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(Journal of Leukocyte Biology. 2003;73:630-638.)
© 2003 by Society for Leukocyte Biology

Stromal cell-derived factor-1/CXCL12 directly enhances survival/antiapoptosis of myeloid progenitor cells through CXCR4 and G{alpha}i proteins and enhances engraftment of competitive, repopulating stem cells

Hal E. Broxmeyer*,{dagger},{ddagger},§, Lisa Kohli*,{ddagger},§, Chang H. Kim, Younghee Lee*,{ddagger},§, Charlie Mantel*,{ddagger},§, Scott Cooper*,{ddagger},§, Giao Hangoc*,{ddagger},§, Montaser Shaheen{dagger},{ddagger},§, Xiaxin Li#,** and D. Wade Clapp#,**

Departments of
* Microbiology/Immunology,
{dagger} Medicine (Hematology/Oncology), and
# Pediatrics (Neonatology),
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stromal cell-derived factor-1 (SDF-1/CXCL12) enhances survival of myeloid progenitor cells. The two main questions addressed by us were whether these effects on the progenitors were direct-acting and if SDF-1/CXCL12 enhanced engrafting capability of competitive, repopulating mouse stem cells subjected to short-term ex vivo culture with other growth factors. SDF-1/CXCL12 had survival-enhancing/antiapoptosis effects on human bone marrow (BM) and cord blood (CB) and mouse BM colony-forming units (CFU)-granulocyte macrophage, burst-forming units-erythroid, and CFU-granulocyte-erythroid-macrophage-megakaryocyte with similar dose responses. The survival effects were direct-acting, as assessed on colony formation by single isolated human BM and CB CD34+++ cells. Effects were mediated through CXCR4 and G{alpha}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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell survival is an extremely important component of blood cell regulation during development and adult life [1 2 3 4 5 ]. Without cytokine-survival signals, hematopoietic stem cells (HSC) and progenitor stem cells undergo apoptosis [6 7 8 9 ], an evolutionarily conserved process by which an individual cell commits to a genetically determined series of events leading to death in a suicidal manner [6 7 8 9 10 11 12 ].

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{alpha} 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 {alpha} (CC) and ß (CXC) chemokine families [21 ]. The effects of SDF-1{alpha} 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?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Heparinized human BM cells were collected from healthy volunteers with informed consent. Heparinized human CB was collected from the placentas of healthy, full-term neonates according to institutional guidelines. Mononuclear cells (MNC) were separated by density gradient centrifugation on Ficoll Hypaque (1.077 g/ml; Pharmacia, Piscataway, NJ). CD34+ cells were positively selected by a MACS CD34+ isolation kit (Miltenyi Biotec, Auburn, CA; purity was more than 90% in each experiment). More highly purified CD34+++ cells (containing the top 20% highest CD34-expressing cells; >=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{alpha} and SDF-1ß, as well as anti-SDF-1 were purchased from R&D Systems (Minneapolis, MN). Purified, biosynthesized SDF-1{alpha} 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{alpha}. 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.

 
Statistical analysis
Statistical significance for the colony assays based on populations of cells was determined using Student’s t-test, and the statistical differences between treatment groups of the single isolated CD34+++ cells were calculated by contingency table analysis using the {chi}2 statistic.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SDF-1/CXCL12 enhances survival of human and mouse MPC after growth factor withdrawal
Myeloid progenitors require cytokines to survive, even in the presence of serum, and these cells undergo apoptosis if certain growth factors are withdrawn from or delayed in their addition to these cells [6 ]. It is possible to assess the effects of individual cytokines for their ability to maintain the survival and subsequent proliferative capacity of MPC subjected to delayed addition of growth factors [37 38 39 ]. As shown in Figure 1 , the survival-enhancing effects of soluble SDF-1 (100 ng/ml) were seen for colony-forming units-granulocyte macrophage (CFU-GM) in low-density human BM for CFU-GM and CFU-granulocyte-erythroid-macrophage-megakaryocyte (GEMM) in CD34+ (time 0 cloning efficiency for all progenitors, 10–20%) and CD34+++ (time 0 cloning efficiency for all progenitors, 50–65%) human CB cells and for CFU-GM in unseparated BM from BDF1, C57Bl/6, BALB/c, and C3H/HeJ mice (Fig. 1) . The survival-enhancing effects of SDF-1 were also seen on CFU-GM, burst-forming units-erythroid (BFU-E), and CFU-GEMM in CD34+++ human BM, CFU-GM, and CFU-GEMM in low-density human CB cells and CFU-GM, BFU-E, and CFU-GEMM in c-kit+Lin- mouse BM cells (data not shown). Thus, the survival-enhancing effects of SDF-1/CXCR4 are active on a broad range of MPC from different tissues (human BM and CB) and different specifies (human and mouse). For all studies, the survival-enhancing effects of SDF-1/CXCL12 were dose-dependent. SDF-1 at concentrations of 100 ng/ml had maximum survival-enhancing effects (P<0.001), and the highest concentration tested (500 ng/ml) had the same effect as 100 ng/ml. SDF-1/CXCL12 (50 ng/ml) gave maximal or close-to-maximal effects (P<0.01), and concentrations of 1–25 ng/ml were without significant enhancing activity (P>05). No significant dose-response differences were detected between human or mouse cells, human BM or CB, or the different progenitor cell types in these populations.



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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.

 
The in vitro survival-enhancing effects of SDF-1 for mouse BM CFU-GM were not duplicated with the following nonspecies-specific chemokines in the concentration ranges of 25–200 ng/ml: rhu or rmu macrophage-inflammatory protein-1{alpha}/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 G{alpha}i-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 G{alpha}i 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.

 
To verify that the survival-enhancing effects of SDF-1/CXCL12 on MPC were acting through CXCR4, a receptor for SDF-1, mouse BM CFU-GM, BFU-E, and CFU-GEMM (Fig. 3) , and low-density human CB CFU-GM and CFU-GEMM (Fig. 4 ) was treated for ~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.

 
Enhanced MPC survival by SDF-1/CXCL12 is apparent at the single-cell level
Studies evaluating the influence of cytokines on purified populations of cells (e.g., CD34+ human cells) have been used to suggest direct-acting effects. However, use of bulk populations of purified cells, even at low cell-plating concentrations, cannot be used to definitively define direct-acting effects, as it is still possible that effects are accessory cell-mediated by phenotypically similar cells. Thus, to more rigorously determine whether the survival-enhancing effect of SDF-1/CXCL12 was a direct-acting one on the progenitors, we evaluated the effects of SDF-1/CXCL12 on single isolated MPC subjected to delayed addition of growth factors. CD34+++ BM and CB cells were sorted by FACS using an autoclone device [30 ] as a single cell into a well containing methylcellulose with 30% FBS in the absence and presence of 100 ng/ml SDF-1/CXCL12 before addition of growth factors at time 0 or after 24 h. As shown in Figure 5 , the survival-enhancing effects of SDF-1/CXCL12 were detected at the level of single-isolated CD34+++ BM CFU-GM and BFU-E plus CFU-GEMM (Fig. 5A) and CB CFU-GEMM (Fig. 5B) . Effects on CB cells were only evaluated after 24 h delayed addition of growth factors. As there was not a decreased survival of single CB CFU-GM after 24 h in the absence of SDF-1/CXCL12, we couldn’t determine an effect of SDF-1/CXCL12 on survival of single CB CFU-GM. However, there was a significant decrease in CB CFU-GEMM with the 24-h delay in addition of growth factors, and this was counter-balanced by SDF-1/CXCL12. As CFU-GEMM, but not BFU-E, is detected when CB cells are stimulated by the combination of Epo, CSFs, and SLF [46 ], we did not evaluate the effect of SDF-1/CXLC12 on survival of CB BFU-E. The survival-enhancing effects of SDF-1/CXCL12 were similar on BM BFU-E and CFU-GEMM (data not shown), and the results of BFU-E and CFU-GEMM were combined in Figure 5A so that erythroid cell-containing colonies in BM and CB could be compared. There were no apparent differences in the survival-enhancing effects of SDF-1/CXCL12 on erythroid-containing colonies from BM and CB growing from single isolated cells.



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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 {chi}2 statistic. NS, Not significant.

 
SDF-1/CXCL12 counteracts the loss in engrafting capacity of long-term, marrow-competitive, repopulating mouse stem cells subjected to short-term in vitro culture
It has recently been shown that SDF-1/CXCL12 enhances the engrafting capability of human lineage-negative CB cells [47 ] and CD34+ populations from G-CSF-mobilized human peripheral blood [48 ] in a xenogenic system using sublethally irradiated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. Engraftment of CD34+ cells from human BM was not enhanced after treatment with SDF-1/CXCL12 [48 ]. Although engraftment of human cells into SCID mice is considered a useful assay to assess human stem cells [49 , 50 ], these assays do not measure the long-term, marrow-competitive, repopulating stem cell that can be assayed in a syngeneic mouse system [51 ]. Also, although it is clear that mouse stem cells can save the life of lethally irradiated mice, it has not been shown that human stem cells can save the life of lethally irradiated SCID mice. Thus, information on the effects of agents/cytokines on mouse stem cells is extremely important. We chose to evaluate the effects of SDF-1/CXCL12 in an assay that might be of practical information. We used a system that evaluated stem cells in a setting similar to that used for mouse preclinical gene transduction analysis. To assess whether SDF-1/CXCL12 would enhance engraftment of HSC, a mouse-competitive, repopulating HSC assay was used [35 ]. Low-density BM cells from CD45.2 C57Bl/6 mice were left for 48 h in FBS with SLF (100 ng/ml) and IL-6 (200 U/ml) with control diluent or SDF-1 (200 ng/ml) or were tested fresh after a 1-h culture in which the nonadherent cells were removed before injection into lethally irradiated C57Bl/6. These test cells, which express the CD45.2 isoantigen, were competed against CD45.1 B6.BoyJ mice, which express the CD45.1 antigen at a 3:1 ratio with 7 x 105 low-density B6.BoyJ-competitor cells. The in vitro culture portion of the studies was done in the presence of SLF and IL-6, as these factors have been used successfully for gene-transduction protocols. The results shown in Figure 6 demonstrate a significant enhancement in CD45.2 cell chimerism for cells treated in culture with IL-6 and SLF for 48 h with SDF-1/CXCL12 compared with those treated with only IL-6 and SLF at the 1-, 4-, and 6-month post-transplant times. Most importantly and of potential future clinical relevance, engraftment of cells treated with SDF-1/CXCL12 for 2 days in the presence of SLF and IL-6 was equivalent at each time point to that of fresh CD45.2 BM cells. The total percentage of CD45.2 plus CD45.1 cells equaled 99–101%, confirming the accuracy of the percent CD45.2 chimerism data. When the data in Figure 6 were used to calculate HSC-competitive RU, the effects of SDF-1/CXCL12 on HSC survival were even more apparent (Fig. 7 ). In the absence of SDF-1/CXCL12, there was a highly significant 47% decrease in HSC RU after 2 days of culture 0, even in the presence of IL-6 and SLF, as assessed 6 months after transplantation. However, there was no significant difference in HSC RU compared with fresh, noncultured cells and full-functional stem-cell activity when SDF-1/CXCL12 was added with IL-6 and SLF for the 2-day culture period. These results demonstrate the enhanced, competitive, repopulation-engrafting capability of murine HSC subjected to culture with SDF-1/CXCL12 versus without SDF-1/CXCL12 for 2 days in the presence of SLF and IL-6.



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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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results support and extend previous studies of others [22 23 24 25 26 ] and ourselves [27 , 29 ] by demonstrating survival-enhancing effects of SDF-1 for a wide array of murine and human MPC. This is in contrast to a reported restriction for SDF-1 survival effects on human CD34+ cells from nonmobilized peripheral blood but not from human BM cells [26 , 52 ]. There are reports of MPC from human CB differing from these cells in human BM in responsiveness to cytokines (reviewed in ref. [53 ]). However, we could detect no obvious differences in sensitivity of the different myeloid progenitors (CFU-GM, BFU-E, CFU-GEMM) from different species or tissues to responsiveness to the survival-enhancing effects of SDF-1 when using unseparated or highly purified populations of cells. We did note that at the single-cell level, delayed addition of growth factors resulted in little or no decrease in survival of CB CFU-GM, in contrast to that of single human BM CFU-GM and populations of purified CD34+++ CB and BM CFU-GM. The reasons for this difference in survival of CFU-GM from single versus populations of CB cells are not clear and require additional study.

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 G{alpha}i 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 G{alpha}i proteins—results consistent with our recent studies demonstrating that transgenic SDF-1/CXCL12 action also acts through G{alpha}i 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.


   ACKNOWLEDGEMENTS
 
These studies were supported by Public Health Service Grants RO1 HL56416, RO1 HL67384, and RO1 DK53674 to H. E. B. and by P60 HL53586 and RO1 HL63219 to D. W. C. C. H. K. is a Special Fellow of the Leukemia and Lymphoma Society. L. K. was supported by NIH T32 Training Grant DK07519 to H. E. B.

Received October 16, 2002; revised February 3, 2003; accepted February 10, 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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