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 progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34(+)Thy-1(+)Lin- cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34(+)Thy-1(+)Lin- cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34(hi) PKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34(+)Thy-1(+)Lin- cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal 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 retained 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 short-term culture of CD34(+)Thy-1(+)Lin- cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.