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M. Xie et al.
lopoiesis,13,14 we hypothesized that G-CSF acts directly on My-bi HSC but not on Ly-bi HSC.
To address this issue, we used a serum-free culture sys- tem, which enabled us to exclude the effect of unknown factors contaminated in the serum.15 To overcome the het- erogeneity of HSC, we performed single-cell culture, sin- gle-cell transplantation, and single-cell reverse transcrip- tion-polymerase chain reaction (RT-PCR) on highly puri- fied HSC and hematopoietic progenitor cells (HPC). Surprisingly, we found that G-CSF increased the number of divisions of Ly-bi HSC and maintained their repopulat- ing activity after transplantation. SCF alone transiently activated My-bi HSC and increased their long-term recon- stitution potential, but SCF + G-CSF did not show any additional effect on that potential. We conclude that G- CSF acts directly on Ly-bi HSC but not on My-bi HSC. This study suggested that My-bi HSC, which are more or less equivalent to long-term HSC, remain in the quiescent state after G-CSF injection in clinical settings.
Methods
Mice
C57BL/6 (CD45.2-B6) mice were purchased from Beijing HFK Bioscience Co. (Beijing, China). C57BL/6 mice congenic for the Ly5 locus (CD45.1-B6) were bred and maintained at the State Key Laboratory of Experimental Hematology. Animal experiments were approved by the Animal Care and Use Committees, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College.
Single-cell sorting
BM cells were isolated from 8- to 10-week old female CD45.1- or CD45.2-B6 mice, and c-Kit-positive cells were enriched using anti-c-Kit antibody-conjugated MACS beads (Miltenyi Biotechnology, catalog n. 130091224). Cell surface markers used for the identification of HSC1, HSC2, HPC1, HPC2, HPC3, and HPC4 are listed in Online Supplementary Table S1. Antibodies used for flow cytometry are listed in Online Supplementary Table S2.
Single-cell culture
Single cells were cultured in serum-free medium, supplemented with 50 ng/mL recombinant mouse SCF (Peprotech, 250-03) plus 50 ng/mL recombinant mouse thrombopoietin (TPO) (Peprotech, 315-14), 10 ng/mL recombinant human G-CSF (Peprotech, 300- 23), or 10 ng/mL recombinant mouse GM-CSF (Peprotech, 315- 03). Cells were cultured for 7 days at 37°C with 5% CO2 in the air. Number of cells per well were counted daily under inverted microscope.
Serial competitive repopulation
Twenty HSC1 or HSC2 cells from CD45.1-B6 mice were cul- tured with cytokines for 7 days, and cells were transplanted into lethally irradiated CD45.2-B6 mice with 5×105 BM competitor cells from CD45.2-B6 mice. As control, 20 freshly isolated HSC1 or HSC2 cells from CD45.1-B6 mice were similarly transplanted. For secondary transplantation of HSC1 cells, 2×107 BM cells from primary recipients were transplanted into lethally irradiated CD45.2-B6 mice. PB cells were analyzed as previously described.16
Single-cell transplantation
Single HSC1 cells from CD45.1-B6 mice were cultured with SCF + TPO for 1 day, and the surviving single HSC1 cells were selected and transplanted into lethally irradiated CD45.2-B6 mice
with 5×105 BM competitor cells from CD45.2-B6 mice (control). For the cultured cell group, single HSC1 cells were cultured with cytokines for 7 days, and cells of each well were transplanted into lethally irradiated CD45.2-B6 mice with 5×105 BM competitor cells from CD45.2-B6 mice. PB cells were analyzed as previously described.16
Single-cell reverse transcription polymerase chain reaction
For single-cell RT-PCR, 48 single HSC1, HSC2, HPC1, HPC2, HPC3, and HPC4 cells were sorted into each well containing RT- STA master mix. For single-cell RT-PCR for cultured cells, single HSC1 cells were cultured with SCF, SCF+G-CSF, and SCF+TPO for 7 days. Single cells were randomly picked up from 48 wells by a micromanipulator and were placed into the RT-STA master mix. Freshly isolated 48 single HSC1 cells were used as a control. PCR was performed as previously described.16 The 48 genes set used for six populations and cultured cells are listed in Online Supplementary Tables S3 and S4, respectively.
Statistical analysis
Statistical significance was assessed with the unpaired t-test and analysis of variance using GraphPad Prism 6.0 (GraphPad).
Results
Definitions of HSC1, HSC2, HPC1, HPC2, HPC3, and HPC4
HSC1 (CD201+CD150+CD48–CD41–CD34–KSL) and HSC2 (CD201+CD150–CD48–CD41–CD34–KSL) cells were defined as shown in Figure 1A. HSC1 were separated from HSC2 by expression of CD150.17,18 We previously showed that HSC1 cells are enriched in long-term (LT, >6 months) My-bi HSC (LT-My-bi HSC), while HSC2 cells are enriched in short-term (ST, <6 months) Ly-bi HSC (ST-Ly-bi HSC).16,18-21 HPC1 (CD201+CD150+CD48– CD41+CD34–KSL), HPC2 (CD150+Flt-3–CD34+KSL), HPC3 (CD150–Flt-3–CD34+KSL), and HPC4 (CD150–Flt- 3+CD34+KSL) cells were defined as shown in Figure 1A and B. HPC1 cells are reportedly enriched in myeloid- restricted repopulating progenitors.19 HPC2, HPC3, and HPC4 cells were enriched in MPP2, MPP3, and MPP4/lym- phoid-primed multipotent progenitor (LMPP), respectively (Online Supplementary Figure S1).
Cytokine receptor expression
Single-cell RT-PCR was performed on HSC1, HSC2, HPC1, HPC2, HPC3, and HPC4 cells to investigate the expression of cytokine receptors. Gene expression data are shown as heatmaps in Online Supplementary Figure S2. Figure 1C shows the percentage of gene-expressing cells. c-Kit and Mpl were detected in most cells examined. M- CSF receptor, Csf1r, was not expressed in >40% of cells in any of the populations examined. GM-CSF receptor com- prises Csf2ra and Csf2rb. Csf2ra was not detected in >15% of cells in any of the populations examined. Csf2rb was also expressed in <15% of HSC1 cells, but was expressed in approximately from 40 to 60% of HSC2, HPC2, HPC3, and HPC4 cells, and in 100% of HPC1. G-CSF receptor, Csf3r, was detected in approximately 30% of HSC1 and HPC1 cells, while it was detected in >50% of HSC2 and HPC2, and >70% of HPC3 and HPC4 cells. Cxcl12 was expressed in approximately from 20 to 40% of all popula- tions. Cxcr4 was expressed in approximately from 30 to
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