THROMBOPOIETIN, KIT-LIGAND, AND FLK2 FLT3 LIGAND TOGETHER INDUCE INCREASED NUMBERS OF PRIMITIVE HEMATOPOIETIC PROGENITORS FROM HUMAN CD34(+)THY-1(+)LIN(-) CELLS WITH PRESERVED ABILITY TO ENGRAFT SCID-HU BONE/

CD34(+)Thy-1(+)Lin(-) cells are enriched for primitive hematopoietic p rogenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic ste m cells (HSC), as defined by the in vivo SCID-hu bone assay. We evalua ted the effects of different cytokine combinations on BM-derived PKH26 -labeled CD34(+)Thy-1(+)Lin(-) cells in 6-day stroma-free cultures. Ne arly all (>95%) of the CD34(+)Thy-1(+)Lin(-) cells divided by day 6 wh en cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34(hi) PKHlo (postdivision) cell populati on retained a high CAFC frequency, a mean 3.2-fold increase of CAFC nu mbers, as well as a capacity for in vivo marrow repopulation similar t o freshly isolated CD34(+)Thy-1(+)Lin(-) cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minima l loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34(hi) PKHhi cells r etained a high frequency of CAFC, whereas the small population of CD34 (hi) PKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for s hort-term culture of CD34(+)Thy-1(+)Lin(-) cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity f or in vivo engraftment. (C) 1998 by The American Society of Hematology .