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, 123 (8), 3420-35

The Nucleotide Sugar UDP-glucose Mobilizes Long-Term Repopulating Primitive Hematopoietic Cells

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The Nucleotide Sugar UDP-glucose Mobilizes Long-Term Repopulating Primitive Hematopoietic Cells

Sungho Kook et al. J Clin Invest.

Abstract

Hematopoietic stem progenitor cells (HSPCs) are present in very small numbers in the circulating blood in steady-state conditions. In response to stress or injury, HSPCs are primed to migrate out of their niche to peripheral blood. Mobilized HSPCs are now commonly used as stem cell sources due to faster engraftment and reduced risk of posttransplant infection. In this study, we demonstrated that a nucleotide sugar, UDP-glucose, which is released into extracellular fluids in response to stress, mediates HSPC mobilization. UDP-glucose-mobilized cells possessed the capacity to achieve long-term repopulation in lethally irradiated animals and the ability to differentiate into multi-lineage blood cells. Compared with G-CSF-mobilized cells, UDP-glucose-mobilized cells preferentially supported long-term repopulation and exhibited lymphoid-biased differentiation, suggesting that UDP-glucose triggers the mobilization of functionally distinct subsets of HSPCs. Furthermore, co-administration of UDP-glucose and G-CSF led to greater HSPC mobilization than G-CSF alone. Administration of the antioxidant agent NAC significantly reduced UDP-glucose-induced mobilization, coinciding with a reduction in RANKL and osteoclastogenesis. These findings provide direct evidence demonstrating a potential role for UDP-glucose in HSPC mobilization and may provide an attractive strategy to improve the yield of stem cells in poor-mobilizing allogeneic or autologous donors.

Figures

Figure 1
Figure 1. HPC mobilization with UDP-Glc.
(A) The effects of doses and injection routes were determined. The effect of UDP-Glc peaked 2–4 hours after UDP-Glc injection, thus PB cells were collected 2–4 hours after the last injection in all experiments unless otherwise stated. (B) B6 mice were injected once daily for 6 days with UDP-Glc (UDP-G, 200 mg/kg) or vehicle (CTL). Single-cell suspensions from spleens were stained for LSK and SLAM LSK and the number of HSPCs enumerated. (C) B6 and BALB/C mice were injected daily with UDP-Glc (200 mg/kg), control mice with vehicle. PB was drawn at the indicated time points and LSK cells quantified. Linc-Kit+ (LK) subsets were quantified in BALB/C mice, which express low or no Sca-1. (D) B6 and BALB/C mice were treated once daily for 6 days with vehicle or UDP-Glc, PB cells harvested, and CFU-C activity measured. (E) Chemotaxis assays were performed using 5-μm-pore filters. Lin bone marrow cells from B6 mice were placed in the upper well (106/well) and CXCL12 (120 ng/ml), UDP-Glc (10 μM), or UTP (10 μM) in lower wells. After 6 hours of incubation, cells were collected from lower wells and LSK cells counted; cell migration was not significantly different at UDP-Glc concentrations of 1, 10, and 50 μM. *P < 0.05, **P < 0.01. Data are mean ± SD and reflect 3 independent experiments in A, C, and D and 2 independent experiments in E (duplicate wells/treatment for D and E).
Figure 2
Figure 2. UDP-Glc mobilizes HSPCs with long-term engraftment potential.
(AD) B6 mice were injected with UDP-Glc (200 mg/kg, 6 days), G-CSF (300 μg/kg, 4 days), G-CSF plus UDP-Glc, or PBS. (A and B) CFU-Cs were counted. (C) Primary stromal cells were prepared as previously described (62). PB cells were harvested and overlaid on irradiated stromal layers in 96-well plates. After 5 weeks, wells containing cobblestone areas were counted as positive wells. (D) Mobilization efficiency was assessed according to the numbers of circulating LSK cells in PB. n > 5 mice/group. (E) PB cells (1.5–2 × 106) were collected from PBS- and UDP-Glc–injected mice and transplanted into lethally irradiated recipient animals (Supplemental Figure 2). The contribution of donor cells was measured by quantifying CD45.1 (vehicle-injected) and CD45.2 (UDP-Glc–injected) cells in recipient PB at the indicated times after transplantation. (F) Sixteen weeks after transplantation, bone marrow cells were analyzed by flow cytometry for LSK and SLAM LSK cells after gating on CD45.1+ (PBS-injected) and CD45.2+ (UDP-Glc–injected) cells. n = 6 mice/group. (G) Donor-derived SLAM LSK cells (CD45.2+) were transplantable from primary recipients into irradiated secondary and tertiary recipient mice. Tertiary recipient animals were examined 5–6 weeks posttransplant. PB of recipient animals (n = 5) was analyzed for multilineage reconstitution and gated for HSPCs. Data are mean ± SD and reflect 3 independent experiments in AC (duplicate wells/treatment in A and B). *P < 0.05, **P < 0.01 vs. control; #P < 0.05, ##P < 0.01.
Figure 3
Figure 3. UDP-Glc causes no significant alterations in PB wbc.
(A) B6 mice were injected as described in Figure 2A. PB mononuclear cells were collected from each treatment group and stained with indicated antibodies, followed by flow cytometry analysis. Data are depicted as the mean number of wbc per milliliter of blood. For lineage marker–expressing cells, a mean value of the cell number ± SD obtained from 3 independent experiments is shown. (B) Csf3r–/– (KO) and WT mice were treated with UDP-Glc or PBS as described above. n > 5 mice/group. Left: Flow cytometry plots show the gating strategy for identification of LSK cells. PB cells were gated on a forward-scatter/side-scatter (FS/SS) dot plot. Lin cells were gated (data not shown), with subsequent gating on c-Kit+Sca-1+ cells. Right: Mice (n > 5/group) were individually analyzed for each group. (C) Csf3r–/– (KO) and WT mice (n = 5/group) were treated as described above. PB was collected and analyzed as described in A. Data are mean ± SD of 3 independent experiments (A and C). *P < 0.05, **P < 0.01; ##P < 0.01 vs. Csfr+/+ CTL.
Figure 4
Figure 4. UDP-Glc–mobilized cells display superior long-term repopulating capacity compared with G-CSF–mobilized cells.
(A) B6 mice were injected with UDP-Glc (200 mg/kg, 6 days) or G-CSF (300 μg/kg, 4 days) and competitive repopulation assay performed (Supplemental Figure 2). UDP-Glc– and G-CSF–mobilized cells (2 × 106 each) were transplanted into conditioned animals and contribution of donor cells measured. (B and C) Bone marrow cells were obtained from recipient animals (n > 3) and analyzed for LSK and SLAM LSK cells after gating on CD45.1+ (G-CSF) and CD45.2+ (UDP-Glc) cells. (D) Bone marrow cells from primary recipients were sorted based on CD45 expression as described in A and transplanted into secondary recipients. Donor cell chimerism in recipient mice was analyzed at the indicated times. (E) Bone marrow SLAM LSK cells of primary recipients were used for serial transplantation into secondary and tertiary recipients and engraftment analyzed in tertiary recipients (n = 5). (F) B6 mice (n = 4–5) were injected with UDP-Glc or G-CSF as in A and PB cells analyzed for LSK and SLAM LSK cells. Data pooled from 2 independent experiments. (G) B6 mice were injected with UDP-Glc (n > 15) or G-CSF (n > 12) as in A. Mononuclear cells obtained from PB were stained for SLAM LSK cells, which were transplanted into primary recipients, then bone marrow cells transplanted into lethally irradiated secondary and tertiary recipients, and engraftment was analyzed in tertiary recipients (as in E; n = 5). Data are mean ± SD. *P < 0.05, **P < 0.01.
Figure 5
Figure 5. UDP-Glc mobilizes distinct subsets of HSPCs in comparison with G-CSF.
(A) The effect of UDP-Glc on cell cycle status of HSPCs was evaluated using LSK cells from vehicle- or UDP-Glc–injected mice. Mice were injected once daily with UDP-Glc or PBS for 6 days as described above. The bone marrow and PB samples were pooled from each group (n > 4) and stained for Ki67 and DAPI. LSK cells were pregated and further analyzed for their cell cycle status. Left: The proportions of cells in the respective cell cycle phases are indicated in percentages. Right: Data are mean ± SD of 3 independent experiments with 4 to 5 mice per group. (B) Recipient animals were transplanted as described in Figure 4A. PB cells from recipients (n > 3) were analyzed at 3–4 months posttransplant. The differentiation potential of UDP-Glc– and G-CSF–mobilized cells was determined using CD11b and B220 as markers of myeloid and lymphoid lineages, respectively. (C) Lineage analysis of donor cells in the blood of recipients. At 5 months posttransplantation, PB mononuclear cells of recipient mice were stained with the indicated lineage markers. Data are presented as the percentage of gated cells positive for each lineage marker. Data are mean ± SD of 2 independent experiments with 3 mice per group. *P < 0.05, **P < 0.01.
Figure 6
Figure 6. A combination of UDP-Glc and G-CSF has an improved mobilization efficacy over the use of each agent alone.
(A) Combinatorial administration schedule: G-CSF was injected daily for 4 consecutive days. UDP-Glc was injected daily for 6 consecutive days. Mice were sacrificed (day 0; SAC) and blood cells analyzed for HSPC activity. (B) Mice were treated as described in A. PB cells were collected from G-CSF– and UDP-Glc/G-CSF–injected mice and transplanted in equal numbers into lethally irradiated recipient animals. The contribution of donor cells in the PB of recipient animals was assessed at the indicated times. (C and D) Eighteen weeks after transplantation, bone marrow cells were obtained from recipient animals (n = 5) and analyzed by flow cytometry for LSK (C) and SLAM LSK (D) cells. Data are mean ± SD. **P < 0.01.
Figure 7
Figure 7. UDP-Glc increases mitochondrial ROS levels and promotes a transient osteoclast differentiation.
(A) Mice were injected with UDP-Glc or UDP-Glc/G-CSF as described in Figure 6A. Lin bone marrow cells (first row) were further gated (second row) to identify LSK cells. The cellular levels of mitochondrial superoxide were determined using MitoSOX-red within LSK cells (third row). Numbers indicate the percentage of gated cells. Data are representative of at least 4 mice analyzed individually per treatment group. (B) Mice were injected with UDP-Glc as described above. Bone marrow cell lysates were analyzed by Western blotting for RANKL expression. Values above each band represent fold difference in RANKL expression relative to control sample (vehicle injected) after normalization to β-actin loading control, as determined by densitometry. (C and D) Mice were treated as described above. (C) Femurs were sectioned longitudinally and immunostained with an antibody to RANKL. (D) Tissue sections were also stained for TRAP activity. Arrowheads indicate TRAP-positive cells. A representative TRAP staining is shown. Scale bar: 50 μM. (E) Bone marrow cells were pretreated with M-CSF and incubated with the indicated concentration of UDP-Glc. TRAP-positive cells were counted from at least 3 wells per treatment group. *P < 0.05, **P < 0.01.
Figure 8
Figure 8. NAC abrogates UDP-Glc–induced mobilization.
(A and B) Mice (n > 4/group) were treated with NAC as described in the Methods. UDP-Glc–mediated LSK (A) and SLAM LSK (B) cell mobilization was significantly suppressed by NAC treatment. The data shown are the mean ± SD. (C and D) Mice were treated as described in A. RANKL expression was determined with Western blotting (C) and immunohistochemistry (D). In Western blot analysis, the numerical values represent the fold change in densitometry data (calculated as described above). Scale bar: 50 μM. (E) Mice were treated as indicated. Arrowheads indicate TRAP-positive cells. A representative TRAP staining is shown. Scale bar: 50 μM. #P < 0.05, ##P < 0.01, **P < 0.01.
Figure 9
Figure 9. Role of osteoclasts in UDP-Glc–mediated HSPC mobilization.
(A) op/op mutant mice (n = 6/group) and littermate controls (n = 10/group) were treated with vehicle or UDP-Glc. HSPC mobilization was assessed by measuring the numbers of LSK and SLAM LSK cells in PB. In the representative images of TRAP staining, arrowheads indicate TRAP-positive cells. Scale bar: 50 μM. (B) P2rx7–/– KO and WT controls (n = 10/group) were treated with vehicle or UDP-Glc. HSPC mobilization was assessed as described in A. In the representative images of TRAP staining, osteoclasts are present at high numbers at 6 weeks of age in P2rx7–/– KO mouse. Data were obtained from 6- to 8-week-old animals. Arrowheads indicate TRAP-positive cells. Scale bar: 50 μM. (C) B6 mice were injected as described for Figure 4A. Bone marrow cells were collected from each treatment group and stained with indicated antibodies. Data represent 3 independent experiments. (D) Bone marrow mononuclear cells were isolated from B6 mice. To determine the protease activity, bone marrow supernatants were analyzed by zymogram analysis as described in the Methods. The intensity of the zymogram bands was analyzed utilizing densitometry, represented as fold change relative to vehicle-treated mice. Similar results were observed in 3 independent experiments. Data are mean ± SD. *P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01 vs. P2rx7+/+ CTL.
Figure 10
Figure 10. Analysis of HSPC migration in response to UDP and UDP-Glc.
(A) Mice were treated as described in Figure 8. Expression levels of CXCR4 were determined in bone marrow and PB cells after gating on LSK or SLAM LSK subsets. Data are from at least 2 independent experiments with 4 mice/group. (B and C) B6 mice (n = 7/group) were treated with UDP-Glc or UDP-Glc/NAC as described in A. PB cells (CD45.2) were collected and injected into recipient mice (CD45.1.2) (6 × 106/mouse), which were conditioned 24 hours before injection. Animals were sacrificed 12–14 hours after injection. (B) Recipient bone marrow cells were analyzed for expression of donor cell marker (CD45.2). (C) CD45.2+ cells were gated and analyzed for the presence of LSK cells. (D) B6 mice (n = 5/group) were injected with UDP (200 mg/kg), UDP-Glc (200 mg/kg), or UDP plus UDP-Glc and PB LSK cells quantified. (E) Chemotaxis assays were performed as described in Figure 1E. Lin bone marrow cells were placed in the upper well (106/well); UDP, UDP-Glc, or UDP plus UDP-Glc (10 μM each) was placed in the lower wells. Cells that migrated to lower wells were collected and stained for Sca-1 and c-Kit. Data represent 2 independent experiments, each with duplicate wells per treatment condition. Data are mean ± SD. *P < 0.05, **P < 0.01.

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