In vivo expansion of purified hematopoietic stem cells transplanted in nonablated W/Wv mice

We have evaluated the in vivo amplification potential of purified murine hematopoietic stem cells, identified as Wheat Germ Agglutinin+ (WGA+), 15-1.1−, Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation into stem cell defective nonmyeloablated W/Wv mice. C57BL Rho-dull cells (250/500 cells/mouse) permanently engrafted nonablated W/Wv mice as defined by the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation. At this time, approximately 61% of Rho-dull cells and all the Rho-bright progenitor and colony forming cells of the engrafted mice were found to be donor-derived by c-Kit genotyping and by their response to stem cell factor (SCF). Retransplantation of 250–1000 Rho-dull cells from primary into secondary W/Wv recipients generated C57BL hematopoiesis in 40%–64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the primary recipients. One and half years after transplantation, the bone marrow of the secondary engrafted animals contained C57BL Rho-dull cells ( 51% by genotype), which were capable of reconstituting tertiary W/Wv recipients. In this respect, 25% of tertiary mice expressed C57BL hematopoiesis when transplanted with 250–1000 Rho-dull cells purified from secondary W/Wv recipients. On the basis of the number of Rho-dull cells purified from a single mouse, we calculate that approximately 7.3×104 Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopulating stem cells, were generated in the marrow of reconstituted primary W/Wv recipients transplanted 1.5 years earlier with 250–500 C57BL Rho-dull cells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals.