Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor.
Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.
Ueda
T
T
Department of Clinical Oncology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
Tsuji
K
K
Yoshino
H
H
Ebihara
Y
Y
Yagasaki
H
H
Hisakawa
H
H
Mitsui
T
T
Manabe
A
A
Tanaka
R
R
Kobayashi
K
K
Ito
M
M
Yasukawa
K
K
Nakahata
T
T
D000818:Animals; D018952:Antigens, CD34; D016026:Bone Marrow Transplantation; D016895:Culture Media, Serum-Free; D006410:Hematopoiesis / Q000187:drug effects*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism* / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D019947:Receptors, Interleukin-6 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility; D019089:Stem Cell Factor / Q000378:metabolism* / Q000494:pharmacology; D013926:Thrombopoietin / Q000378:metabolism* / Q000494:pharmacology; D014183:Transplantation, Heterologous
D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D007377:Interleukin-3; D015850:Interleukin-6; D008565:Membrane Proteins; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D013926:Thrombopoietin