i proteins and enhances engraftment of competitive, repopulating stem cells
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,
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Departments of
* Microbiology/Immunology,
Medicine (Hematology/Oncology), and
# Pediatrics (Neonatology),
Walther Oncology Center and
** Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis;
Walther Cancer Institute, Indianapolis, Indiana; and
¶ Laboratory of Immunology and Hematopoiesis, Department of Veterinary Pathology, Purdue University, West Lafayette, Indiana
Correspondence: Hal E. Broxmeyer, Ph.D., Walther Oncology Center, Indiana University School of Medicine, 1044 West Walnut Street, R4-302, Indianapolis, IN 46202. E-mail: hbroxmey{at}iupui.edu
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i proteins. Moreover, SDF-1/CXCL12 greatly enhanced the engrafting capability of mouse long-term, marrow-competitive, repopulating stem cells cultured ex vivo with interleukin-6 and steel factor for 48 h. These results extend information on the survival effects mediated through the SDF-1/CXCL12–CXCR4 axis and may be of relevance for ex vivo expansion and gene-transduction procedures.
Key Words: hematopoietic progenitor and stem cells • chemokines • cytokines • apoptosis
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Stromal cell derived factor-1 (SDF-1), a chemokine designated CXCL12 [13
], is an extremely important molecule for hematopoiesis. When genes for SDF-1 or its receptor CXCR4 are functionally deleted in mice, the mice die before birth [14
15
16
]. Two alternate splicing variants, SDF-1 and SDF-1ß, were isolated as orphan clones from bone marrow (BM) stromal cells [17
, 18
]. The mature human and mouse SDF-1 proteins are almost identical except for a conservative change in one amino acid [19
]. SDF-1/CXCL12 is the only CXC chemokine thus far identified whose genomic location is on human chromosome 10 [20
]. It is considered a "primordial" chemokine, based on sequence similarities to the (CC) and ß (CXC) chemokine families [21
]. The effects of SDF-1 and SDF-1ß are identical.
SDF-1/CXCL12 has been shown to have survival-enhancing effects for CD4+ T cells [22 ], dendritic cells [23 ], leukemia B cells [24 ], early cells of the T-lymphoid series [25 ], myeloid progenitor cells (MPC) in CD34+ cell populations in steady-state, nonmobilized human peripheral blood [26 ] and cord blood (CB) [27 ], and MPC in mouse BM [28 , 29 ]. Our present report focuses on aspects of the survival-enhancing/antiapoptotic effects of SDF-1/CXCL12 not previously reported. The main questions addressed in this study include: Is the enhancing effect a direct-acting one on the progenitors, as evaluated by the rigorous criteria of actions on single isolated cells? Does the antiapoptotic effect extend to populations of phenotypically defined stem cells? Does SDF-1/CXCL12 enhance engraftment of long-term, marrow-competitive, repopulating stem cells from mouse BM in the context of an ex vivo culture situation used for gene transduction?
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98% pure CD34+) were isolated by fluorescein-activated cell sorter (FACS) [30
]. Mouse cells were obtained from femurs of B2D6F1 (BDF1), C57Bl/6, BALB/c, and C3H/HeJ mice purchased from Harlan Laboratories (Indianapolis, IN) and Jackson Laboratories (Bar Harbor, ME). Congenic C57Bl/6 (CD45.2+) and B6.SJL-Ptrca Pep3b/BoyJ (B6.BoyJ; CD45.1+) mice were purchased from Jackson Laboratories and were maintained in our animal facility. c-kit+Lin- mouse marrow cells were obtained as described previously [31
].
Cytokines, antibodies, and other agents
Purified recombinant preparations of SDF-1 and SDF-1ß, as well as anti-SDF-1 were purchased from R&D Systems (Minneapolis, MN). Purified, biosynthesized SDF-1 was obtained from Dr. Ian Clark-Lewis (University of British Columbia, Vancouver, Canada). The effects and dose-response curves for all three preparations were exactly the same in our assays. Most of the studies used the biosynthesized SDF-1. Purified recombinant human and murine (rhu and rmu) preparations of steel factor and human interleukin (IL)-3 and IL-6 were purchased from R&D Systems. Purified recombinant preparations of human and murine granulocyte macrophage-colony stimulating factor (GM-CSF) were kind gifts of Immunex Corp. (Seattle, WA), and purified rhu erythropoietin (Epo) was purchased from Amgen Corp. (Thousand Oaks, CA). Pokeweed mitogen mouse spleen cell-conditioned medium (PWMSCM) was prepared as described [32
]. AMD3100 (a specific antagonist of SDF-1 binding to CXCR4) was a kind gift of AnorMed Corp. (Langley, British Columbia, Canada). Antibody to human CXCR4 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-mouse CXCR4 [33
] was a kind gift from Millenium Corp. (Cambridge, MA). Anti-c-kit, anti-activated caspase-3, and antibodies to CD3E, CD4, CD8a, Gr-1, B220, Mac-1, and isotype controls were purchased from PharMingen (San Diego, CA).
MPC assays
Magnetic bead-separated human BM or CB CD34+ (103 cells/ml), FACS-sorted CD34+++ (100–150 cells/ml), or low-density cells (5x104 cells/ml) were plated, or CD34+++ cells were sorted as a single cell/well by an autoclone devise in 0.9% methylcellulose culture medium with 30% fetal bovine serum (FBS; Hyclone Inc., Logan, UT), the combination of human GM-CSF (10 ng/ml), IL-3 (10 ng/ml), steel factor (50 ng/ml), and Epo (1 U/ml). Unseparated mouse BM cells (5x104 ml) were plated in methylcellulose culture with the combination of 5% vol/vol PWMSCM, murine steel factor (50 ng/ml), human Epo (1 U/ml), and hemin (0.1 mM; Sigma Chemical Co., St. Louis, MO). More details can be found elsewhere [34
]. For the delayed addition of growth factors, cells were plated with or without SDF-1 at time (T) 0, and the human or murine growth factors were added, respectively, to human or murine cells at time 0, 24, or 48 h after the start of the cultures (T=0 h). Colonies were scored at 14 days (for human cells) or 7 days (for mouse cells) after the addition of growth factors. Plates were incubated at 5% CO2 in lowered (5%) oxygen in a humidified atmosphere.
Competitive long-term mouse BM stem-cell assay
BM cells were flushed from the femurs and tibias of 6- to 8-week-old B6.BoyJ mice using Iscove’s modified Dulbecco’s media (IMDM; Gibco-BRL, Gaithersburg, MD) containing 5% fetal calf serum (FCS; Hyclone Laboratories). Low-density MNC (LDMNC) were prepared by centrifugation on Ficoll-Hypaque (density 1.119; Sigma Chemical Co.). B6.BoyJ LDMNC were used as competitor cells. Cells were maintained in 10% FBS and the indicated growth factors at a concentration of 1 x 106 cells/ml.
Twenty-seven 8- to 10-week-old female C57Bl/6 mice were lethally irradiated (1100 cGy split dose) before transplantation as described previously [35 ]. CD45.2 C57BL/6 test cells (2.1 million) were mixed with 7.5 x 105 freshly isolated B6.BoyJ low-density competitor cells. All test populations were mixed with a common pool of competitor cells. Each cell mixture was resuspended in 0.5 ml IMDM/2% FCS and injected into the tail vein of six lethally irradiated recipients. Six mice were transplanted with only competitor cells to evaluate any potential late contribution of endogenous hematopoiesis from the irradiated, recipient animals. Three mice were irradiated only to be certain that the irradiation dose was lethal.
Tail vein blood samples (100 µl) were obtained post-transplantation for analysis of chimerism. Peripheral blood cells were incubated in red blood cell lysis buffer (0.16 M NH4Cl, 0.1 M KHCO3, 0.1 mM EDTA) for 5 min at 4°C. The cells were washed twice, resuspended in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA), and aliquoted into seven individual tubes for antibody staining. Each sample was stained with CD45.1 fluorescein isothiocyanate (FITC; B6.BoyJ strain) and CD45.2 FITC (C57Bl/6 strain) at 4°C for 20 min. Samples were washed twice and resuspended in PBS/0.1% BSA before analysis by fluorescence cytometry. Defined mixtures of C57Bl/6 and B6.BoyJ cells were stained with CD45.1 FITC and CD45.2 FITC, individually as controls to assist with appropriate gate settings and to assure that no errors occurred during antibody staining. A total of 5000 events were collected from each sample. All data were analyzed using CELLQuest software (Becton Dickinson, San Jose, CA). Instrument settings and gates used to analyze data were identical from month to month and between genotypes. An unpaired Student’s t-test was used to determine whether significant differences existed in chimerism between genotypes. Competitive stem-cell repopulating units (RU) were calculated based on the formula: 10 x % donor chimerism/100 - % donor chimerism.
Apoptosis
These studies were done (as described in the legend to Fig. 2 ) using activated capase-3 [29
, 36
] as a measure of apoptosis.
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Figure 2. Influence of soluble rSDF-1 on apoptosis of c-kit+/c-kit+Lin- mouse BM cells subjected to delayed addition of growth factors (GF). (A) Apoptosis was measured by activated caspase-3 expression using multivariate intracellular flow cytometry methods [39
]. BM from normal mice was highly enriched for c-kit+ cells using a magnetic bead separation kit. The c-kit+ cells were incubated in serum at 37°C for 72 h under one of three conditions. Condition 1: with GF (10 ng/ml rmuGM-CSF, 50 ng/ml rmuSLF, 100 ng/ml rhuFlt3L); condition 2: without GF; condition 3: with exogenous SDF-1 (100 ng/ml). After 72 h, GF were added to the cells in conditions 2 and 3 at the concentrations listed for condition 1 (delayed addition). The cells were incubated for another 24 h. Cells were then stained with FITC-conjugated antibodies to lineage markers (CD3e, CD4, CD8a, Gr-1, B220, and Mac-1) or an isotype control and were then stained intracellularly with biotin-conjugated antibody against activated caspase-3 and a phycoerythrin-conjugated streptavidin secondary antibody as described [39
]. Cellular debris and dead cells were gated out by forward/side laser-light scatter (FSC) during data accumulation with a FACscan (Becton Dickenson) flow cytometer. Specific fluorescence data were further analyzed by gating on Lin- cells. These events (c-kit+Lin-) are displayed as dot plots of FSC versus intracellular-activated caspase-3 level. It was found that apoptosis analysis under these conditions was facilitated by presentation in this manner, because of a clearer separation of active caspase-3-positive and -negative populations. The dot plots show the c-kit+Lin- cells with two regions marked. The medium gray region designates cells that stained dimly for activated caspase-3, which are nonapoptotic cells based on isotype-nonspecific control antibody staining. The black region designates cells that stain more brightly for activated caspase-3 and are apoptotic cells. The percentage for gated Lin- cells that fall into each of the two marked regions is shown next to that population. This is one representative experiment. (B) Average results ± 1SD for c-kit+Lin- cells for the number (n) of experiments shown. The statistical comparisons are shown in the box to the right. WT, wild-type controls.
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2 statistic. |
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Figure 1. Influence of SDF-1 on survival of human and murine MPC after delayed addition of growth factors. The results shown are the mean ± 1SEM of the number (N) of experiments shown in parentheses. Human MPC were stimulated with rhuEpo (1 U/ml), rhuIL-3 (10 ng/ml), rhuGM-CSF (10 ng/ml), and rhu steel locus factor (SLF; 50 ng/ml) in methylcellulose cultures and colonies scored 14 days after the addition of growth factors. Mouse CFU-GM were scored 7 days after the addition of rmuGM-CSF (10 ng/ml) and rmuSLF (50 ng/ml) to agar cultures. *, P < 0.002, compared with minus SDF-1 at T = 24 h.
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/CCL3, rmu homologue of monocyte chemoattractant protein-1 (MCP-1)/CCL2, rhuMCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, rmuMCP-5/CCL12, rhu regulated on activation, normal T expressed and secreted/CCL5, or rhuIL-8/CXCL8 (data not shown).
SDF-1/CXCL12 enhancement of MPC survival is antiapoptotic, pertussis toxin (PTX)-sensitive and blocked by anti-CXCR4, a selective antagonist of CXCR4, and anti-SDF-1
To determine whether the SDF-1/CXCL12 survival-enhancing effects on the myeloid progenitors reflected antiapoptotic effects, we evaluated the effects of SDF-1/CXCL12 on apoptosis (as assessed by activated caspase-3) in populations of c-kit+Lin- BM cells using multivariate flow cytometric analysis. As shown in Figure 2
, SDF-1/CXCL12 significantly decreased apoptosis of c-kit+Lin- mouse BM cells in the absence of added growth factors. As c-kit+Lin- cells are also highly enriched in stem cells, it is likely that SDF-1/CXCL12 also decreased apoptosis of stem cells.
Chemokine receptors are linked to heterotrimeric G-proteins, and a number of chemokine functions are Gi-linked and PTX-sensitive [19
, 40
41
42
]. As seen in Figure 3
, SDF-1/CXCL12 enhancement of the survival of CFU-GM, BFU-E, and CFU-GEMM from mouse BM cells after delayed growth factor addition is blocked by treatment of the cells with PTX, which did not have an effect on day 0 colony formation in the absence or presence of SDF-1. The percent survival for CFU-GM, BFU-E, and CFU-GEMM was, respectively, 101 ± 4, 102 ± 7, and 102 ± 5 without SDF-1 and 101 ± 1, 98 ± 2, and 104 ± 3 with SDF-1 added at day 0 (P>0.05 for all). PTX also significantly blocked the survival-enhancing effects of SDF-1 on low-density and CD34+ human BM CFU-GM, BFU-E, and CFU-GEMM (data not shown).
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Figure 3. Influence of antagonists of Gi proteins and CXCR4 and blocking antibodies to CXCR4 or SDF-1 on SDF-1 enhanced survival of C3H/HeJ mouse BM MPC to delayed addition of growth factors. Unseparated mouse BM cells were pretreated as shown in the figure before plating in the absence or presence of SDF-1 (100 ng/ml) and adding rhuEpo, rmuSLF, PWMSCM, and hemin at 0 and 24 h. Colonies were scored 7 days after addition of growth factor. The average results ± 1SD are for the number of experiments shown. a, P < 0.001, compared with the T = 0, minus SDF-1 media control; b, P < 0.01, compared with minus SDF-1 within the same time point.
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30 min with AMD3100, a specific antagonist of SDF-1 binding to CXCR4 [43
44
45
], or antibody to CXCR4. AMD3100 is not species-specific. Anti-mouse CXCR4 was used on mouse cells, and anti-human CXCR4 was used on human cells. We had previously verified that AMD3100 blocked the functional activity of SDF-1/CXCL12 by showing that the SDF-1/CXCL12-induced activation of mitogen-activated protein kinase activity in the human factor-dependent cell line MO7e was blocked by treatment of the cells with AMD3100 [27
]. The treated cells were then plated in semi-solid methylcellulose culture medium without washing in the absence or presence of SDF-1/CXCL12 (100 ng/ml) with growth factors added at day 0 or 1. AMD3100 at 1 µM or 10 µg/ml anti-CXCR4 blocked the survival-enhancing effects of SDF-1/CXCL12 on mouse BM and human CB MPC. Tenfold dilution of AMD3100 or anti-CXCR4 did not block the survival-enhancing effects (data not shown). Neither AMD3100 nor anti-CXCR4 had an effect on day 0 colony formation in the absence or presence of SDF-1, as shown in Figure 4
for human cells; percent-survival under these conditions ranged from 98 to 106 for mouse progenitors (P>0.05). The survival-enhancing effect of SDF-1/CXCL12 was neutralized by anti-SDF-1/CXCL12 for mouse progenitors (Fig. 3)
and human progenitors (data not shown). Anti-SDF-1/CXCL12 had no effect on day 0 colony formation in the absence or presence of SDF-1/CXCL12 with percent-survival under these conditions ranging from 100 to 107 (P>0.05).
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Figure 4. Influence of SDF-1 on survival of human CB CFU-GM (A) and CFU-GEMM (B) in the presence of control diluent, AMD3100, or anti-CXCR4 antibody. Low-density human CB cells were preincubated with control medium or an isotype-antibody control, a specific antagonist of SDF-1 binding to CXCR4 (AMD3100; AnorMed Corp.), or a neutralizing antibody to human CXCR4 before plating in the absence or presence of SDF-1 (100 ng/ml) and adding human growth factors, as in the legend to Figure 1
, at 0 and 24 h. Colonies were scored 14 days after the addition of growth factors. The average results ± 1SEM are for four experiments CFU-GM (A) and for two experiments for CFU-GEMM (B). a, P < 0.01, compared with the T = 0, minus SDF-1 media control; b, P < 0.02, compared with minus SDF-1 within the same time point.
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Figure 5. Influence of SDF-1 on survival of human MPC after delayed growth factor addition to wells with single CD34+++ cells. Wells contained 30% FBS minus/plus 100 ng/ml SDF-1. A combination of growth factors [rhuEpo (2 U/ml), rhuGM-CSF (10 ng/ml), rhuIL-3 (10 ng/ml), and rhuSLF (50 ng/ml)] was placed into wells at time 0 or after 24 or 48 h incubation. The percent wells with a colony were scored 14 days after addition of growth factors. The number of wells evaluated per group was for A: T = 0 (±SDF-1) 288 each and T = 24 and 48 h (±SDF-1) 576 each; for B: 488 each ± SDF-1 for the T = 0 and 24 h time points. The statistical differences shown between treatment groups were calculated by contingency table analysis using the 2 statistic. NS, Not significant.
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Figure 6. Influence of SDF-1 on the engrafting capability of competitive, repopulating mouse HSC cultured for 48 h in the presence of SLF and IL-6. Results are based on six recipient mice per group for each time point. Results are given as mean ± 1SD. **, P < 0.01, and *, P < 0.05, of day 2 SDF-1-treated cells compared with day 2 vehicle-treated cells. The results of 2-day vehicle-treated cells to that of day 0 controls were P < 0.01 at each time point, and there was no significant difference between the 2-day SDF-1-treated cells and the day 0 controls; P > 0.05.
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Figure 7. Influence of SDF-1 on numbers of mouse HSC-competitive RU. Results (mean±1SD) are calculated from the data shown in Figure 6
. *, P < 0.05, and **, P < 0.01, in group D2 vehicle control compared with groups D2 SDF-1 and D0 vehicle control. No significant difference was noted between groups D2 SDF-1 and D0 vehicle.
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Importantly and for the first time, the SDF-1/CXCL12 survival-enhancing effects on the myeloid progenitors were proven to be direct-acting on the progenitors by studies at the single-cell level with CD34+++ human BM and CB cells, the most rigorous means to definitively prove direct action. Inactivation of the SDF-1/CXCL12-enhancing effects with specific neutralizing antibodies to SDF-1/CXCL12 and CXCR4 and with an antagonist to SDF-1/CXCL12 binding to CXCR4 confirms that the effects seen are a result of SDF-1/CXCL12 and not a contaminant. However, these results do not rule out the possibility that SDF-1/CXCL12 is interactive with other cytokines in this enhanced cell survival. We have shown that SDF-1/CXCL12 can synergize with other cytokines to enhance survival of myeloid progenitors [27 , 29 ], and others have shown that populations of cells highly enriched for MPC produce and secrete a number of cytokines [54 ], including those we have shown that synergize with SDF-1/CXCL12 to enhance progenitor cell survival.
There is some promiscuity for chemokines and different receptors, and many chemokines can bind and act through a number of different chemokine receptors. The specificity of SDF-1/CXCL12 for CXCR4 is supported by the nearly identical phenotypes of SDF-1-/- and CXCR4-/- mice [14 15 16 ]. However, there have been rumors, not yet substantiated, that SDF-1/CXCL12 may also act through non-CXCR4 receptors. That the complete SDF-1/CXCL12 survival-enhancing effects we noted were mediated through CXCR4 was proven by use of antibodies to CXCR4 and also by a specific antagonist of SDF-1/CXCL12 binding to CXCR4.
Many chemokine actions, especially those involving chemotaxis of leukocytes, are mediated through PTX-sensitive Gi proteins; SDF-1-mediated chemotaxis of myeloid progenitors is so mediated (reviewed in refs. [19
, 40
41
42
]). Other survival-enhancing studies with SDF-1/CXCL12 [22
23
24
25
26
27
] had not addressed whether G-proteins were involved in these actions. We have now shown that SDF-1 enhancement of the survival of normal myeloid progenitors is also mediated through PTX-sensitive Gi proteins—results consistent with our recent studies demonstrating that transgenic SDF-1/CXCL12 action also acts through Gi proteins.
SDF-1/CXCL12 was shown to be antiapoptotic by evaluation of activated caspase-3 in phenotypically defined populations of mouse c-kit+Lin- BM cells, which are highly enriched for MPC and stem cells [31 ]. That antiapoptosis induced by SDF-1/CXCL12 occurred is certainly consistent with the knowledge that withdrawal of growth factors sets into motion the process of apoptosis [6 7 8 9 ]. Our previous studies had shown that using APO2.7 monoclonal antibodies to quantitate apoptotic cells, SDF-1/CXCL12 survival-enhancing effects on CD34+++ human CB cells and the human factor-dependent cell line MO7e are synergistic with other survival-enhancing/antiapoptotic cytokines, and intracellular mediators of antiapoptosis are induced by SDF-1/CXCL12 alone and in synergy with these other cytokines [27 ]. Also, using activated caspase-3 analysis, transgenic SDF-1/CXCL12 enhanced survival of phenotypically defined populations of stem/progenitor cells [29 ]. Activated caspase is considered a primary marker for apoptotic cells [55 ] and has been used previously by many investigators to assess apoptosis of different cell populations. However, we are cognizant that no one marker or method should be considered all-inclusive. Considering the small numbers of c-kit+Lin- cells we had to evaluate, we felt that flow cytometric measurement of active caspase-3-containing cells was our best choice for assessing apoptosis of c-kit+Lin- cells, as it involves fewer manipulations (and thus, lower cell loss) than other procedures used to measure apoptosis.
We were also able to demonstrate that SDF-1/CXCL12 could, in an ex vivo environment, enhance the engrafting capability of mouse long-term, marrow-competitive, repopulating HSC cultured with IL-6 and steel factor. A current problem with gene transfer/gene therapy protocols is loss of stem cells or stem-cell activity during ex vivo transduction methodologies. Even in the presence of IL-6 and steel factor—two potent cytokines used successfully for gene transfer of mouse cells—there is a significant loss in engrafting capability as highlighted in Figures 6 and 7 . In our studies, this loss was counterbalanced by inclusion of SDF-1/CXCL12 during the 48-h ex vivo culture of BM cells with IL-6 and steel factor. Thus, these results extend studies evaluating human stem-cell populations in a xenogeneic transplant model [47 , 48 ] by demonstrating that ex vivo treatment with SDF-1/CXCL12 enhances the engrafting capability of long-term, marrow-competitive, repopulating stem cells in a mouse syngeneic system. Although the human-to-mouse xenogeneic assay is considered to detect human stem cells [49 , 50 ], it is not yet clear what subset(s) of human stem cells are being identified in this assay, as the human-to-mouse system is not a competitive, repopulating assay, it involves only sublethally irradiated mice, and it has not been demonstrated that human cells can rescue a lethally irradiated mouse, a "hallmark" characteristic for stem cells of the earliest, most immature type. Although our results are consistent with a survival-enhancing effect of SDF-1/CXCL12 on competitive, repopulating stem cells, especially in the context of our data showing SDF-1/CXCL12 antiapoptotic effects on c-kit+Lin- cells, which are highly enriched for competitive, repopulating stem cells, and SDF-1-enhanced survival/antiapoptosis of MPC, it is possible that the enhanced engraftment we noted was a result of SDF-1/CXCL12-induced, enhanced homing and/or survival, as the SDF-1/CXCR4 axis has been implicated in chemotaxis/homing of stem cells (ref. [56 ] and reviewed in refs. [19 , 40 41 42 , 50 ]). SDF-1 may have enhanced the homing capability of the stem cells and thereby, the engrafting capacity of the cultured cells. There is some very limited evidence that short SDF-1/CXCL12 pretreatment may enhance homing of human cells into NOD/SCID mice [48 ]. However, this particular study used very short-term exposure (a few hours) of cells to SDF-1/CXCL12 and low concentrations of SDF-1/CXCL12. At the concentrations of SDF-1/CXCL12 that we used, these investigators [48 ] found suppression not enhancement of engraftment. Thus, we presently favor the interpretation that ex vivo treatment of stem cells with SDF-1/CXCL12 in the presence of IL-6 and steel factor probably enhances at least survival and thus engraftment of the stem cells. Not answered in our current work is whether SDF-1/CXCL12 alone, in the absence of other exogenously added cytokines, will enhance the engrafting capability of the mouse long-term, marrow-competitive, repopulating HSC. Regardless, our studies demonstrate a highly significant and potentially relevant enhancement of SDF-1/CXCL12 for stem cells above that seen with IL-6 and SLF. These effects may be applicable for ex vivo maintenance and expansion of HSC and progenitor stem cells for clinical transplantation and also for gene transfer/gene therapy protocols that use ex vivo culture.
Received October 16, 2002; revised February 3, 2003; accepted February 10, 2003.
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in the inflammatory component of allergic airway disease J. Immunol. 165,499-502 on the survival of human bone marrow cells responding to hematopoietic colony stimulating factors J. Immunol. 142,4329-4334[Abstract] or anti-CXCR4 antibody enhances migration and NOD/SCID repopulating potential Exp. Hematol. 30,1061-1069[CrossRef][Medline]This article has been cited by other articles:
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A. L. Moharita, M. Taborga, K. E. Corcoran, M. Bryan, P. S. Patel, and P. Rameshwar SDF-1{alpha} regulation in breast cancer cells contacting bone marrow stroma is critical for normal hematopoiesis Blood, November 15, 2006; 108(10): 3245 - 3252. [Abstract] [Full Text] [PDF]
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A. Foudi, P. Jarrier, Y. Zhang, M. Wittner, J.-F. Geay, Y. Lecluse, T. Nagasawa, W. Vainchenker, and F. Louache Reduced retention of radioprotective hematopoietic cells within the bone marrow microenvironment in CXCR4-/- chimeric mice Blood, March 15, 2006; 107(6): 2243 - 2251. [Abstract] [Full Text] [PDF]
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R. Gupta, S. Karpatkin, and R. S. Basch Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase Blood, March 1, 2006; 107(5): 1837 - 1846. [Abstract] [Full Text] [PDF]
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M. Berthebaud, C. Riviere, P. Jarrier, A. Foudi, Y. Zhang, D. Compagno, A. Galy, W. Vainchenker, and F. Louache RGS16 is a negative regulator of SDF-1-CXCR4 signaling in megakaryocytes Blood, November 1, 2005; 106(9): 2962 - 2968. [Abstract] [Full Text] [PDF]
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H. E. Broxmeyer, C. M. Orschell, D. W. Clapp, G. Hangoc, S. Cooper, P. A. Plett, W. C. Liles, X. Li, B. Graham-Evans, T. B. Campbell, et al. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist J. Exp. Med., April 18, 2005; 201(8): 1307 - 1318. [Abstract] [Full Text] [PDF]
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 L. J. Bendall, R. Baraz, J. Juarez, W. Shen, and K. F. Bradstock Defective p38 Mitogen-Activated Protein Kinase Signaling Impairs Chemotaxic but not Proliferative Responses to Stromal-Derived Factor-1{alpha} in Acute Lymphoblastic Leukemia Cancer Res., April 15, 2005; 65(8): 3290 - 3298. [Abstract] [Full Text] [PDF]
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 M. A. Jaleel, A. C. Tsai, S. Sarkar, P. V. Freedman, and L. P. Rubin Stromal cell-derived factor-1 (SDF-1) signalling regulates human placental trophoblast cell survival Mol. Hum. Reprod., December 1, 2004; 10(12): 901 - 909. [Abstract] [Full Text] [PDF]
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 K. W. Christopherson II, G. Hangoc, C. R. Mantel, and H. E. Broxmeyer Modulation of Hematopoietic Stem Cell Homing and Engraftment by CD26 Science, August 13, 2004; 305(5686): 1000 - 1003. [Abstract] [Full Text] [PDF]
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E. J. C. Rombouts, B. Pavic, B. Lowenberg, and R. E. Ploemacher Relation between CXCR-4 expression, Flt3 mutations, and unfavorable prognosis of adult acute myeloid leukemia Blood, July 15, 2004; 104(2): 550 - 557. [Abstract] [Full Text] [PDF]
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S. Tavor, I. Petit, S. Porozov, A. Avigdor, A. Dar, L. Leider-Trejo, N. Shemtov, V. Deutsch, E. Naparstek, A. Nagler, et al. CXCR4 Regulates Migration and Development of Human Acute Myelogenous Leukemia Stem Cells in Transplanted NOD/SCID Mice Cancer Res., April 15, 2004; 64(8): 2817 - 2824. [Abstract] [Full Text] [PDF]
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J. Kahn, T. Byk, L. Jansson-Sjostrand, I. Petit, S. Shivtiel, A. Nagler, I. Hardan, V. Deutsch, Z. Gazit, D. Gazit, et al. Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation Blood, April 15, 2004; 103(8): 2942 - 2949. [Abstract] [Full Text] [PDF]
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A. Avigdor, P. Goichberg, S. Shivtiel, A. Dar, A. Peled, S. Samira, O. Kollet, R. Hershkoviz, R. Alon, I. Hardan, et al. CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to bone marrow Blood, April 15, 2004; 103(8): 2981 - 2989. [Abstract] [Full Text] [PDF]
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 M. H. Cottler-Fox, T. Lapidot, I. Petit, O. Kollet, J. F. DiPersio, D. Link, and S. Devine Stem Cell Mobilization Hematology, January 1, 2003; 2003(1): 419 - 437. [Abstract] [Full Text] [PDF]
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