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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 CD34hiPKHlo (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 CD34hi PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34hiPKHlo 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.

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