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 microns, 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 (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)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, 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, MIP1 alpha, and IL-1 alpha. 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 MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.
Wagner
J E
JE
Department of Pediatrics, University of Minnesota, Minneapolis, USA.
Collins
D
D
Fuller
S
S
Schain
L R
LR
Berson
A E
AE
Almici
C
C
Hall
M A
MA
Chen
K E
KE
Okarma
T B
TB
Lebkowski
J S
JS
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D000954:Antigens, Surface / Q000032:analysis*; D001854:Bone Marrow Cells; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D005434:Flow Cytometry; D016298:Hematopoietic Cell Growth Factors / Q000378:metabolism* / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology; D006801:Humans; D016130:Immunophenotyping; D007163:Immunosorbent Techniques; D051379:Mice; D016513:Mice, SCID; D018121:Receptors, Cytokine / Q000032:analysis*; D014183:Transplantation, Heterologous
D015703:Antigens, CD; D018952:Antigens, CD34; D000954:Antigens, Surface; D016298:Hematopoietic Cell Growth Factors; D018121:Receptors, Cytokine