after 24 weeks, 10 had over 50% donor-derived cells in peripheral blood. Two general pat terns of long-term reconstitution were observed: one in which many don or-derived cells were observed 5 to 6 weeks after reconstitution and a nother in which donor-derived cells were rare initially but expanded w ith time. This result suggests that two classes of long-term repopulat ing hematopoietic stem cells exist, differing in their ability to func tion early in the course of transplantation. Alternatively, distinct a natomic sites of engraftment may dictate these two outcomes from a sin gle type of cell. As an approach to measure the extent of self-renewal by the injected cells, recipients of five or 200 stem cells were kill ed 8 to 13 months after the transplants, and Thy-1,1(low)Lin(neg)Ly-6A /E(+) progeny of the original injected cells were isolated for a secon d transplant. While a numerical expansion of cells expressing the cell surface phenotype of stem cells was observed, along with activity in the colony-forming unit-spleen assay, the expanded cells were vastly i nferior in radioprotection and long-term reconstitution assays when co mpared with cells freshly isolated from normal animals. This result de monstrates that in stem cell expansion experiments, cell surface antig en expression is not an appropriate indicator of stem cell function.