Isolation of Small, Primitive Human Hematopoietic Stem Cells: Distribution of Cell Surface Cytokine Receptors and Growth in SCID-Hu Mice

Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 μm, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte-macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCIO) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in Go/G, phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-la, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1a, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1a, and IL-1