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. 2000 Apr;105(7):1013-21.
doi: 10.1172/JCI8583.

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor

T Ueda  1 , K Tsuji, H Yoshino, Y Ebihara, H Yagasaki, H Hisakawa, T Mitsui, A Manabe, R Tanaka, K Kobayashi, M Ito, K Yasukawa, T Nakahata
Affiliations

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor

T Ueda et al. J Clin Invest. 2000 Apr.

Abstract

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.

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Figures

Figure 1
Figure 1
The expansion of total cells (a) and HPCs (b) by SCF+FL, SCF+FL+TPO, and SCF+FL+TPO+IL-6/sIL-6R. A total of 2 × 104 CB CD34+ cells from 3 samples were cultured, and the numbers of total cells and colony-forming cells in the clonal culture were analyzed at day 7 of culture. AStatistically different from data corresponding to the fresh CB CD34+ cells (P < 0.01, Student’s t test).
Figure 2
Figure 2
The repopulating ability of fresh CB CD34+ cells and the cells cultured with SCF+FL, SCF+FL+TPO, and SCF+FL+TPO+IL-6/sIL-6R. A total of 2 × 104 CB CD34+ cells and their progenies were transplanted into NOD/SCID mice, and the proportion of human CD45+ cells in recipient BM cells was analyzed by flow cytometry 12 weeks after the transplantation.
Figure 3
Figure 3
The expression of CD10, CD19, CD3, CD13, CD33, and CD34 on human CD45+ cells (shown in Figure 2) repopulating in BM of NOD/SCID mice engrafted with fresh CB CD34+ cells and the cells cultured with SCF+FL, SCF+FL+TPO, and SCF+FL+TPO+IL-6/sIL-6R.
Figure 4
Figure 4
The comparison of the proportions of human CD45+ cells in BM cells of NOD/SCID mouse recipients transplanted with fresh CB CD34+ cells from 12 different samples and their progenies cultured with SCF+FL+TPO+IL-6/sIL-6R. NOD/SCID mice were injected with 1 × 104 to 2 × 104 fresh CB CD34+ and their progenies, and the proportions of human CD45+ cells in recipient BM cells were analyzed by flow cytometry 10–12 weeks after the transplantation. The percentages of CD45+ cells in BM cells of the recipients transplanted with the cultured cells were higher than those with fresh CB CD34+ cells (P < 0.01, Mann-Whitney U test). EX, experiment.
Figure 5
Figure 5
The frequencies of human HSCs capable of repopulating in NOD/SCID mice in fresh CB CD34+ cells (n = 52) and the cells cultured with SCF+FL+TPO+IL-6/sIL-6R (n = 38). They were estimated as 1 in 39,386 and 1 in 9,484, respectively, by a limiting dilution method.
Figure 6
Figure 6
The effects of IL-3 on the expansion of total cells (a), HPCs (b), and LTR-HSCs (c). A total of 1 × 104 CB CD34+ cells were cultured with SCF+FL+TPO+IL-6/sIL-6R in the presence or absence of IL-3, and the numbers of total cells and colony-forming cells were analyzed at day 7 of culture. Cells cultured from 6 different samples were also transplanted into NOD/SCID mice (n = 12), and the proportions of human CD45+ cells in recipient BM cells were analyzed by flow cytometry 12 weeks after the transplantation. (c) The effects of the addition of IL-3 on the proportions of CD45+ cells in recipient BM cells in 4 samples whose progenies cultured with SCF+FL+TPO+IL-6/sIL-6R exhibited repopulating ability. The percentages of CD45+ cells in BM cells of the recipients transplanted with the cells cultured with IL-3 were lower than those without IL-3 (P < 0.01, Mann-Whitney U test). EX, experiment.
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