ENRICHMENT OF HUMAN HEMATOPOIETIC STEM-CELL ACTIVITY IN THE CD34(-1(+)LIN(-) SUBPOPULATION FROM MOBILIZED PERIPHERAL-BLOOD()THY)

The number of CD34(+) cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose ch emotherapy and hematopoietic growth factors. These so-called periphera l blood stem cell grafts are now frequently used for autologous transp lantation of patients with malignancies. In this report, we address th e question of whether true long-term repopulating pluripotent hematopo ietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM -CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization. W e have examined the presence of stem cells in mobilized peripheral blo od (MPB) by using an antibody to the human Thy-1 molecule to stain the CD34(+)Lineage(-) (Lin(-)) population. The kinetics of mobilization o f CD34(+)Thy-1(+) Lin(-) cells into peripheral blood were studied, and the percentage of cells with this phenotype was found to vary widely depending on the day of leukapheresis. A CD34(+)Thy-1(+)Lin(-) cell po pulation, potentially containing PHSCs, was isolated by fluorescence a ctivated cell sorting (FAGS) and analyzed for activity. The multilinea ge differentiative capacity of this candidate stem cell-containing pop ulation in MPB was determined using an in vitro long-term culture syst em, in which cobblestone area formation was used as a means of detecti ng PHSCs. We also measured repopulating capacity by using two in vivo models in which severe combined immunodeficiency (SCID)hu mice were im planted with human fetal bone or thymus grafts. Using these assays, we show that the highest frequency of cobblestone area forming cells (CA FC) after 7 weeks of culture was observed in a subpopulation of CD34()Lin(-) cells, which expressed low levels of Thy-1. This cell populati on was capable of producing both B and myeloid cells, and maintaining CD34(+)Lin(-) cells in these long term cultures. Moreover, the CD34(+) Thy-1(+)Lin(-) cell subset possessed a higher ability to engraft and t o demonstrate multilineage differentiative potential at 8 weeks in the SCID-hu bone assay. However, in the SCID-hu thymus model, both Thy-1( +) and Thy-1(-) subpopulations were capable of donor T-cell engraftmen t at 6 weeks, suggesting the presence of cells capable of initiating T lymphopoiesis in both populations. In summary, contained within the C D34(+)Thy-1(+)Lin(-) cell subset is a population that possesses in vit ro and in vivo long-term hematopoietic activity, with the ability to g ive rise to B, T, and myeloid cells. Thus, this population includes ce lls with features consistent with those of long-term repopulating mult ilineage potential hematopoietic stem cells. Moreover, from kinetic an alysis, high levels of CD34(+)Thy-1(+)Lin(-) cells may be present only transiently in apheresis samples, despite continued presence of total CD34(+) cells. (C) 1995 by The American Society of Hematology.