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

We have evaluated the in vivo amplification potential of purified murine he matopoietic stem cells, identified as Wheat Germ Agglutinin(+) (WGA(+)), 15 -1.1(-), Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation int o stem cell defective nonmyeloablated W/W-v mice. C57BL Rho-dull cells (250 / 500 cells/mouse) permanently engrafted nonablated W/W-v mice as defined b y the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation, At this time, approximat ely 61% of Rho-dull cells and all the Rho-bright progenitor and colony form ing cells of the engrafted mice were found to be donor-derived by c-Kit gen otyping and by their response to stem cell factor (SCF). Retransplantation of 250-1000 Rho-dull cells from primary into secondary W/W-v recipients gen erated C57BL hematopoiesis in 40%-64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the prim ary recipients. One and half years after transplantation, the bone marrow o f the secondary engrafted animals contained C57BL Rho-dull cells (congruent to 51% by genotype), which were capable of reconstituting tertiary W/W-v r ecipients, In this respect, 25% of tertiary mice expressed C57BL hematopoie sis when transplanted with 250-1000 Rho-dull cells purified from secondary W/W-v recipients. On the basis of the number of Rho-dull cells purified fro m a single mouse, we calculate that approximately 7.3x10(4) Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopu lating stem cells, were generated in the marrow of reconstituted primary W/ W-v recipients transplanted 1.5 years earlier with 250-500 C57BL Rho-dull c ells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals. (C) 1999 International Society for Experimental He matology. Published by Elsevier Science Inc.