Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0).
An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G(1) fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G(0)/G(1) and S/G(2)/M. Interestingly, the G(0) cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle-associated change in in vivo stem cell homing, the cultured G(0)/G(1) and S/G(2)/M CD34(+) CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor beta(1) that increased the G(0)/G(1) fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle-associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo-manipulated grafts. (Blood. 2000;96:4185-4193)
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Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
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D000818:Animals; D016465:Bone Marrow Purging; D002453:Cell Cycle; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000637:transplantation; D003114:Colony-Forming Units Assay; D016895:Culture Media, Serum-Free; D056743:Cyclin D3; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics; D005312:Fetal Blood / Q000166:cytology*; D016195:G2 Phase; D018507:Gene Expression Regulation, Developmental; D006085:Graft Survival; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000145:classification / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000235:genetics; D007231:Infant, Newborn; D039041:Integrin alpha4beta1; D016023:Integrins / Q000096:biosynthesis / Q000235:genetics; D007377:Interleukin-3 / Q000494:pharmacology; D019394:Ki-67 Antigen / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000494:pharmacology; D008677:Metaphase; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011828:Radiation Chimera; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics; D017469:Receptors, Fibronectin / Q000096:biosynthesis / Q000235:genetics; D016201:Receptors, Lymphocyte Homing / Q000096:biosynthesis / Q000235:genetics; D016192:Resting Phase, Cell Cycle; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase; D019089:Stem Cell Factor / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014183:Transplantation, Heterologous
C534696:CCND3 protein, human; C534697:Ccnd3 protein, mouse; D016895:Culture Media, Serum-Free; D056743:Cyclin D3; D016213:Cyclins; D018960:Hyaluronan Receptors; D039041:Integrin alpha4beta1; D016023:Integrins; D007377:Interleukin-3; D019394:Ki-67 Antigen; D008565:Membrane Proteins; D019718:Receptors, CXCR4; D017469:Receptors, Fibronectin; D016201:Receptors, Lymphocyte Homing; D019089:Stem Cell Factor; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C084881:flt3 ligand protein; D016179:Granulocyte Colony-Stimulating Factor