OBJECTIVES: The purpose of this study was to investigate cell fates and long-term repopulating potential of a primitive hematopoietic stem cell (HSC) population (i.e., FR25Lin(-) cells) in vitro. MATERIALS AND METHODS: FR25Lin(-) cells were isolated by elutriation and cell sorting and cultured with a combination of cytokines for 7 days. Utilizing the membrane dye PKH-26, cultured cells were separated into two subsets based on their proliferation rates and assayed for progenitors and HSC. RESULTS: Fresh FR25Lin(-) cells were mostly quiescent; however, some of this population entered cell cycle after cytokine exposure reaching a peak 4 to 5 days after culture. Two subsets of cultured cells were isolated: 1) cells that had divided several times (PKH(dull) cells) and 2) cells that remained undivided or divided only once or twice (PKH(bright) cells). The PKH(dull) cells accounted for 94% of total viable cells in culture after 5 days. The PKH(dull) subset contained all the multi-potential in vivo progenitors (CFU-S) and 10 times more committed progenitors (CFU-C). Quantitative analysis of HSC engraftment from the PKH(bright) subset demonstrated stem cell maintenance. For the PKH(dull) subset, on day 5, HSC numbers increased. By day 7, increased differentiation in the PKH(dull) population supports expanding differentiation divisions. CONCLUSIONS: Our primitive HSC population underwent different types of cell divisions stimulated by cytokines, resulting in subsets with different self-renewal and differentiation potentials. This in vitro/in vivo model provides a useful tool for studies of early events during HSC self-renewal and differentiation.
OBJECTIVES: The purpose of this study was to investigate cell fates and long-term repopulating potential of a primitive hematopoietic stem cell (HSC) population (i.e., FR25Lin(-) cells) in vitro. MATERIALS AND METHODS: FR25Lin(-) cells were isolated by elutriation and cell sorting and cultured with a combination of cytokines for 7 days. Utilizing the membrane dye PKH-26, cultured cells were separated into two subsets based on their proliferation rates and assayed for progenitors and HSC. RESULTS: Fresh FR25Lin(-) cells were mostly quiescent; however, some of this population entered cell cycle after cytokine exposure reaching a peak 4 to 5 days after culture. Two subsets of cultured cells were isolated: 1) cells that had divided several times (PKH(dull) cells) and 2) cells that remained undivided or divided only once or twice (PKH(bright) cells). The PKH(dull) cells accounted for 94% of total viable cells in culture after 5 days. The PKH(dull) subset contained all the multi-potential in vivo progenitors (CFU-S) and 10 times more committed progenitors (CFU-C). Quantitative analysis of HSC engraftment from the PKH(bright) subset demonstrated stem cell maintenance. For the PKH(dull) subset, on day 5, HSC numbers increased. By day 7, increased differentiation in the PKH(dull) population supports expanding differentiation divisions. CONCLUSIONS: Our primitive HSC population underwent different types of cell divisions stimulated by cytokines, resulting in subsets with different self-renewal and differentiation potentials. This in vitro/in vivo model provides a useful tool for studies of early events during HSC self-renewal and differentiation.