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 contract, 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 (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34⁺ cells were in G₀/G₁ phase, whereas greater proportions of the two larger CD34⁺ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1α, IL- 3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1α, 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, MIP1α, and IL-1α. Collectively, these results indicate that the Fr 25/29 CD34⁺ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1α and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.