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M. Xie et al.
50% of HPC1, 3, and 4 cells, and in 70% of HPC2 cells. Notably, Cxcr4 was expressed in very few HSC1 and 2 cells.
Effect of granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor on the division of single hematopoietic stem cells and hematopoietic progenitor cells
We next compared the effects of SCF alone, SCF + G- CSF, SCF + GM-CSF, and SCF + TPO on these six popula- tions by single-cell culture. Figure 2A shows the percent- age of cells that underwent divisions. Figure 2B shows the cell number per well at day 7 of culture. SCF supported the survival of a proportion of HSC1, HPC1, HPC2, and HPC3 cells, and induced their division 1-2 times. However, SCF alone did not support the survival of most HSC2 and HPC4 cells. SCF + G-CSF did not support division of HSC1 more than SCF alone. However, SCF + G-CSF sig- nificantly increased the number of divisions in HSC2, HPC1, HPC2, HPC3, and HPC4 cells, leading to a signifi- cant increase in the cell number per well. SCF + GM-CSF did not support the division of HSC1, HSC2, HPC1, and HPC2 cells but significantly increased the number of divi- sions of HPC3 and HPC4 cells, leading to an increase in the cell number per well. SCF + TPO significantly increased the number of divisions and cells per well in HSC1, HSC2, HPC1, HPC2, and HPC3 cells, but not in HPC4 cells. These data suggested that the target cells of G- CSF, GM-CSF, SCF, and TPO were different among HSC and HPC: SCF acted directly on HSC1, HPC1, HPC2, and HPC3 cells, but not on HSC2 and HPC4 cells. TPO acted on HSC1, HSC2, HPC1, HPC2, and HPC3 cells, but not on HPC4 cells. G-CSF acted directly on HSC2 and HPC1-4 cells, but not on HSC1 cells. GM-CSF acted directly on HPC3 and HPC4 cells.
Effect of granulocyte colony-stimulating factor on reconstitution potential
To examine the effect of the cytokines on the reconsti- tution potential in HSC1 and HSC2 cells, we performed competitive repopulation assay. Figure 3A shows the per- centage of CD45.1 cells in the myeloid, B-cell, CD4 T-cell, and CD8 T-cell lineages after transplantation with HSC1 cells. Compared with freshly isolated cells, the levels of reconstitution of each lineage in the SCF culture were sig- nificantly increased after secondary transplantation. The levels of myeloid and B-cell lineages in SCF + G-CSF or SCF + GM-CSF cultures were also significantly increased after secondary transplantation. However, when we com- pared the reconstitution levels among cultured cells, there was no significant difference between SCF + G-CSF or GM-CSF and SCF cultures, suggesting that SCF, but nei- ther G-SCF nor GM-CSF, increased the long-term reconsti- tution potential in HSC1 cells. The levels of reconstitution of each lineage in SCF + TPO culture were significantly increased in the early months after primary transplanta- tion, suggesting that this combination of cytokines increased the short-term reconstitution potential in HSC1 cells.
Figure 3B shows the percentage of CD45.1 cells in myeloid, B-cell, CD4 T-cell and CD8 T-cell lineages after transplantation with HSC2 cells. Freshly isolated HSC2 cells showed B-lymphoid-biased reconstitution. The level of B-cell lineage reconstitution in SCF and SCF + TPO cul- tures was significantly lower than that in freshly isolated
cells, whereas that in SCF + G-CSF culture was compara- ble with that in freshly isolated cells. Taken together, these data suggested that SCF alone was sufficient to increase the long-term multilineage reconstitution potential. SCF + TPO increased the short-term multilineage reconstitution potential. SCF + G-CSF did not enhance the long-term myeloid lineage reconstitution potential but maintained the short-term lymphoid reconstitution potential.
Transplantation of clonally cultured cells
To further clarify the effect of G-CSF on HSC, we per- formed clonal transplantation assay. Eleven mice in the control group, 10 mice in the SCF group, 12 mice in the SCF + G-CSF group, and 4 mice in the SCF + TPO group were reconstituted (Figure 4). The percentage of chimerism and its lineage composition in single HSC1 cells varied from one another as reported.16,21 Similar to freshly isolated HSC1 cells, after one day culture single HSC1 cells showed a varying degree of reconstitution, indicating the heterogeneity of HSC.
We used the published criteria of My-bi, Bala, and Ly-bi HSC,11,12 and LT- and ST-HSC.22 In the control group of mice, 6 LT-My-bi HSC, 2 ST-Ly-bi HSC, and 3 HPC were detected (Figure 4A and Online Supplementary Table S5). After culture with SCF for 7 days, 1 LT-My-bi HSC, 1 ST- Bala HSC, 4 ST-Ly-bi HSC, and 4 HPC were detected (Figure 4B and Online Supplementary Table S6). After cul- ture with SCF + G-CSF, 2 LT-My-bi HSC, 1 ST-My-bi HSC, 5 ST-Ly-bi HSC, and 4 HPC were detected (Figure 4C and Online Supplementary Table S7). After culture with SCF + TPO, 3 ST-Ly-bi HSC and 1 HPC were detected (Figure 4D and Online Supplementary Table S8). These data showed no difference in reconstitution potential between SCF and SCF + G-CSF cultures, but the significant reduc- tion in reconstitution potential after culture with SCF + TPO.
To be more precise, LT-My-bi HSC activity was detect- ed in the mouse transplanted with three cells from SCF culture (#1 mouse), and similarly, in the mice transplanted with three cells from SCF + G-CSF culture (#1 and #2 mice) (Figure 4B and C and Online Supplementary Tables S6 and S7). ST-Ly-bi but not LT-My-bi HSC activity was detected in mice transplanted with >50 cells from SCF + TPO culture (#1, 2, and 3 mice) (Figure 4D and Online Supplementary Tables S8). These data suggested the similar effects of SCF and SCF + G-CSF on LT-My-bi HSC and the differentiation effect of SCF + TPO, associated with a number of divisions, on LT-My-bi HSC.
Gene expression of cultured cells
We examined the expression of cytokine receptors in day 7 cultured cells by single-cell RT-PCR. Gene expres- sion data are shown as heatmaps in Online Supplementary Figure S3. Figure 5A depicts the gene expression in individ- ual cells. Figure 5B depicts the relative expression level of genes. Consistent with the data in Figure 1C, both c-Kit and Mpl were expressed in the majority of freshly isolated HSC1 cells, while Csf3r was expressed in approximately 30% of the cells. After culture with SCF, the percentage of c-Kit- and Mpl-expressing cells slightly decreased but their relative expression levels significantly increased. However, neither the percentage of Csf3r-expressing cells nor the relative expression level of Csf3r changed. Interestingly, the percentage of Csf3r-expressing cells increased after culture with SCF + G-CSF or TPO. The rel-
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