URIDINE DIPHOSPHATE COMPOUNDS AS MOBILIZERS OF HEMATOPOIETIC PROGENITOR CELLS
The present invention provides for compositions and methods for administering one or more UDP compound, alone or in combination with another hematopoietic progenitor cell mobillizing compound (for example, but not limited to, G-CSF), to mobilize hematopoietic progenitor cells for transplant or other purposes. The methods of the invention may be particularly advantageous as applied to improve the stem cell yield in so-called “poor mobilizing” patients.
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This application is a division of U.S. patent application Ser. No. 14/495,663, filed Sep. 24, 2014, which is a continuation of PCT/US13/034452, filed Mar. 28, 2013, and claims priority to U.S. Provisional Application No. 61/618,173 filed Mar. 30, 2012, the contents of each of which are incorporated by reference in their entirety herein, and priority to each of which is claimed.
GRANT INFORMATIONThis invention was made with government support under grant number W81XWH-09-1-0364 awarded by the Department of Defense. The government has certain rights in the invention.
1. INTRODUCTIONThe present invention relates to the use of uridine diphosphate compounds and particularly uridine diphosphate glucose in methods for mobilizing hematopoietic progenitor cells from the bone marrow, alone or together with another mobilizing agent such as granulocyte colony stimulating factor, AMD-3100, cyclophosphamide or fucoidan.
2. BACKGROUND OF THE INVENTIONHematopoietic stem progenitor cells (HSPCs) are normally present in very small numbers in the circulating blood. However, in response to stress or injury, HSPCs are primed to migrate out of their niche into the peripheral blood. HSPCs have been developed as an alternative to bone marrow harvest for transplant. Because they exhibit faster engraftment and reduced risk of posttransplant infection, mobilized HSPCs are now more commonly used as stem cell sources.
Uridine diphosphate-glucose (“UDP-glucose”) is a nucleotide sugar which is released into extracellular fluids in response to various stressors (Lazarowski et al., “Release of cellular UDP-glucose as a potential extracellular signaling molecule,” Mol Pharmacol 63, 1190-1197, 2003). UDP is a potent agonist of the human P2Y14 receptor (Carter et al., “Quantification of Gi-mediated inhibition of adenylyl cyclase activity reveals that UDP is a potent agonist of the human P2Y14 receptor,” Mol. Pharmacol. 76(6):1341-8, 2009) and has been reported to be associated with a number of physiologic effects, including inotropic effects in cardiac myocytes mediated by P2Y6 receptors via an IP3-dependent pathway (Wihlborg et al., “Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphate (UDP) via P2Y2 and P2Y6 receptors on cardiomyocytes and release of UTP in man during myocardial infarction,” Circ. Res. 98(7):970-6, 2006).
3. SUMMARY OF THE INVENTIONThe present invention relates to the use of UDP compounds (including UDP itself, UDP-sugars such as UDP-glucose, and others) to mobilize hematopoietic progenitor cells such as HSPCs from the bone marrow into the peripheral circulation of a subject.
It is based, at least in part, on the discovery that UDP-glucose is a mediator of HSPC mobilization. Specifically, it was discovered that UDP-glucose-mobilized HSPCs differentiated into multi-lineage blood cells and achieved long-term repopulation in lethally irradiated animals. The lymphoid-biased differentiation and ability to preferentially support long term repopulation of UDP-glucose mobilized HSPCs is superior to that of G-CSF mobilized HSPCs. It was further discovered that co-administration of UDP-Glucose and G-CSF led to a synergistic enhancement of HSPC mobilization.
Accordingly, the present invention provides for compositions and methods for administering one or more UDP compound, alone or in combination with another hematopoietic progenitor cell mobilizing compound (for example, but not limited to, G-CSF), to mobilize hematopoietic progenitor cells for transplant or other purposes. The methods of the invention may be particularly advantageous as applied to improve the stem cell yield in so-called “poor mobilizing” patients.
(B-C) Mice were treated as described in
(D) Mice were treated as indicated. Arrowheads (black color) indicate TRAP-positive cells. A representative TRAP staining is shown. Scale bar, 50 μM.
For clarity and not by way of limitation, this detailed description is divided into the following subsections:
(i) UDP compounds;
(ii) non-UDP HPC mobilizing compounds;
(iii) pharmaceutical compositions; and
(iv) methods of use.
5.1 UDP CompoundsUDP compounds include UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-glucuronic acid, UDP, P536 (Alcino et al., “Activity of P536, an Analog of UDP-Glucose, Against Trypanosoma cruzi,” Antimicrob. Agents and Chemother. 32(9), 1412-1415, 1988), UDP-6S-6C-methylglucose and UDP-6R-6C-methylglucose (Campbell and Tanner, “UDP-Glucose Analogues as Inhibitors and Mechanistic Probes of UDP-Glucose Dehydrogenase,” J. Org. Chem., 64(26), 9487-9492, 1999), MRS 2690 (a P2Y14 receptor agonist available from Tocris. Co.), competitive antagonists of UDP-glucose set forth in Fricks et al., “UDP Is a Competitive Antagonist at the Human P2Y14 Receptor,” J. Pharmacol. Exp. Ther. 325(2), 588-594 (2008), and UDP-containing compounds that inhibit UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay set forth in Hamel et al., J. Biomol. Screen. 16 (9), 1098-1105 (2011) (including compound A, described therein). A mixture of any two or more of the above may also be administered and is within the scope of “UDP compound”.
The present invention provides for a pharmaceutical composition comprising a UDP compound, together with a suitable pharmaceutical carrier. In one non-limiting embodiment, the pharmaceutical composition is formulated for subcutaneous administration, for example having a pH of between about 5 and 8 and optionally containing a suitable pharmaceutical buffer.
5.2 Non-UDP HSPC Mobilizing CompoundsAn HSPC mobilizing compound is a compound that promotes a mobilization or relocation of hematopoietic stem progenitor cells such as HSPCs from the bone marrow to the peripheral circulation in a subject. Non-limiting examples of HSPC mobilizing compounds that are not UDP compounds include G-CSF, poly-(1,6)-β-D-glucopyranosyl-(1,3)-β-D glucopyranose (PGG) β-glucan (Cramer et al., “Mobilization of Hematopoietic Progenitor Cells by Yeast-Derived β-Glucan Requires Activation of Matrix Metalloproteinase-9,” Stem Cells 26, 1231-1240, 2008), AMD3100 (Cashen et al., “AMD3100: CXCR4 antagonist and rapid stem cell-mobilizing agent,” Future Oncol. 3 (1):19-27, 2007), cyclophosphamide, fucoidan, and mixtures thereof.
5.3 Pharmaceutical CompositionsIn non-limiting embodiments, the present invention provides for pharmaceutical compositions for use in a method of mobilizing or relocating hematopoietic progenitor cells such as HSPCs from the bone marrow into the peripheral circulation of a subject in need of such treatment, comprising administering to the subject a UDP compound, a non-UDP HSPC mobilizing compound, and a combination thereof. UDP compounds and non-UDP HSPC mobilizing compounds are set forth in the preceding sections.
In certain non-limiting embodiments, the pharmaceutical composition may comprise one or more UDP compounds selected from the group consisting of UDP, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-glucuronic acid, P536, UDP-6S-6C-methylglucose, UDP-6R-6C-methylglucose, MRS 2690, and mixtures thereof.
In a further non-limiting embodiment, the pharmaceutical composition may comprise one or more UDP compounds, as described above, and one or more non-UDP HSPC mobilizing compounds, selected from the group consisting of G-CSF, poly-(1,6)- -D-glucopyranosyl-(1,3)- -Dglucopyranose (PGG)-glucan, AMD3100, cyclophosphamide, fucoidan, and mixtures thereof.
In certain non-limiting embodiments, the pharmaceutical composition may also contain one or more pharmaceutically acceptable carriers and excipients which can be in solid or liquid form.
5.4 Methods of UseThe present invention provides for a method of mobilizing or relocating (or promoting mobilization or relocation of) hematopoietic progenitor cells such as HSPCs (including HSPCs of lymphoid lineage) from the bone marrow into the peripheral circulation of a subject in need of such treatment, comprising administering, to the subject, an effective amount of a UDP compound, as described above.
The subject may be a human or non-human subject. The subject may be in need of such treatment in view of an anticipated, contemporaneous or past bone marrow toxic event such as radiation or chemotherapy or other toxic exposure (or to rescue people from radiation accidents or terrorist attack (dirty bomb)). The subject may be in need of such treatment in view of a desire to collect HSPCs from the peripheral circulation of the subject for the purpose of transplantation, where the transplantation may be autologous or allogeneic. In one non-limiting embodiment, the subject is being prepared to donate hematopoietic stem progenitor cells. In one non-limiting embodiment, the subject is a chemotherapy patient. In one non-limiting embodiment, the subject suffers from lymphopenia. In non-limiting embodiments, the subject may suffer from long term bone marrow failure (BMF) or Fanconi's anemia (FA).
The UDP compound may be administered by any suitable route, including but not limited to subcutaneous, intramuscular, intravenous, intraperitoneal, oral, rectal, or any other route known in the art.
In certain non-limiting embodiments, the UDP compound may be administered once a day, once every other day, once every third day, or once a week, or twice a day, twice every other day, or twice a week, during the treatment period.
In certain non-limiting embodiments, the treatment period may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, two weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, three weeks, or one month, or between 3 and 14 days, or between 3 and 10 days, until a target level of HSPCs in the peripheral circulation has been reached. The treatment period may optionally be repeated after an interval of non-treatment, said interval of non-treatment being, in certain non-limiting embodiments, be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, two weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, three weeks, one month, 5 weeks, 6 weeks, or until a triggering event, such as the administration of chemotherapy, occurs.
In certain non-limiting embodiments, the UDP compound may be administered
as part of a regimen with one or more other non-UDP compound HSPC mobilizing compounds. The UDP compound and the other HSPC mobilizing compound may be administered concurrently or at different times over a period when they each can have an effect on the bone marrow (for example, function complementarily).
In non-limiting embodiments, the UDP compound is administered at a dosage of 0.1-500 mg/kg or at a dosage of 1-100 mg/kg or at a dosage of 0.1-20 mg/kg or at a dosage of 0.01-1 mg/kg. In one non-limiting embodiment, the UDP compound is UDP-glucose, administered to a human at a dosage of 1-100 mg/kg or at a dosage of 0.1-20 mg/kg. In one non-limiting embodiment, the UDP compound is UDP-glucose, administered to a human at a dosage of 0.01-300 mg/kg or at a dosage of 1-100 mg/kg or at a dosage of 0.1-20 mg/kg or at a dosage of 0.01-1 mg/kg.
In one non-limiting embodiment, UDP-glucose or UDP is administered to a subject daily over a treatment period between 3 and 10 days. In one non-limiting embodiment, UDP-glucose or UDP is administered to a human subject daily over a treatment period between 3 and 10 days. In one non-limiting embodiment, UDP-glucose or UDP is administered to a human subject, subcutaneously, daily over a treatment period between 3 and 10 days.
In one non-limiting embodiment, UDP-glucose or UDP is administered as part of a treatment regimen with G-CSF, where the two agents are administered simultaneously or not simultaneously, but where they are both administered over the treatment period. In non-limiting embodiments, G-CSF may be administered subcutaneously either as a bolus or by continuous infusion or by any other route used in the art for administering G-CSF. In non-limiting embodiments, G-CSF may be administered at a daily dose of between 1 and 15 micrograms/kg per day or between 1 and 10 micrograms/kg per day or 5 or 10 micrograms/kg per day, for example but not by way of limitation, once a day, once every other day, once every third day, or once a week, or twice a day, twice every other day, or twice a week, during the treatment period. Preferably G-CSF is given in at least four consecutive daily injections (Broxmeyer et al., “Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist,” J. Exp. Med. 201, 1307-1318, 2005) although other regimens may be used. For example, but not by limitation, the treatment period may be daily treatment for 5 consecutive days, 6 consecutive days or 7 consecutive days. In one non-limiting embodiment, G-CSF treatment is started 1, 2 or 3 days after the first administration of UDP compound. In other non-limiting embodiments where UDP-glucose and UDP are administered in a treatment regimen with G-CSF, G-CSF may be administered at a daily dose of between 0.01 and 120 micrograms/kg/day, or between 5 and 20 micrograms/kg per day, for example but not by way of limitation, once a day, once every other day, once every third day, or once a week, or twice a day, twice every other day, or twice a week, during the treatment period.
6. Example 1 6.1 Materials and MethodsAnimals and Treatment.
We used 4-6 weeks old C57BL6 and BALB/C in all the experiments. Mice received subcutaneous injections of UDP-glucose (200 mg/kg, Sigma) dissolved in sterile endotoxin-free PBS. G-CSF (Neupogen, Amgen) was administered daily at a dose of 300 μg/kg subcutaneously for 4 consecutive days as previously described (Broxmeyer et al., 2005). For the combination group, mice were injected UDP-Glc at 200 mg/kg subcutaneously for 6 consecutive days (from day 0 to day 5), accompanied by 300 μg/kg subcutaneous injections of G-CSF (from day 2 to day 5). Antioxidant, N-acetyl-L-cysteine (Sigma-Aldrich), was administered subcutaneously at 100 mg/kg/day. Bone marrow cells were obtained from both femur and tibia and used for flow cytometry and Western blot analysis. All animal studies were conducted after review by the University of Pittsburgh's Institutional Animal Care and Use Committee and in accordance with the University of Pittsburgh's Policy on the Care, Welfare and Treatment of Laboratory Animals.
Colony Forming Cell (CFC) Assay and Cobblestone Area Forming Cell (CAFC) Assay.
Mobilized mononuclear peripheral blood cells (1×106) and spleen cells (0.5×106) were seeded for CFC assay. The number of BFU-E, CFU-GM and CFU-GEMM colonies was counted using standard criteria. CAFC assay was performed in duplicate using MyeloCult M5300 (StemCell Technologies) as described previously (Ploemacher et al., 1989). After 5 weeks, wells containing cobblestone areas were counted as positive wells.
Transplantation.
For competitive repopulation assays, an equal number of peripheral blood cells mobilized by each agent (PBS vs. UDP-Glc; UDP-Glc vs. G-CSF; G-CSF vs. UDP-Glc/G-CSF) were transplanted into conditioned recipient mice (CD45.1.2., 9.5-10Gy). Although we used CD45 congenic animals (B6) in competitive repopulation assay, in order to confirm that our results are not due to potential variability resulting from the disparity between the CD45.1 and CD45.2, the results were further confirmed by injecting mobilizers the other way around (ex; inject G-CSF into CD45.1 and UDP-Glc to CD45.2 mice, and vice versa). The ratio of CD45.1/CD45.2 cells in recipient's peripheral blood was determined at various times after transplantation.
Flow Cytometry Analysis.
The relative contributions of UDP-Glc, G-CSF, and UDP-Glc/G-CSF-mobilized peripheral blood cells to recipient blood and bone marrow were assessed by flow cytometry analysis using anti-CD45.1 and anti-CD45.2 antibodies (eBioscience). Peripheral blood Lin-/Sca-1+/c-Kit+ (LSK) and CD150+CD48-(SLAM) LSK cells were phenotyped using the following antibodies: lineage markers PE-Cy7-conjuaged anti-CD3, anti-CD4, anti-CD8, anti-CD45R, anti-CD11b, anti-Gr-1, and anti-TER-119 (eBioscience); PE-conjugated anti-Sca-1 (eBioscience); APC-conjugated anti-c-Kit (eBioscience); perCP/Cy5.5-conjuated anti-CD150 (BioLegend); pacific Blue™-conjugated anti-CD48 (BioLegend). The percentage of bone marrow LSK and SLAM LSK cells derived from UDP-Glc- and G-CSF-mobilized cells was analyzed among gated either the CD45.1 or CD45.2 compartment. Mitochondrial superoxide level was measured using MitoSox™ (Invitrogen) within LSK cells, according to manufacturer's instructions.
Western Blot Analysis.
Equal amounts (20 μg/sample) of protein extracts were loaded on 15% SDS-PAGE and blotted onto polyvinyl difluoride membranes. The blots were probed with primary antibody specific for goat polyclonal RANKL (Santa Cruz Biotechnology) and mouse monoclonal β-actin (Sigma-Aldrich) overnight at 4° C.
TRAP Staining and Immunohistochemistry.
Femurs dissected from treated mice were fixed in 4% paraformaldehyde solution in phosphate buffered saline (PBS, pH7.2) for 2 days and then decalcified in 10% EDTA (pH 7.5) for 10 days. After decalcification, they were embedded in paraffin and longitudinally cut to 5 μm thickness. For identification of osteoclasts, the sections were deparaffinized, dehydrated, and stained using TRAP staining kit (B-Bridge International, Inc.) according to the manufacturer's instructions. For immunohistochemical staining of RANKL, after dehydration, the sections were immunolabeled overnight with goat polyclonal antibody against mouse RANKL (Santa Cruz Biotechnology, 1:50) at 4° C. Subsequently, they were incubated with biotinylated goat-specific secondary antibody (Vector Laboratories) followed by DAB staining according to the manufacturer's instructions (Vector Laboratories).
Statistical Analysis.
All the data were expressed as the mean±standard deviation (SD). A one-way ANOVA was used for multiple comparisons using SPSS version 16.0 software. A P value <0.05 was considered statistically significant.
6.2 ResultsUDP-Glc Promotes Mobilization of Hematopoietic Stem Progenitor Cells.
To test whether in vivo administration of exogenous UDP-Glc may mimic stress conditions and trigger HSPC mobilization, we injected UDP-Glc into mice and assessed its ability to mobilize HSPCs that are capable of forming colonies (CFU-Cs). Spleen cells from UDP-Glc-treated mice showed an increase in the number of CFU-GM (
UDP-Glc Mobilizes Long-Term Repopulating Hematopoietic Stem Progenitor Cells.
G-CSF is the most commonly used cytokine for mobilization of HSPCs in the clinic. We thus determined the mobilizing capability of UDP-Glc in comparison with G-CSF. G-CSF was administered as described in Broxmeyer et al., 2005 and Wright et al., 2001. UDP-Glc was significantly less efficient than G-CSF in mobilizing CFU-Cs to peripheral blood (
Neither phenotypic analysis nor in vitro HSPC assays necessarily accurately reflect stem progenitor cell activity in vivo (Park et al., 2008). To assess the functional properties of UDP-Glc-mobilized HSPCs in vivo, we performed competitive repopulation assays, where the equal number of blood cells, from either control or UDP-Glc-treated mice, was transplanted into conditioned recipient mice. UDP-Glc-mobilized cells showed a significant repopulation advantage compared to vehicle-treated blood cells over a 3-month period post-transplant (
The maintenance of stem cell pool and generation of functional mature blood cells depend on close interaction with specialized microenvironments or niches in bone marrow (Purton and Scadden, 2006). Therefore, the engraftment of HSPCs to bone marrow more accurately represents clinical outcome in clinical protocols. We thus assessed whether donor-derived HSPCs are sustainable in the bone marrow of recipient animals for an extended period after transplantation. Sixteen weeks after transplantation, we could readily detect HSPC population (LSK and SLAM LSK cells) derived from UDP-Glc-mobilized cells in the bone marrow of recipient animals (
Next, we compared the HSPC mobilizing capability of UDP-Glc with that of G-CSF using competitive repopulation assay. At one month following transplantation, G-CSF-mobilized cells displayed a considerable competitive advantage over UDP-Glc-mobilized cells (
We assessed whether this was because the recipient's bone marrow niches were predominantly occupied by UDP-Glc-mobilized cells. To this end, we analyzed the bone marrow of recipient animals at 18 weeks after transplantation. A significantly higher portion of LSK and SLAM LSK cells in recipient bone marrow were derived from UDP-Glc-treated mice at 18 weeks after transplantation (
The preferential engraftment of long-term repopulating cells with UDP-Glc-mobilized cells may indicate the possibility that UDP-Glc mobilizes a more primitive subset of HSPCs such as SLAM LSK cells than G-CSF. UDP-Glc promoted LSK cell mobilization into the peripheral blood, with efficacy similar to that of G-CSF (0.048% vs. 0.058%) (
Serial transplantation represents the gold standard for assessing the long-term repopulation abilities. In order to further compare the long-term repopulation abilities of UDP-Glc- and G-CSF-mobilized HSPCs, we performed serial transplantation experiments under competitive settings. Primary recipients were transplanted with UDP-Glc (CD45.2)- and G-CSF (CD45.1)-mobilized peripheral blood cells as shown in
Administration of G-CSF promotes cell cycle entry by quiescent HSC in both mice and baboon (Steinman, 2002). Unlike G-CSF, UDP-Glc does not appear to function as a potent mitogen for HSPCs. Therefore, it is conceivable that UDP-Glc releases HSPCs from the niche without disruption of their cell cycle quiescence, and this may improve the long-term engraftment ability of UDP-Glc-mobilized HSPCs. Indeed, UDP-Glc did not result in significant changes in the G2/M or S phase of the cell cycle. Rather, UDP-Glc-treated mice showed an increased quiescent G0 fraction of HSPCs in their bone marrow (61% vs. 72%) (
It is also interesting to note that UDP-Glc-mobilized cells, as compared to G-CSF-mobilized counterparts, exhibited a differentiation pattern skewed toward the lymphoid lineage in recipient mice (
The Combination of UDP-Glc with G-CSF Improves Hematopoietic Stem Progenitor Cell Mobilization.
There is a keen interest in improving the mobilizing effects of G-CSF (Broxmeyer et al., 2005). Therefore, we investigated possible functional synergies between UDP-Glc and G-CSF. The mobilizing effect of UDP-Glc peaked 2-3 hours after the sixth daily consecutive injection (
UDP-Glc Mobilizes Hematopoietic Stem Progenitor Cells Through the Alterations of the Osteoblast/Osteoclast Balance Mediated by ROS.
It has been recently proposed that Reactive Oxygen Species (ROS) signaling is closely associated with HSPC mobilization (Dar et al., 2011; Tesio et al., 2011). We therefore examined whether UDP-Glc modulates the level of intracellular ROS levels. Since mitochondria are a major source of ROS, we measured the levels of mitochondrial superoxide in LSK cells. Upon UDP-Glc treatment, ROS levels were significantly increased in LSK cells (
To investigate if the elevated ROS levels are indeed potential mediators of the UDP-Glc-mediated HSPC mobilization, an antioxidant, N-Acetylcysteine (NAC), was administered. NAC was able to significantly abrogate the LSK cell mobilization induced by UDP-Glc (
Without being bound to any particular theory, it is plausible that UDP-Glc increases ROS levels, and this in turn enhances RANKL-induced osteoclast differentiation, leading to HSPC mobilization.
Interestingly, while the combination of UDP-Glc and G-CSF augmented HSPC mobilization, it significantly reduced ROS levels compared to UDP-Glc alone (
Mobilized HSPCs could regenerate a complete hematopoietic system for cancer patients with hematolymphoid malignancies or solid tumors, yet, more than 20 percent of patients fail to mobilize sufficient stem cells for transplantation (Schmitz et al., 1996). These so-called “poor mobilizer patients” include patients who were previously treated with intensive radiation and chemotherapy; those who have genetic disorders such as Fanconi's anemia; and those who are over 60 years of age (Broxmeyer et al., 2005; Cottler-Fox et al., 2003). A combination of G-CSF with cytotoxic agents improves HSPC mobilization in the poor mobilizer patients, but often is accompanied by serious side effects (Hornung and Longo, 1992). Such limitations necessitate the discovery of novel mobilizing regimens that permit tailoring therapy on an individual basis.
In this study, we identified UDP-Glc as a novel mobilizer of HSPCs and investigated the phenotypic and functional features of UDP-Glc-mobilized cells.
Following administration of UDP-Glc, the blood contained increased numbers of HSPCs including CFU-GM, BFU-E and CFU-GEMM. However, UDP-Glc-mobilized cells had a significantly lower capacity to form in vitro colonies compared to G-CSF-mobilized cells. This indicates that UDP-Glc is not as efficient as G-CSF in mobilizing lineage committed progenitor cells. In contrast, UDP-Glc and G-CSF exhibited an approximately equivalent level of CAFC activity, suggesting that UDP-Glc preferentially mobilize the more primitive subset of HSPCs.
Functional characteristics of HSPC, such as homing, engraftment, cell cycle status and self renewal vary according to their tissue of origin (Chitteti et al., 2011; Lapid et al., 2008). Indeed, circulating blood stem cells cannot compete effectively against bone marrow-derived stem cells for long-term multilineage repopulation (Micklem et al., 1975). Therefore, when mobilized cells are assessed for their functional activity, it is more legitimate to compare cells from same tissue origin, i.e. G-CSF-mobilized peripheral blood vs. UDP-Glc-mobilized peripheral blood.
To this end, we adapted a competitive repopulation assay in which a mixture of equal numbers of UDP-Glc- and G-CSF-mobilized blood cells are transplanted into conditioned recipients, which allows a direct comparison of UDP-Glc-mobilized cells to G-CSF-mobilized cells under the same microenvironment. Using the donor chimerism analysis at several time points following transplantation, we found that G-CSF-mobilized cells were predominant during the early post-transplantation period, which probably reflects the superior ability of G-CSF to mobilize HSPCs and/or short-term repopulating cells. However, as post-transplant time passed, UDP-Glc-mobilized cells out-competed G-CSF-mobilized cells for the repopulation of recipient animals.
While long-term and short-term HSPCs show a similar multilineage potential, their self renewal capacity is different. Therefore, one of the most important aspects of stem cell mobilization is whether cells mobilized by “mobilizers” have a long-term repopulating ability. UDP-Glc-mobilized peripheral blood contained a greater numbers of SLAM LSK cells than G-CSF-mobilized cells, which could provide a potential explanation for their superior long term repopulating ability (
It is known that quiescent HSCs have higher long-term repopulating abilities than HSCs in active cell cycle (Passegue et al., 2005). Since UDP-Glc did not disrupt cell cycle quiescence of HSPC (
Peripheral blood cells mobilized by a combination of G-CSF and UDP-Glc consistently out-compete G-CSF-mobilized cells throughout the whole post-transplantation period, indicating that the combination regimen enhances both short- and long-term repopulating capacity of the mobilized cells. In this context, UDP-Glc can also be viewed as a complementary regimen that potentiates the long-term repopulating capacity of G-CSF mobilization.
HSPC mobilization is a dynamic, cyclical, and multistage process. The molecular mechanisms that are responsible for HSPC mobilization are complex. Redox signaling plays a central role in regulating HSPC mobilization (Tesio et al., 2011), because many of the cytokines, chemokines and adhesion molecules associated with HSPC mobilization are regulated through a redox-regulated process (Lekli et al., 2009). Mice treated with UDP-Glc expressed high levels of mitochondrial superoxide in their HSPCs. Lowering these ROS levels by antioxidants significantly reduced the mobilizing effect of UDP-Glc and this coincided with the reduction in RANKL and osteoclastogenesis. These results, therefore, suggest that ROS play a role in mediating the UDP-Glc-induced HSPC mobilization through an increase of RANKL expression and osteoclast activity. The other mechanisms for UDP-Glc-induced mobilization would be an indirect effect involving activation of neutrophils with the subsequent release of proteases (Pruijt et al., 2002), since the increased levels of proteases can attack several target proteins, including CXCR4, SDF-1, or VCAM-1, leading to inactivation of CXCR4/CXCL12- or VCAM-1/VLA-4-dependent signals and thus cell migration out of BM. This is however unlikely to be the scenario, because UDP-Glc-mobilized HSPCs appear to favor differentiation of lymphoid rather than myeloid lineage (
UDP-Glc is a natural product, so that it may mitigate many of the side effects which are often associated with other synthetic mobilizers. Indeed, none of the UDP-Glc-treated animals showed signs of side effects such as spleen enlargement (See
- Abbracchio, M. P., and Burnstock, G. (1998). Purinergic signalling: pathophysiological roles. Jpn J Pharmacol 78, 113-145.
- Alcino, A., Fresno, M., and Alarcon, B. (1988) Activity of P536, an Analog of UDP-Glucose, Against Trypanosoma cruzi. Antimicrob. Agents and Chemother. 32(9), 1412-1415.
- Arase, T., Uchida, H., Kajitani, T., Ono, M., Tamaki, K., Oda, H., Nishikawa, S., Kagami, M., Nagashima, T., Masuda, H., et al. (2009). The UDP-glucose receptor P2RY14 triggers innate mucosal immunity in the female reproductive tract by inducing IL-8. J Immunol 182, 7074-7084.
- Bai, X. C., Lu, D., Liu, A. L., Zhang, Z. M., Li, X. M., Zou, Z. P., Zeng, W. S., Cheng, B. L., and Luo, S. Q. (2005). Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast. J Biol Chem 280, 17497-17506.
- Barsony, J., Sugimura, Y., and Verbalis, J. G. (2011). Osteoclast response to low extracellular sodium and the mechanism of hyponatremia-induced bone loss. J Biol Chem 286, 10864-10875.
- Brautigam, V. M., Dubyak, G. R., Crain, J. M., and Watters, J. J. (2008). The inflammatory effects of UDP-glucose in N9 microglia are not mediated by P2Y14 receptor activation. Purinergic Signal 4, 73-78.
- Broxmeyer, H. E., Orschell, C. M., Clapp, D. W., Hangoc, G., Cooper, S., Plett, P. A., Liles, W. C., Li, X., Graham-Evans, B., Campbell, T. B., et al. (2005). Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 201, 1307-1318.
- Campbell R. and Tanner, M. (1999). UDP-Glucose Analogues as Inhibitors and Mechanistic Probes of UDP-Glucose Dehydrogenase. J. Org. Chem., 64(26), 9487-9492.
- Carter R L, Fricks I P, Barrett M O, Burianek L E, Zhou Y, Ko H, Das A, Jacobson K A, Lazarowski E R, Harden T K. (2009) Quantification of Gi-mediated inhibition of adenylyl cyclase activity reveals that UDP is a potent agonist of the human P2Y14 receptor. Mol Pharmacol. 76(6):1341-8.
- Cashen, A. F. , Nervi, B. and Dipersio, J. (2007) AMD3100: CXCR4 antagonist and rapid stem cell-mobilizing agent. Future Oncol. 3 (1):19-27.
- Chen, Y., Corriden, R., Inoue, Y., Yip, L., Hashiguchi, N., Zinkernagel, A., Nizet, V., Insel, P. A., and Junger, W. G. (2006). ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors. Science 314, 1792-1795.
- Chitteti, B. R., Liu, Y., and Srour, E. F. (2011). Genomic and proteomic analysis of the impact of mitotic quiescence on the engraftment of human CD34+ cells. PLoS One 6, e17498.
- Cottler-Fox, M. H., Lapidot, T., Petit, I., Kollet, O., DiPersio, J. F., Link, D., and Devine, S. (2003). Stem cell mobilization. Hematology Am Soc Hematol Educ Program, 419-437.
- Cramer, D., Wagner, S., Li, B., Liu, J., Hansen, R., Reca, R., Wu, W., Surma, E. Z., Laber, D. A., Ratajczak, M. Z., and Yan, J., (2008). Mobilization of Hematopoietic Progenitor Cells by Yeast-Derived-Glucan Requires Activation of Matrix Metalloproteinase-9. Stem Cells 26, 1231-1240.
- D′Addio, A., Curti, A., Worel, N., Douglas, K., Motta, M. R., Rizzi, S., Dan, E., Taioli, S., Giudice, V., Agis, H., et al. (2011). The addition of plerixafor is safe and allows adequate PBSC collection in multiple myeloma and lymphoma patients poor mobilizers after chemotherapy and G-CSF. Bone Marrow Transplant 46, 356-363.
- Dar, A., Schajnovitz, A., Lapid, K., Kalinkovich, A., Itkin, T., Ludin, A., Kao, W. M., Battista, M., Tesio, M., Kollet, O., et al. (2011). Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells. Leukemia.
- Di Virgilio, F., Chiozzi, P., Ferrari, D., Falzoni, S., Sanz, J. M., Morelli, A., Torboli, M., Bolognesi, G., and Baricordi, O. R. (2001). Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood 97, 587-600.
- Eigenbrodt, E., Reinacher, M., Scheefers-Borchel, U., Scheefers, H., and Friis, R. (1992). Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells. Crit Rev Oncog 3, 91-115.
- Fricks, I. P., Maddileti, S., Carter, R. L., Lazarowski, E. R., Nicholas, R. A., Jacobson, K. A., and Harden, T. K. (2008) UDP Is a Competitive Antagonist at the Human P2Y14 ReceptorJ Pharmacol Exp Ther. 325(2), 588-594.
- Greenbaum, A. M., and Link, D. C. (2011). Mechanisms of G-CSF-mediated hematopoietic stem and progenitor mobilization. Leukemia 25, 211-217.
- Hamel et al., (2011) J Biomol Screen 16 (9), 1098-1105.
- Hartmann, O., Le Corroller, A. G., Blaise, D., Michon, J., Philip, I., Norol,
- F., Janvier, M., Pico, J. L., Baranzelli, M. C., Rubie, H., et al. (1997). Peripheral blood stem cell and bone marrow transplantation for solid tumors and lymphomas: hematologic recovery and costs. A randomized, controlled trial. Ann Intern Med 126, 600-607.
- Hill, G. R., Olver, S. D., Kuns, R. D., Varelias, A., Raffelt, N. C., Don, A. L., Markey, K. A., Wilson, Y. A., Smyth, M. J., Iwakura, Y., et al. (2010). Stem cell mobilization with G-CSF induces type 17 differentiation and promotes scleroderma. Blood 116, 819-828.
- Hornung, R. L., and Longo, D. L. (1992). Hematopoietic stem cell depletion by restorative growth factor regimens during repeated high-dose cyclophosphamide therapy. Blood 80, 77-83.
- Kollet, O., Dar, A., Shivtiel, S., Kalinkovich, A., Lapid, K., Sztainberg, Y., Tesio, M., Samstein, R. M., Goichberg, P., Spiegel, A., et al. (2006). Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat Med 12, 657-664.
- Lapid, K., Vagima, Y., Kollet, O., and Lapidot, T. (2008). Egress and mobilization of hematopoietic stem and progenitor cells.
- Lazarowski, E. R., Shea, D. A., Boucher, R. C., and Harden, T. K. (2003). Release of cellular UDP-glucose as a potential extracellular signaling molecule. Mol Pharmacol 63, 1190-1197.
- Lekli, I., Gurusamy, N., Ray, D., Tosaki, A., and Das, D. K. (2009). Redox regulation of stem cell mobilization. Can J Physiol Pharmacol 87, 989-995.
- Linden, J. (2006). Cell biology. Purinergic chemotaxis. Science 314, 1689-1690.
- Liu, F., Poursine-Laurent, J., and Link, D. C. (2000). Expression of the G-CSF receptor on hematopoietic progenitor cells is not required for their mobilization by G-CSF. Blood 95, 3025-3031.
- Majolino, I., Aversa, F., Bacigalupo, A., Bandini, G., Arcese, W., and Reali, G. (1995). Allogeneic transplants of rhG-CSF-mobilized peripheral blood stem cells (PBSC) from normal donors. GITMO. Gruppo Italiano Trapianto di Midollo Osseo. Haematologica 80, 40-43.
- Micklem, H. S., Anderson, N., and Ross, E. (1975). Limited potential of circulating haemopoietic stem cells. Nature 256, 41-43.
- Mollee, P., Pereira, D., Nagy, T., Song, K., Saragosa, R., Keating, A., and Crump, M. (2002). Cyclophosphamide, etoposide and G-CSF to mobilize peripheral blood stem cells for autologous stem cell transplantation in patients with lymphoma. Bone Marrow Transplant 30, 273-278.
- Neben, S., Marcus, K., and Mauch, P. (1993). Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor. Blood 81, 1960-1967.
- Park, C. Y., Majeti, R., and Weissman, I. L. (2008). In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood. Nat Protoc 3, 1932-1940.
- Passegue, E., Wagers, A. J., Giuriato, S., Anderson, W. C., and Weissman, I. L. (2005). Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J Exp Med 202, 1599-1611.
- Platzbecker, U., Prange-Krex, G., Bornhauser, M., Koch, R., Soucek, S., Aikele, P., Haack, A., Haag, C., Schuler, U., Berndt, A., et al. (2001). Spleen enlargement in healthy donors during G-CSF mobilization of PBPCs. Transfusion 41, 184-189.
- Ploemacher, R. E., van der Sluijs, J. P., Voerman, J. S., and Brons, N. H. (1989). An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74, 2755-2763.
- Pruijt, J. F., Verzaal, P., van Os, R., de Kruijf, E. J., van Schie, M. L., Mantovani, A., Vecchi, A., Lindley, I. J., Willemze, R., Starckx, S., et al. (2002). Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice. Proc Natl Acad Sci US A 99, 6228-6233.
- Pulliam, A. C., Hobson, M. J., Ciccone, S. L., Li, Y., Chen, S., Srour, E. F., Yang, F. C., Broxmeyer, H. E., and Clapp, D. W. (2008). AMD3100 synergizes with G-CSF to mobilize repopulating stem cells in Fanconi anemia knockout mice. Exp Hematol 36, 1084-1090.
- Purton, L. E., and Scadden, D. T. (2006). Osteoclasts eat stem cells out of house and home. Nat Med 12, 610-611.
- Rossi, L., Manfredini, R., Bertolini, F., Ferrari, D., Fogli, M., Zini, R., Salati, S., Salvestrini, V., Gulinelli, S., Adinolfi, E., et al. (2007). The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration. Blood 109, 533-542.
- Sak, K., Boeynaems, J. M., and Everaus, H. (2003). Involvement of P2Y receptors in the differentiation of haematopoietic cells. J Leukoc Biol 73, 442-447.
- Schmitz, N., Linch, D. C., Dreger, P., Goldstone, A. H., Boogaerts, M. A., Ferrant, A., Demuynck, H. M., Link, H., Zander, A., and Barge, A. (1996). Randomised trial of filgrastim-mobilised peripheral blood progenitor cell transplantation versus autologous bone-marrow transplantation in lymphoma patients. Lancet 347, 353-357.
- Scrivens, M., and Dickenson, J. M. (2005). Pharmacological effects mediated by UDP-glucose that are independent of P2Y14 receptor expression. Pharmacol Res 51, 533-538.
- Steinman, R. A. (2002). Cell cycle regulators and hematopoiesis. Oncogene 21, 3403-3413.
- Tesio, M., Golan, K., Corso, S., Giordano, S., Schajnovitz, A., Vagima, Y., Shivtiel, S., Kalinkovich, A., Caione, L., Gammaitoni, L., et al. (2011). Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling. Blood 117, 419-428.
- Tricot, G., Jagannath, S., Vesole, D., Nelson, J., Tindle, S., Miller, L., Cheson, B., Crowley, J., and Barlogie, B. (1995). Peripheral blood stem cell transplants for multiple myeloma: identification of favorable variables for rapid engraftment in 225 patients. Blood 85, 588-596.
- Wihlborg A K, Balogh J, Wang L, Boma C, Dou Y, Joshi B V, Lazarowski E, Jacobson K A, Amer A, Erlinge D. (2006). Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphate (UDP) via P2Y2 and P2Y6 receptors on cardiomyocytes and release of UTP in man during myocardial infarction. Circ Res. 98(7):970-6
- Wright, D. E., Cheshier, S. H., Wagers, A. J., Randall, T. D., Christensen, J. L., and Weissman, I. L. (2001). Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle. Blood 97, 2278-2285.
- Yeoh, J. S., Ausema, A., Wierenga, P., de Haan, G., and van Os, R. (2007). Mobilized peripheral blood stem cells provide rapid reconstitution but impaired long-term engraftment in a mouse model. Bone Marrow Transplant 39, 401-409.
Animals and Treatment.
Mice received subcutaneous injections of UDP-glucose (200 mg/kg, Sigma) dissolved in sterile endotoxin-free PBS. G-CSF (Neupogen, Amgen) was administered daily at a dose of 300 mg/kg subcutaneously for 4 consecutive days as previously described (10). For the combination group, mice were injected UDP-Glc at 200 mg/kg subcutaneously for 6 consecutive days (from day 0 to day 5), accompanied by 300 μg/kg subcutaneous injections of G-CSF (from day 2 to day 5). Antioxidant, N-acetyl-L-cysteine (Sigma-Aldrich), was administered subcutaneously at 100 mg/kg/day. Bone marrow cells were obtained from both femur and tibia and used for flow cytometry and Western blot analysis. All animal studies were conducted after review by the University of Pittsburgh's Institutional Animal Care and Use Committee and in accordance with the University of Pittsburgh's Policy on the Care, Welfare and Treatment of Laboratory Animals.
Colony Forming Cell (CFC) Assay and Cobblestone Area Forming Cell (CAFC) Assay.
Mobilized mononuclear peripheral blood cells (1×106) and spleen cells (0.5×106) were seeded for CFC assay. The number of BFU-E, CFU-GM and CFU-GEMM colonies was counted using standard criteria. CAFC assay was performed in duplicate using MyeloCult M5300 (StemCell Technologies) as described previously (58). After 5 weeks, wells containing cobblestone areas were counted as positive wells.
Transplantation.
For competitive repopulation assays, an equal number of peripheral blood cells mobilized by each agent (PBS vs. UDP-Glc; UDP-Glc vs. G-CSF; G-CSF vs. UDP-Glc/G-CSF) were transplanted into conditioned recipient mice (CD45.1.2., 9.5-10Gy). Although we used CD45 congenic animals (B6) in competitive repopulation assay, in order to confirm that our results are not due to potential variability resulting from the disparity between the CD45.1 and CD45.2, the results were further confirmed by injecting mobilizers the other way around (ex; inject G-CSF into CD45.1 and UDP-Glc to CD45.2 mice, and vice versa). The ratio of CD45.1/CD45.2 cells in recipient's peripheral blood was determined at various times after transplantation. When we transplant sorted CD45.1+ SLAM LSK or CD45.2+ SLAM LSK cells into lethally irradiated animals, 1-2×106 peripheral blood cells (CD45.1.2) were co-administered whose contribution to recipient hematopoietic reconstitution is minimal.
Flow Cytometry Analysis.
The relative contributions of UDP-Glc, G-CSF, and UDP-Glc/G-CSF-mobilized peripheral blood cells to recipient blood and bone marrow were assessed by flow cytometry analysis using anti-CD45.1 and anti-CD45.2 antibodies (eBioscience). Peripheral blood Lin-/Sca-1+/c-Kit+(LSK) and CD150+CD48-(SLAM) LSK cells were phenotyped using the following antibodies: lineage markers PE-Cy7-conjuaged anti-CD3, anti-CD4, anti-CD8, anti-CD45R, anti-CD11 b. anti-Gr-1, and anti-TER-119 (eBioscience); PE-conjugated anti-Sca-1 (eBioscience); APC-conjugated anti-c-Kit (eBioscience); perCP/Cy5.5-conjuated anti-CD150 (BioLegend); pacific Blue™-conjugated anti-CD48 (BioLegend). The percentage of bone marrow LSK and SLAM LSK cells derived from UDP-Glc- and G-CSF-mobilized cells was analyzed among gated either the CD45.1 or CD45.2 compartment. Mitochondrial superoxide level was measured using MitoSox™ (Invitrogen) within LSK cells, according to manufacturer's instructions.
Western Blot Analysis.
Equal amounts (20 μg/sample) of protein extracts were loaded on 15% SDS-PAGE and blotted onto polyvinyl difluoride membranes. The blots were probed with primary antibody specific for goat polyclonal RANKL (Santa Cruz Biotechnology) and mouse monoclonal β-actin (Sigma-Aldrich) overnight at 4° C. Densitometric analysis was performed using Un-scan-IT image analysis software.
TRAP Staining and Immunohistochemistry:
Femurs dissected from treated mice were fixed in 4% paraformaldehyde solution in phosphate buffered saline (PBS, pH7.2) for 2 days and then decalcified in 10% EDTA (pH 7.5) for 10 days. After decalcification, they were embedded in paraffin and longitudinally cut to 5 μm thickness. For identification of osteoclasts, the sections were deparaffinized, dehydrated, and stained using TRAP staining kit (B-Bridge International, Inc.) according to the manufacturer's instructions. For the in vitro osteoclast differentiation assay, bone marrow cells (2×105) were pretreated with 20 ng/ml M-CSF (eBioscience) for 3 days and further cultured 4 days with various concentrations (0-200 M) of UDP-Glc. After seven days of incubation, the cells were stained and counted as described above. For immunohistochemical staining of RANKL, after dehydration, the sections were immunolabeled overnight with goat polyclonal antibody against mouse RANKL (Santa Cruz Biotechnology, 1:50) at 4° C. Subsequently, they were incubated with biotinylated goat-specific secondary antibody (Vector Laboratories) followed by DAB staining according to the manufacturer's instructions (Vector Laboratories).
Chemotaxis Assay.
For chemotactic assays, lineage-depleted cells (106/well) were placed in the upper chamber. UDP, UTP and UDP-G (10 μM, Sigma) were placed to the bottom chamber with or without CXCL12 (120 ng/ml, Peprotech). After 6 hours incubation, migrated cells were stained with FITC-conjugated anti-mouse Sca-1 (eBioscience) and APC-conjugated anti-mouse c-Kit (eBioscience). Flow cytometry was used to enumerate migrated cells.
Zymographic Analysis.
For zymography, bone marrow supernatants were loaded on 10% pre-casted polyacrylamide gel with gelatin for MMP-2 and MMP-9, and 12.5% pre-casted polyacrylamide gel (BIO-RAD) with casein for neutrophil elastase (NE) and cathepsin G (CG) under non-reducing conditions. After electrophoresis, the gels were washed in zymogram renaturation buffer (2.5% Triton X-100) and then incubated overnight at 37° C. in zymogram development buffer (BIO-RAD). The gels were then stained with 0.5% Coomassie blue solution and destained with 30% ethanol and 10% acetic acid. Proteinase activity was determined by colorless zones against a blue background.
Statistical Analysis.
All the data were expressed as the mean±standard deviation (SD). A one-way ANOVA was used for multiple comparisons using SPSS version 16.0 software. A P value <0.05 was considered statistically significant.
7.2 RESULTSUDP-Glc Promotes Mobilization of Hematopoietic Stem Progenitor Cells.
To investigate whether in vivo administration of exogenous UDP-Glc may mimic stress conditions to trigger HSC/HPC mobilization, we injected UDP-Glc into mice and assessed for its ability to mobilize HPCs that are capable of forming colonies (CFU-Cs). Spleen cells from UDP-Glc-treated mice showed an increase in the number of colony-forming unit-granulocyte-macrophage (CFU-GM) (
Since mobilized HSPCs are routinely harvested from the peripheral blood in the clinic, we quantified HSPCs in the peripheral blood of UDP-Glc-treated mice. There was a notable increase in the frequency of LSK cells in the circulation after 6 daily single UDP-Glc injections in B6 mice, which is one of the most difficult mouse strain to be mobilized (25) (
It is known that uridine-5′-triphosphate (UTP) is functionally associated with human HSPC migration and their engraftment (18, 19). We thus examined whether UDP-Glc possesses a similar activity on mouse HSPCs. SDF-la (CXCL12), as anticipated, potently chemoattracted mouse HSPCs (LSK) cells (
UDP-Glc Mobilizes Long-Term Repopulating Hematopoietic Stem Progenitor Cells.
G-CSF is the most commonly used cytokine for mobilization of HSPCs in the clinic. We thus determined the mobilizing capability of UDP-Glc in comparison with G-CSF. G-CSF was administered as described in the previous study (10, 11). As shown in
Neither phenotypic analysis nor in vitro HPC assays necessarily accurately reflect stem progenitor cell activity in vivo (26). To assess the functional properties of UDP-Glc-mobilized HSPCs in vivo, we performed competitive repopulation assays, where the equal number of blood cells, from either control or UDP-Glc-treated mice, was transplanted into conditioned recipient mice (
The maintenance of stem cell pool and generation of functional mature blood cells depend on close interaction with specialized microenvironments or niches in bone marrow (27). Therefore, the engraftment of HSPCs to bone marrow more accurately represents clinical outcome in clinical protocols. We thus assessed whether donor-derived HSPCs are sustainable in the bone marrow of recipient animals for an extended period after transplantation. Sixteen weeks after transplantation, we could readily detect HSPC population (LSK and SLAM LSK cells) derived from UDP-Glc-mobilized cells in the bone marrow of recipient animals (
Of note, despite the significant increases in peripheral HSPCs, there was no significant change in the number of white blood cells (WBC) in mice treated with UDP-Glc (
A similar pattern in response to UDP-Glc was observed in G-CSFR deficient mice (Csf3r−/−). Despite the fact that G-CSFR deficient mice are neutrophenic at baseline (28), UDP-Glc was still able to induce a statistically significant mobilization of LSK cells in Csf3r−/− mice (
UDP-Glucose Mobilizes Distinct Subsets of Hematopoietic Stem Progenitor Cells in Comparison with G-CSF.
Next, we compared the HSPC mobilizing capability of UDP-Glc with that of G-CSF using competitive repopulation assay. At one month following transplantation, G-CSF-mobilized cells displayed a considerable competitive advantage over UDP-Glc-mobilized cells (
We assessed whether this was because recipient's bone marrow niches were predominantly occupied by UDP-Glc-mobilized cells. To this end, we analyzed the bone marrow of primary recipient animals at 18 weeks after transplantation. A significantly higher portion of LSK and SLAM LSK cells in recipient bone marrow were derived from UDP-Glc-treated mice (
Serial transplantation represents the gold standard for assessing the long-term repopulation abilities. In order to further compare the long-term repopulation abilities of UDP-Glc- and G-CSF-mobilized HSPCs, we performed serial transplantation experiments under competitive settings. Primary recipients (CD45.1.2) were transplanted with UDP-Glc (CD45.2)- and G-CSF (CD45.1)-mobilized peripheral blood cells as shown in
The preferential engraftment of long-term repopulating cells with UDP-Glc-mobilized cells may indicate the possibility that UDP-Glc mobilizes a more primitive subset of HSPCs such as SLAM LSK cells than G-CSF. UDP-Glc promoted LSK cell mobilization into the peripheral blood, with efficacy similar to that of G-CSF (0.048% vs. 0.058%) (
It is known that G-CSF administration promotes cell cycle entry by quiescent bone marrow HSC in both mice and baboon (29). Unlike G-CSF, UDP-Glc does not appear to function as a potent mitogen for bone marrow HSPCs (
It is also noteworthy that UDP-Glc-mobilized cells, as compared to G-CSF-mobilized counterparts, exhibited a differentiation pattern skewed toward the lymphoid lineage in recipient mice (
The Combination of UDP-Glc with G-CSF Improves Hematopoietic Stem Progenitor Cell Mobilization.
There is a keen interest in improving the mobilizing effects of G-CSF (10). Therefore, we investigated possible functional synergies between UDP-Glc and G-CSF. The mobilizing effect of UDP-Glc peaked 2-4 hours after the sixth daily consecutive injection (
UDP-Glc Mobilizes Hematopoietic Stem Progenitor Cells Through the Alterations of the Osteoblast/Osteoclast Balance Mediated by Mitochondrial Superoxide.
It has been recently proposed that Reactive Oxygen Species (ROS) signaling is closely associated with HSPC mobilization (30, 31). We examined whether UDP-Glc modulates the level of intracellular ROS levels in HSPCs. Since mitochondria are a major source of ROS, we measured the levels of mitochondrial superoxide in LSK cells. Upon UDP-Glc treatment, superoxide levels were significantly increased in LSK cells (
To investigate if the elevated superoxide levels are indeed potential mediators of the UDP-Glc-mediated HSPC mobilization, an antioxidant, N-Acetylcysteine (NAC), was administered. NAC was able to significantly abrogate the LSK and SLAM LSK cell mobilization induced by UDP-Glc (
Controversy still exists regarding the role of osteoclasts in regulating HSPC mobilization (34-37) raising a question as to whether osteoclasts indeed play an essential role in UDP-Glc-mediated HSPC mobilization. To address this question, we first utilized the osteopetrotic (op/op) mouse model. Mice homozygous for the op mutation exhibit a severe deficiency of osteoclasts so that this strain can serve as a model to investigate the role of osteoclasts in UDP-Glc-mediated HSPC mobilization (38). Administration of UDP-Glc into littermate control mice (CTL; +/op) induced osteoclastogenesis and promoted the mobilization of LSK cells and SLAM LSK cells (
To further study the impact of osteoblasts/osteoclasts in UDP-Glc-mediated HSPC mobilization, P2X7 deficient mice were analyzed. Deficiency of P2X7 in mice results in impaired bone formation and excessive bone resorption (39). In accordance with this finding, a significantly increased numbers of osteoclasts were detected in non-treated P2X7 KO mice (
Meanwhile, since the proteolytic enzymes produced by monocytes/granulocytes are also important contributors mediating HSPCs mobilization (40), we examined whether UDP-Glc could induce the release of proteases from monocytes/granulocytes. As previously documented, there were overall increases in the percent of CD11b+ and/or Gr-1+ cells in the bone marrow of G-CSF-treated mice (hatched bars in
We then determined the effect of UDP-Glc on the expression of CXCR4, which plays a key role in homing and mobilization of HSPCs (41). UDP-Glc-treated mice displayed no significant change in the percentage of CXCR4 expressing cells in their bone marrow LSK and SLAM LSK cells (
It is previously known that UDP antagonizes the action of UDP-Glc (42). We thus evaluated the effects of UDP on UDP-Glc-mediated HSPCs migration. UDP, alone, did not elicit any appreciable change in the number of LSK cells in the peripheral circulation (
Mobilized HSPCs could regenerate a complete hematopoietic system for cancer patients with hematolymphoid malignancies or solid tumors. Yet more than 20 percent of patients fail to mobilize sufficient stem cells for transplantation (43). These include patients who were previously treated with intensive radiation and chemotherapy; those who have genetic disorders such as Fanconi's anemia; and those who are over 60 years of age (10, 44). A combination of G-CSF with cytotoxic agents improves HSPC mobilization in the poor mobilizer patients, but often accompanies serious side effects (45). Such limitations necessitate the discovery of novel mobilizing regimens, so that it may be used to tailor therapy on an individual basis. Data presented in the current study establish a novel aspect of a nucleotide sugar, UDP-Glc, in the HSPC mobilization.
Functional characteristics of HSPCs, such as homing, engraftment, cell cycle status and self-renewal activity vary according to their tissue of origin (13, 46). For example, circulating blood stem cells can't compete effectively against bone marrow-derived stem cells for long-term multilineage repopulation (47). Therefore, when mobilized cells are assessed for their functional activity, it is more legitimate to compare cells from same tissue origin, i.e. G-CSF to compare cells from same tissue origin, i.e. G-CSF-mobilized peripheral blood vs. UDP-Glc-mobilized peripheral blood. To this end, we adapted a competitive repopulation assay in which a mixture of equal numbers of UDP-Glc- and G-CSF-mobilized blood cells are transplanted into conditioned recipients (
While UDP-Glc-mobilized cells had a lower capacity to form in vitro colonies compared with G-CSF-mobilized cells, serial transplantation experiments showed that UDP-Glc-mobilized cells have greater capacity than G-CSF-mobilized cells to engraft lethally irradiated recipients, suggesting that UDP-Glc preferentially mobilizes long-term self-renewing HSPCs. UDP-Glc-mobilized peripheral blood contained a greater numbers of SLAM LSK cells than G-CSF-mobilized cells (
Cytokine-induced stem cell mobilization is often accompanied by profound changes in number and composition of accessory cells contained within the PBSC collection (48). In contrast, UDP-Glc did not cause any noticeable quantitative changes in the accessory cell compartment (
The molecular mechanisms that are responsible for HSPC mobilization are complex and confounding. Redox signaling plays a central role in regulating HSPC mobilization (30), because many of the cytokines, chemokines and adhesion molecules associated with HSPC mobilization are regulated through a redox-regulated process (50). Mice treated with UDP-Glc expressed high levels of mitochondrial superoxide in their HSPCs. Lowering these mitochondrial superoxide levels by antioxidants significantly reduced the mobilizing effect of UDP-Glc and this coincided with the reduction in RANKL and osteoclastogenesis (
It has been shown that osteoclasts mediate HSPC egress from the endosteal osteoblastic niche by degrading endosteal components (34, 35, 37). However, other lines of evidence indicate that osteoclasts are dispensable for HSC mobilization (36). UDP-Glc mobilization was not achievable in mouse models of osteopetrosis (Op/Op), suggesting that osteoclast formation is required for UDP-Glc-mediated HSPC mobilization. Meanwhile, P2X7 knockout mice, which display excessive osteoclast resorption activity at 6-8 weeks of age (39), showed higher number of circulating LSK cells than WT mice even under steady-state conditions. UDP-Glc does not seem to further increase osteoclast formation in P2X7 KO mice that were already osteoporotic, and no significant change was observed in the number of peripheral LSK cells in UDP-Glc-injected P2X7 KO mice. Taken together, it is conceivable that the extent of osteoclast formation in response to UDP-Glc is functionally associated with the ability of UDP-Glc to mobilize HSPCs.
The other mechanisms for UDP-Glc-induced mobilization would be an indirect effect involving activation of neutrophils with the subsequent release of proteases (51): increased levels of proteases can attack several target proteins, including CXCR4, CXCL12 (SDF-1α), or VCAM-1, leading to inactivation of CXCR4/CXCL12- or VCAM-1/VLA-4-dependent signals and thus cell migration out of bone marrow. Unlike G-CSF, however, UDP-Glc had no effect on granulocyte and monocyte mobilization (
UDP-Glc treatment increased CXCR4-cell surface expression in peripheral HSPCs but not in bone marrow HSPCs (
UDP-Glc is known to bind the P2RY14 receptor. It is therefore of interest to investigate whether UDP-Glc triggers HSPC mobilization through P2RY14 receptor-dependent or -independent mechanisms (or both). While this area warrants further study using animal models such as conditional P2ry14 knockout animals, there are contradictory reports that UDP-Glc is not a functionally relevant ligand at P2RY14 receptor (52, 53). It is also noteworthy that HSPC mobilization is often mediated through multiple trans-acting signals rather than ligand-receptor interactions (54, 55). Recent studies established the biological significance of extracellular nucleotides in migration and engraftment of human HSPCs (18, 19): UTP has the capacity to chemoattract human CD34+ cells and enhances engraftment of human HSPCs. Because chemokine/chemokine receptor axes play critical role in HSPC mobilization, it draws attention that P2RY14 also encodes a 7-transmembrane G-protein coupled receptor (GPCR) with a chemokine receptor signature (e.g., DRY motif in the 3rd intracellular domain) (56). However, UDP-Glc doesn't appear to have chemoattractive properties on mouse LSK cells. Rather, UDP, which antagonizes the action of UDP-Glc (42), has chemotactic activity and attracts mouse LSK cells (
Quiescent HSCs have higher long-term repopulating abilities than HSCs in active cell cycle (57). Since UDP-Glc does not affect cell cycle quiescence of bone marrow-resident HSPCs (
UDP-Glc is a naturally occurring metabolite in the human body, so that it may mitigate many of the side effects which are often associated with other synthetic mobilizers. Indeed, none of the UDP-Glc-treated animals showed signs of side effects such as spleen enlargement (
Considering high cost, side effects and ineffectiveness of conventional mobilization regimens, there is a compelling need to seek alternative mobilization regimen. UDP-Glc mobilizes functionally distinct subsets of HSPCs compared to those mobilized by G-CSF, suggesting the possibility that the combination regimen can enhances both short- and long-term repopulating capacity of the mobilized cells. In this context, UDP-Glc can be utilized as a complement regimen that potentiates the long-term repopulating capacity of G-CSF mobilized HSPCs. Therefore, on the basis of our observations, UDP-Glc mobilization, either alone or combined to G-CSF, could potentially provide a scientific basis for improving transplantation outcomes. Moreover, UDP-Glc minimally affects the immune cell content of the mobilized cells and this may alter the likelihoods of graft failure, GVHD and GVL. The small size of UDP-Glc offers other tangible advantages over other protein-based mobilizers, including easy access to intracellular targets and low cost and ease of production as well as oral bioavailability. Administration of UDP-Glc appeared to be well tolerated at high levels, suggesting the potential suitability as a therapeutic agent in man.
7.4 REFERENCES
- 1. Hartmann, O., Le Corroller, A. G., Blaise, D., Michon, J., Philip, I., Norol, F., Janvier, M., Pico, J. L., Baranzelli, M. C., Rubie, H., et al. 1997. Peripheral blood stem cell and bone marrow transplantation for solid tumors and lymphomas: hematologic recovery and costs. A randomized, controlled trial. Ann Intern Med 126:600-607.
- 2. Tricot, G., Jagannath, S., Vesole, D., Nelson, J., Tindle, S., Miller, L., Cheson, B., Crowley, J., and Barlogie, B. 1995. Peripheral blood stem cell transplants for multiple myeloma: identification of favorable variables for rapid engraftment in 225 patients. Blood 85:588-596.
- 3. Neben, S., Marcus, K., and Mauch, P. 1993. Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor. Blood 81:1960-1967.
- 4. Majolino, I., Aversa, F., Bacigalupo, A., Bandini, G., Arcese, W., and Reali, G. 1995. Allogeneic transplants of rhG-CSF-mobilized peripheral blood stem cells (PBSC) from normal donors. GITMO. Gruppo Italiano Trapianto di Midollo Osseo. Haematologica 80:40-43.
- 5. Platzbecker, U., Prange-Krex, G., Bornhauser, M., Koch, R., Soucek, S., Aikele, P., Haack, A., Haag, C., Schuler, U., Berndt, A., et al. 2001. Spleen enlargement in healthy donors during G-CSF mobilization of PBPCs. Transfusion 41:184-189.
- 6. Yeoh, J. S., Ausema, A., Wierenga, P., de Haan, G., and van Os, R. 2007. Mobilized peripheral blood stem cells provide rapid reconstitution but impaired long-term engraftment in a mouse model. Bone Marrow Transplant 39:401-409.
- 7. Hill, G. R., Olver, S. D., Kuns, R. D., Varelias, A., Raffelt, N. C., Don, A. L., Markey, K. A., Wilson, Y. A., Smyth, M. J., Iwakura, Y., et al. 2010. Stem cell mobilization with G-CSF induces type 17 differentiation and promotes scleroderma. Blood 116:819-828.
- 8. Pulliam, A. C., Hobson, M. J., Ciccone, S. L., Li, Y., Chen, S., Srour, E. F., Yang, F. C., Broxmeyer, H. E., and Clapp, D. W. 2008. AMD3100 synergizes with G-CSF to mobilize repopulating stem cells in Fanconi anemia knockout mice. Exp Hematol 36:1084-1090.
- 9. D′Addio, A., Curti, A., Worel, N., Douglas, K., Motta, M. R., Rizzi, S., Dan, E., Taioli, S., Giudice, V., Agis, H., et al. 2011. The addition of plerixafor is safe and allows adequate PBSC collection in multiple myeloma and lymphoma patients poor mobilizers after chemotherapy and G-CSF. Bone Marrow Transplant 46:356-363.
- 10. Broxmeyer, H. E., Orschell, C. M., Clapp, D. W., Hangoc, G., Cooper, S., Plett, P. A., Liles, W. C., Li, X., Graham-Evans, B., Campbell, T. B., et al. 2005. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 201:1307-1318.
- 11. Wright, D. E., Cheshier, S. H., Wagers, A. J., Randall, T. D., Christensen, J. L., and Weissman, I. L. 2001. Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle. Blood 97:2278-2285.
- 12. Mollee, P., Pereira, D., Nagy, T., Song, K., Saragosa, R., Keating, A., and Crump, M. 2002. Cyclophosphamide, etoposide and G-CSF to mobilize peripheral blood stem cells for autologous stem cell transplantation in patients with lymphoma. Bone Marrow Transplant 30:273-278.
- 13. Lapid, K., Vagima, Y., Kollet, O., and Lapidot, T. 2008. Egress and mobilization of hematopoietic stem and progenitor cells.
- 14. Di Virgilio, F., Chiozzi, P., Ferrari, D., Falzoni, S., Sanz, J. M., Morelli, A., Torboli, M., Bolognesi, G., and Baricordi, O. R. 2001. Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood 97:587-600.
- 15. Sak, K., Boeynaems, J. M., and Everaus, H. 2003. Involvement of P2Y receptors in the differentiation of haematopoietic cells. J Leukoc Biol 73:442-447.
- 16. Linden, J. 2006. Cell biology. Purinergic chemotaxis. Science 314:1689-1690.
- 17. Chen, Y., Corriden, R., Inoue, Y., Yip, L., Hashiguchi, N., Zinkernagel, A., Nizet, V., Insel, P. A., and Junger, W. G. 2006. ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors. Science 314:1792-1795.
- 18. Rossi, L., Manfredini, R., Bertolini, F., Ferrari, D., Fogli, M., Zini, R., Salati, S., Salvestrini, V., Gulinelli, S., Adinolfi, E., et al. 2007. The extracellular nucleotide UTP is a potent inducer of hematopoietic stem cell migration. Blood 109:533-542.
- 19. Lemoli, R. M., Ferrari, D., Fogli, M., Rossi, L., Pizzirani, C., Forchap, S., Chiozzi, P., Vaselli, D., Bertolini, F., Foutz, T., et al. 2004. Extracellular nucleotides are potent stimulators of human hematopoietic stem cells in vitro and in vivo. Blood 104:1662-1670.
- 20. Abbracchio, M. P., and Burnstock, G. 1998. Purinergic signalling: pathophysiological roles. Jpn J Pharmacol 78:113-145.
- 21. Lazarowski, E. R., Shea, D. A., Boucher, R. C., and Harden, T. K. 2003. Release of cellular UDP-glucose as a potential extracellular signaling molecule. Mol Pharmacol 63:1190-1197.
- 22. Arase, T., Uchida, H., Kajitani, T., Ono, M., Tamaki, K., Oda, H., Nishikawa, S., Kagami, M., Nagashima, T., Masuda, H., et al. 2009. The UDP-glucose receptor P2RY14 triggers innate mucosal immunity in the female reproductive tract by inducing IL-8. J Immunol 182:7074-7084.
- 23. Eigenbrodt, E., Reinacher, M., Scheefers-Borchel, U., Scheefers, H., and Friis, R. 1992. Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pools found in tumor cells. Crit Rev Oncog 3:91-115.
- 24. He, S., Kim, I., Lim, M. S., and Morrison, S. J. 2011. Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors. Genes Dev 25:1613-1627.
- 25. Roberts, A. W., Foote, S., Alexander, W. S., Scott, C., Robb, L., and Metcalf, D. 1997. Genetic influences determining progenitor cell mobilization and leukocytosis induced by granulocyte colony-stimulating factor. Blood 89:2736-2744.
- 26. Park, C. Y., Majeti, R., and Weissman, I. L. 2008. In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood. Nat Protoc 3:1932-1940.
- 27. Purton, L. E., and Scadden, D. T. 2006. Osteoclasts eat stem cells out of house and home. Nat Med 12:610-611.
- 28. Liu, F., Wu, H. Y., Wesselschmidt, R., Kornaga, T., and Link, D. C. 1996. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice. Immunity 5:491-501.
- 29. Steinman, R. A. 2002. Cell cycle regulators and hematopoiesis. Oncogene 21:3403-3413.
- 30. Tesio, M., Golan, K., Corso, S., Giordano, S., Schajnovitz, A., Vagima, Y., Shivtiel, S., Kalinkovich, A., Caione, L., Gammaitoni, L., et al. 2011. Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling. Blood 117:419-428.
- 31. Dar, A., Schajnovitz, A., Lapid, K., Kalinkovich, A., Itkin, T., Ludin, A., Kao, W. M., Battista, M., Tesio, M., Kollet, O., et al. 2011. Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells. Leukemia.
- 32. Bai, X. C., Lu, D., Liu, A. L., Zhang, Z. M., Li, X. M., Zou, Z. P., Zeng, W. S., Cheng, B. L., and Luo, S. Q. 2005. Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast. J Biol Chem 280:17497-17506.
- 33. Barsony, J., Sugimura, Y., and Verbalis, J. G. 2011. Osteoclast response to low extracellular sodium and the mechanism of hyponatremia-induced bone loss. J Biol Chem 286:10864-10875.
- 34. Kollet, O., Dar, A., Shivtiel, S., Kalinkovich, A., Lapid, K., Sztainberg, Y., Tesio, M., Samstein, R. M., Goichberg, P., Spiegel, A., et al. 2006. Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat Med 12:657-664.
- 35. Calvi, L. M., Adams, G. B., Weibrecht, K. W., Weber, J. M., Olson, D. P., Knight, M. C., Martin, R. P., Schipani, E., Divieti, P., Bringhurst, F. R., et al. 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425:841-846.
- 36. Miyamoto, K., Yoshida, S., Kawasumi, M., Hashimoto, K., Kimura, T., Sato, Y., Kobayashi, T., Miyauchi, Y., Hoshi, H., Iwasaki, R., et al. 2011. Osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization. J Exp Med 208:2175-2181.
- 37. Takamatsu, Y., Simmons, P. J., Moore, R. J., Morris, H. A., To, L. B., and Levesque, J. P. 1998. Osteoclast-mediated bone resorption is stimulated during short-term administration of granulocyte colony-stimulating factor but is not responsible for hematopoietic progenitor cell mobilization. Blood 92:3465-3473.
- 38. Yoshida, H., Hayashi, S., Kunisada, T., Ogawa, M., Nishikawa, S., Okamura, H., Sudo, T., Shultz, L. D., and Nishikawa, S. 1990. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345:442-444.
- 39. Ke, H. Z., Qi, H., Weidema, A. F., Zhang, Q., Panupinthu, N., Crawford, D. T., Grasser, W. A., Paralkar, V. M., Li, M., Audoly, L. P., et al. 2003. Deletion of the P2X7 nucleotide receptor reveals its regulatory roles in bone formation and resorption. Mol Endocrinol 17:1356-1367.
- 40. Levesque, J. P., Hendy, J., Takamatsu, Y., Williams, B., Winkler, I. G., and Simmons, P. J. 2002. Mobilization by either cyclophosphamide or granulocyte colony-stimulating factor transforms the bone marrow into a highly proteolytic environment. Exp Hematol 30:440-449.
- 41. Peled, A., Petit, I., Kollet, O., Magid, M., Ponomaryov, T., Byk, T., Nagler, A., Ben-Hur, H., Many, A., Shultz, L., et al. 1999. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 283:845-848.
- 42. Fricks, I. P., Maddileti, S., Carter, R. L., Lazarowski, E. R., Nicholas, R. A., Jacobson, K. A., and Harden, T. K. 2008. UDP is a competitive antagonist at the human P2Y14 receptor. J Pharmacol Exp Ther 325:588-594.
- 43. Schmitz, N., Linch, D. C., Dreger, P., Goldstone, A. H., Boogaerts, M. A., Ferrant, A., Demuynck, H. M., Link, H., Zander, A., and Barge, A. 1996. Randomised trial of filgrastim-mobilised peripheral blood progenitor cell transplantation versus autologous bone-marrow transplantation in lymphoma patients. Lancet 347:353-357.
- 44. Cottler-Fox, M. H., Lapidot, T., Petit, I., Kollet, O., DiPersio, J. F., Link, D., and Devine, S. 2003. Stem cell mobilization. Hematology Am Soc Hematol Educ Program:419-437.
- 45. Hornung, R. L., and Longo, D. L. 1992. Hematopoietic stem cell depletion by restorative growth factor regimens during repeated high-dose cyclophosphamide therapy. Blood 80:77-83.
- 46. Chitteti, B. R., Liu, Y., and Srour, E. F. 2011. Genomic and proteomic analysis of the impact of mitotic quiescence on the engraftment of human CD34+ cells. PLoS One 6:e17498.
- 47. Micklem, H. S., Anderson, N., and Ross, E. 1975. Limited potential of circulating haemopoietic stem cells. Nature 256:41-43.
- 48. Schwarzenberger, P., Huang, W., Oliver, P., Byrne, P., La Russa, V., Zhang, Z., and Kolls, J. K. 2001. 11-17 mobilizes peripheral blood stem cells with short- and long-term repopulating ability in mice. J Immunol 167:2081-2086.
- 49. Li, J. M., Giver, C. R., Lu, Y., Hossain, M. S., Akhtari, M., and Waller, E. K. 2009. Separating graft-versus-leukemia from graft-versus-host disease in allogeneic hematopoietic stem cell transplantation. Immunotherapy 1:599-621.
- 50. Lekli, I., Gurusamy, N., Ray, D., Tosaki, A., and Das, D. K. 2009. Redox regulation of stem cell mobilization. Can J Physiol Pharmacol 87:989-995.
- 51. Pruijt, J. F., Verzaal, P., van Os, R., de Kruijf, E. J., van Schie, M. L., Mantovani, A., Vecchi, A., Lindley, I. J., Willemze, R., Starckx, S., et al. 2002. Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice. Proc Natl Acad Sci USA 99:6228-6233.
- 52. Brautigam, V. M., Dubyak, G. R., Crain, J. M., and Watters, J. J. 2008. The inflammatory effects of UDP-glucose in N9 microglia are not mediated by P2Y14 receptor activation. Purinergic Signal 4:73-78.
- 53. Scrivens, M., and Dickenson, J. M. 2005. Pharmacological effects mediated by UDP-glucose that are independent of P2Y14 receptor expression. Pharmacol Res 51:533-538.
- 54. Liu, F., Poursine-Laurent, J., and Link, D. C. 2000. Expression of the G-CSF receptor on hematopoietic progenitor cells is not required for their mobilization by G-CSF. Blood 95:3025-3031.
- 55. Greenbaum, A. M., and Link, D. C. 2011. Mechanisms of G-CSF-mediated hematopoietic stem and progenitor mobilization. Leukemia 25:211-217.
- 56. Lee, B. C., Cheng, T., Adams, G. B., Attar, E. C., Miura, N., Lee,
- S. B., Saito, Y., Olszak, I., Dombkowski, D., Olson, D. P., et al. 2003. P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrow hematopoietic stem cells. Genes Dev 17:1592-1604.
- 57. Passegue, E., Wagers, A. J., Giuriato, S., Anderson, W. C., and Weissman, I. L. 2005. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J Exp Med 202:1599-1611.
- 58. Ploemacher, R. E., van der Sluijs, J. P., Voerman, J. S., and Brons, N. H. 1989. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74:2755-2763.
- 59. Cho, J., Shen, H., Yu, H., Li, H., Cheng, T., Lee, S. B., and Lee, B. C. 2011. Ewing sarcoma gene Ews regulates hematopoietic stem cell senescence. Blood 117:1156-1166.
Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.
Claims
1. A pharmaceutical composition comprising a uridine diphosphate (UDP) compound and a second hematopoietic cell mobilizing compound.
2. A pharmaceutical composition comprising a uridine diphosphate (UDP) compound and a second hematopoietic cell mobilizing compound, in an amount effective to mobilize hematopoietic stem cells from the bone into peripheral circulation of a subject in need thereof.
3. The pharmaceutical composition of claim 1, where the UDP compound is UDP-glucose.
4. The pharmaceutical composition of claim 1, where the UDP compound is UDP.
5. The pharmaceutical composition of claim 1, where the UDP compound is selected from the group consisting of UDP-galactose, UDP-N-acetylglucosamine, UDP-glucuronic acid, 5′-O-[[[[(2″,3″,4″,6″-tetra-O-benzoyl-α-D-glucopyranosy)oxy]carbonyl]amino]sulfonyl]uridine (P536), UDP-6S-6C-methylglucose, UDP-6R-6C-methylglucose, Diphosphoric acid 1-α-D-glucopyranosyl ester 2-[(4′-methylthio)uridin-5″-yl] ester disodium salt (MRS 2690), a mixture of one or more of the aforelisted compounds with UDP glucose or UDP, and a mixture of two or more of the foregoing.
6. The pharmaceutical composition of claim 1, where the second hematopoietic cell mobilizing compound is granulocyte colony stimulating factor.
7. The pharmaceutical composition of claim 1, where the second hematopoietic cell mobilizing compound is selected from the group consisting of poly-(1,6)-β-D-glucopyranosyl-(1,3)-β-D glucopyranose (PGG) β-glucan, 1,1′-[1,4-phenylenebis(methylene)]bis [1,4,8,11-tetraazacyclotetradecane] (AMD3100), cyclophosphamide, fucoidan, and a mixture thereof.
8. The pharmaceutical composition of claim 1, where the subject in need thereof is going to have radiation therapy or chemotherapy.
9. The pharmaceutical composition of claim 1, where the subject in need thereof has received chemotherapy.
10. The pharmaceutical composition of claim 1, where the subject in need thereof is being prepared to donate hematopoietic stem progenitor cells.
11. The pharmaceutical composition of claim 1, where the subject in need thereof has been exposed to a toxic level of radiation.
12. The pharmaceutical composition of claim 1, where the subject in need thereof suffers from long term bone marrow failure (BMF) or Fanconi's anemia (FA).
Type: Application
Filed: Aug 4, 2016
Publication Date: Nov 24, 2016
Applicant: University of Pittsburgh - Of the Commonwealth System of Higher Education (Pittsburgh, PA)
Inventor: Byeong-Chel Lee (Pittsburgh, PA)
Application Number: 15/228,878