Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor (original) (raw)
Long-term repopulating ability of the cells cultured with single cytokines or 2-cytokine combinations. We initially examined NOD/SCID mice-repopulating ability of variable doses of fresh CB CD34+ cells. As shown in Table 1, none of 14 NOD/SCID mice transplanted with less than 5 × 103 cells revealed successful engraftment. Three (18.8%) of 16 and 3 (33.3%) of 9 recipients transplanted with 1 × 104 and 2 × 104 cells, respectively, were successfully engrafted. When 4 × 104 CD34+ cells were transplanted, the high engraftment rate was obtained (66.7%). Then, 1 × 104 to 2 × 104 CB CD34+ cells and their progenies in the culture were transplanted for analysis sensitive to the expansion of NOD/SCID-repopulating cells in the present study.
Evaluation of initial cell dose of fresh CD34+ cells
We first examined the expansion of NOD/SCID-repopulating cells by SCF, FL, TPO, IL-6, IL-6/sIL-6R, or IL-3 alone. When the cells cultured from 2 × 104 CB CD34+ cells with each of these cytokines for 1 week were injected into 3 NOD/SCID mice, no recipients revealed successful reconstitution of human hematopoietic cells, although 1 of 3 recipients transplanted with the initial CB CD34+ cells did. We next evaluated the long-term repopulating ability of the cells cultured with various combinations of 2 cytokines among these cytokines. Only 2 of 3 recipients transplanted with the cells cultured with SCF+FL for 1 week were successfully engrafted, whereas all 3 transplants with the initial 2 × 104 CB CD34+ cells failed. On the basis of these findings, we determined to compare the hematopoietic activities of the fresh CB CD34+ cells and the cells cultured with SCF+FL, SCF+FL+TPO, and SCF+FL+TPO+IL-6/sIL-6R.
Reconstitution by the cells cultured from CB CD34+ cells with SCF, FL, TPO, and IL-6/sIL-6R in NOD/SCID mice. When 2 × 104 CB CD34+ cells were cultured with SCF+FL, SCF+FL+TPO, and SCF+FL+TPO+IL-6/sIL-6R for 1 week, total cell numbers increased 7.2-, 17.3-, and 45.4-fold, respectively (n = 3) as shown in Figure 1a. Morphological observation of cytospin preparations showed that there were no significant differences among the compositions of the cells cultured with the combinations, all of which contained 70–80% blastic cells. HPCs in the culture with SCF+FL did not increase, but those with SCF+FL+TPO and SCF+FL+TPO+IL-6/sIL-6R increased 3.1- and 6.1-fold, respectively. The increase of multipotential HPCs was most significant in the culture with SCF+FL+TPO+IL-6/sIL-6R (3.5-fold).
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).
We then transplanted the cells cultured from 1 × 104 to 2 × 104 CB CD34+ cells with the 3 combinations for 1 week into NOD/SCID mice and analyzed the recipient BM cells by flow cytometry 10–12 weeks after the transplantation to examine the long-term repopulating ability of the cultured cells. Figure 2 shows a representative result of flow cytometric analysis of BM cells in recipients engrafted with the progenies of 2 × 104 CB CD34+ cells obtained from the same sample. The recipient mouse transplanted with 2 × 104 CD34+ cells possessed 12.0% human CD45+ cells in BM. When NOD/SCID mice were transplanted with the cells cultured with SCF+FL and SCF+FL+TPO, 23.6% and 39.0% of BM cells were CD45 positive, respectively. In the recipient transplanted with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R, 68.5% were CD45+ cells. The expression of lineage markers on the reconstituted human CD45+ cells was analyzed (Figure 3). All the recipients transplanted with the cells cultured under the 3 conditions possessed CD13+ and CD33+ myeloid cells, CD19+ and/or CD10+ B-cells, and CD34+ immature cells, but not CD3+ T-cells, in BM CD45+ cells, as did the recipient engrafted with the initial CD34+ cells.
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.
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.
Table 2 summarizes the results of the transplantation of the cultured cells. Six (24.0%) of 25 NOD/SCID mice transplanted with 1 × 104 to 2 × 104 CB CD34+ cells showed successful engraftment. When the cells generated from 1 × 104 to 2 × 104 CB CD34+ cells in 1-week culture were transplanted, the rate of success increased. In the transplantation of the cells cultured with SCF+FL and SCF+FL+TPO, 7 (43.8%) of 16 and 7 (50.0%) of 14 recipients, respectively, were engrafted. The recipients transplanted with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R showed the highest engraftment rate; 13 (81.2%) of 16 mice showed successful engraftment. The percentages of human CD45+ cells in BM cells of the recipients transplanted with 1 × 104 to 2 × 104 CB CD34+ cells and the cells cultured with SCF+FL were similarly about 2%, but higher in the mice transplanted with the cells cultured with SCF+FL+TPO and SCF+FL+TPO+IL-6/sIL-6R (6.7% and 11.5%, respectively).
Reconstitution of NOD/SCID mice transplanted with fresh and expanded CD34+ cells
Although B cells were the most predominantly repopulated in the recipients engrafted with the fresh CB CD34+ cells, there were no significant differences in the reconstitution of each lineage of cells among the recipients engrafted with the cultured cells. Interestingly, the proportions of human CD34+ cells in BM cells were higher in the recipients engrafted with the cultured cells than those with the initial CB cells, being only 4.9 ± 3.3% in the latter. In particular, CD34+ cells comprised 21.5 ± 2.3% of the BM cells in the recipients transplanted with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R. On the basis of the data, it was estimated that 30.5-fold of CD34+ cells were present in the recipients transplanted with SCF+FL+TPO+IL-6/sIL-6R, compared with those with fresh CB CD34+ cells.
Hematopoietic activity of the cells engrafted in NOD/SCID mice. Because BM cells of the recipient mice contained a number of human CD34+ cells, the human hematopoietic activity of the reconstituted cells was examined. When 1.5 × 105 BM cells of NOD/SCID mice untransplanted were incubated in the methycellulose clonal culture with human SCF, IL-3, GM-CSF, and EPO, no colonies were formed, although only a small number of mouse macrophage clusters and erythroid bursts were detected. However, BM cells of the engrafted recipients shown in Figures 2 and 3 produced human hematopoietic colonies, whose numbers depended on the proportion of human CD34+ cells in each BM (Table 3). The confirmation that these colonies derived from human HPCs was obtained by the detection of human ALU sequences in DNA extracted from the colonies by PCR analysis. We randomly chose the colonies in the culture of BM cells of the mice engrafted with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R, individually lifted the colonies from culture medium, and extracted the DNA. DNA from all of 5 G colonies, 5 M colonies, 5 GM colonies, 5 E bursts and 3 MIX colonies contained human ALU sequences (data not shown).
Formation of human hematopoietic colonies from BM cells of NOD/SCID mice engrafted with fresh CB CD34+ cells and their progenies
We also monitored the presence of human CD45+ cells in PB of 3 recipients engrafted with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R until 6 months after the transplantation (Table 4). Although the proportion of CD45+ cells in PB was always lower than in BM, a stable number of CD45+ cells were detected for 6 months in all the engrafted recipients examined.
The percentage of CD45+ cells in PB of the engrafted recipients
Expansion of human HSCs capable of repopulating in NOD/SCID mice. The results just discussed suggest the efficient expansion of LTR-HSCs in the cultures, especially by SCF+FL+TPO+IL-6/sIL-6R. We then compared the proportions of human CD45+ cells in BM cells of NOD/SCID mice engrafted with 1 × 104 to 2 × 104 fresh CB CD34+ cells from 12 different samples (1 mouse per sample) and their progenies cultured with SCF+FL+TPO+IL-6/sIL-6R (Figure 4). Although BM cells of 8 of 12 recipients transplanted with fresh cells contained less than 1% CD45+ cells, the reconstitution of more than 1% CD45+ cells was obtained in BM cells of 10 mice transplanted with their progenies. In all 12 samples, the proportion of CD45+ cells in recipient BM cells increased by the culture with SCF+FL+TPO+IL-6/sIL-6R for 1 week (mean: 10-fold; range: 1.5- to 30.3-fold; P < 0.01 by Mann-Whitney U test). Given that this finding further supported the expansion of LTR-HSCs, the expansion rate was calculated using a limiting dilution method. As shown in Figure 5, the frequency of LTR-HSCs was calculated as 1 in 39,386 in fresh CB CD34+ cells. On the other hand, the frequency of LTR-HSCs in the cells cultured with SCF+FL+TPO+IL-6/sIL-6R was calculated as 1 in 9,484. Accordingly, the expansion of LTR-HSCs by SCF+FL+TPO+IL-6/sIL-6R was estimated at 4.2-fold.
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.
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.
Next, to examine the time required for the HSC expansion by SCF+FL+TPO+IL-6/sIL-6R, 1 × 104 CB CD34+ cells and their progenies cultured for 4, 7, and 14 days were transplanted into NOD/SCID mice. In 4 independent experiments, no mice transplanted with the fresh CB CD34+ cells and the cells cultured for 4 days possessed more than 1% of CD45+ cells in BM 12 weeks after the transplantation. However, all 4 mice transplanted with the cells cultured for 7 days were successfully engrafted. The cells cultured for 14 days did not repopulate in any of the 4 NOD/SCID mice. Thus, 7 days seemed to be an optimal duration for the HSC expansion by SCF+FL+TPO+IL-6/sIL-6R.
Reconstitution by the cells cultured with SCF, FL, TPO, and IL-6/sIL-6R under serum-free conditions. To exclude the effects of an unknown factor(s) in FBS on the HSC expansion by SCF+FL+TPO+IL-6/sIL-6R, we transplanted 1 × 104 CB CD34+ cells and their progenies cultured with the cytokine combination under serum-free conditions into 5 NOD/SCID mice. Although no mice transplanted with the fresh CB CD34+ cells were successfully engrafted, 3 mice (60%) transplanted with the cultured cells showed the reconstitution of human hematopoietic cells in BM. Flow cytometric analysis of BM cells of the 3 mice showed that human CD19+ cells, CD13+ cells, and CD33+ cells comprised 86.0 ± 7.5%, 7.8 ± 5.4%, and 9.4 ± 5.4%, respectively, of the CD45+ cell population, a similar distribution to that in the recipients successfully engrafted with the cells cultured under serum-containing conditions. This result indicates that no other cytokines are needed for the HSC expansion by SCF, FL, TPO, and IL-6/sIL-6R.
Inhibitory effect of IL-3 on the expansion of human HSCs. Finally, we examined the effect of IL-3 on the expansion of human hematopoietic cells. As shown in Figure 6, a and b, the addition of IL-3 to the culture supplemented with SCF+FL+TPO+IL-6/sIL-6R increased the numbers of total cells and HPCs after 1 week. Then, to examine the effect of IL-3 on the expansion of LTR-HSCs, the cells in the culture of 1 × 104 CB CD34+ cells obtained from 6 different samples with SCF+FL+TPO+IL-6/sIL-6R in the presence or absence of IL-3 were transplanted into NOD/SCID mice. Although 4 of 6 recipients transplanted with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R possessed more than 1% of human CD45+ cells in BM cells, no mice with the cells cultured with SCF+FL+TPO+IL-6/sIL-6R+IL-3 were successfully engrafted. Figure 6c summarizes the effect of the addition of IL-3 on the long-term repopulating ability of HSCs in the 4 samples that showed successful engraftment. In the transplantation of the cells cultured with IL-3, the percentage of human CD45+ cells in BM cells was less than 0.07% in all 4 recipients (P < 0.01; Mann-Whitney U test). When 5 × 104 CD34+ cells from 2 samples cultured with SCF+FL+TPO+IL-6/sIL-6R in the presence or absence of IL-3 were transplanted, all the mice were successfully engrafted, but the percentages of CD45+ cells in BM of the recipients with the cells cultured with IL-3 were lower than those without IL-3 (41.3% and 49.5%, and 63.1% and 69.3%, respectively). These results indicate that IL-3 expands human mature blood cells and HPCs but has an inhibitory effect on the expansion of LTR-HSCs.
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.