Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online August 25, 2005
Stem Cells Vol. 24 No. 1 January 2006, pp. 55 -64
doi:10.1634/stemcells.2005-0082; www.StemCells.com
© 2006 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0082v1
24/1/55    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, K.
Right arrow Articles by Yuen, P. M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, K.
Right arrow Articles by Yuen, P. M. P.

TISSUE-SPECIFIC STEM CELLS

Small Peptide Analogue of SDF-1{alpha} Supports Survival of Cord Blood CD34+ Cells in Synergy with Other Cytokines and Enhances Their Ex Vivo Expansion and Engraftment into Nonobese Diabetic/Severe Combined Immunodeficient Mice

Karen Lia, Carmen Ka Yee Chuena, Shuk Man Leea, Ping Lawb, Tai Fai Foka, Pak Cheung Nga, Chi Kong Lia, Donald Wongb, Ahmed Merzoukb, Hassan Salarib, Goldie Jia-Shi Gua, Patrick Man Pan Yuena

a Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong, People’s Republic of China;
b Chemokine Therapeutics Corporation, Vancouver, British Columbia, Canada

Key Words. SDF-1 • Cord blood CD34+ cells • Ex vivo expansion • Engraftment • Nonobese diabetic/severe combined immunodeficient mice • CXCR4

Correspondence: Karen Li, Ph.D., Associate Professor, Department of Paediatrics, The Chinese University of Hong Kong, 6th Floor, Clinical Sciences Block, Prince of Wales Hospital, Shatin, NT, Hong Kong, People’s Republic of China. Telephone: 852-2632-2859; Fax: 852-2636-0020; e-mail: lipang{at}cuhk.edu.hk


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The SDF-1/CXCR4 axis has been implicated in the chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. We studied the effects of a SDF-1 peptide analogue CTCE-0214 on the survival of cord blood CD34+ cells in culture, expansion, and engraftment of expanded cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Our results demonstrated that CTCE-0214 synergized with thrombopoietin (TPO), stem cell factor (SCF), or flt-3 ligand (FL) on the survival of stem and progenitor cells in culture. Adding CTCE-0214 at a low concentration (0.01 ng/ml) for 4 days together with TPO, SCF, and FL significantly enhanced ex vivo expansion of CD34+ cells to subsets of primitive (CD34+CD38– cells, colony-forming unit-mixed [CFU-GEMMs]), erythroid (CFU-Es), myeloid (CFU-GMs), and megakaryocytic (CD61+CD41+ cells, CFU-MKs) progenitors, as well as their multilineage engraftment in NOD/SCID mice. Interestingly, the short exposure of expanded cells to CTCE-0214 (100 and 500 ng/ml) for 4 hours did not increase the quantity of progenitor cells but enhanced their engraftment capacity. The proportion of CD34+ cells expressing surface CXCR4 was decreased, but the overall number of this population increased upon expansion. The small peptide analogue of SDF-1 could be developed for ex vivo expansion and improving engraftment of cord blood transplantation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Stromal cell–derived factor-1{alpha} (SDF-1 or CXCL12) is a member of the CXC chemokine family that binds to the G-protein–coupled receptor CXCR4. The SDF-1/CXCR4 axis is essential in mouse development because the disruption of either gene leads to hematopoietic, cardiovascular, and cerebellar defects as well as embryonic lethality [1, 2]. SDF-1 has been implicated in a variety of activities of the hematopoietic stem and progenitor cells, including homing/engraftment [3–6], mobilization [7, 8], and expansion [9, 10]. It is also known to be involved in the internalization of HIV [11, 12] and other functions of the immune system [13–15]. CXCR4 is widely expressed by malignant cells and may confer an invasive phenotype in the metastasis process [16, 17]. Recently, a small molecule antagonist of SDF-1{alpha} (AMD-3100) has been used to mobilize hematopoietic progenitor cells in normal donors for potential allogeneic transplantation [18], as well as in patients with non-Hodgkin’s lymphoma or multiple myeloma for autologous transplantation [19].

The function of SDF-1{alpha} can be reproduced by small peptide agonists [20]. Small peptides have many advantages over the native molecule, including ease of manufacturing, stability, and reduced immunogenicity. Earlier studies demonstrated that these agonist peptides can mobilize hematopoietic progenitor cells in mice [21, 22], promote chemotaxis of human peripheral blood (PB) CD34+ cells from healthy donors with or without mobilization using granulocyte-colony stimulating factor [22], and enhance megakaryopoiesis after administration of human progenitor cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice [21].

One of the major limitations on using cord blood for transplantation, especially of adult patients, is the quantity of hematopoietic stem and progenitor cells in the graft. The CD34+ cell dose was the one factor consistently identified as significantly associated with the slow rate of engraftment, risk of treatment-related mortality, and survival [23]. It is possible that SDF-1{alpha} can be helpful for cord blood transplantation in two aspects: expanding the quantities of primitive stem and progenitor cells and increasing the homing potential of the infused cells into the hematopoietic niche. In this study, we investigated both possibilities by studying the effects of a peptide SDF-1 agonist, CTCE-0214, in maintaining the survival of human cord blood hematopoietic progenitor cells, expanding these cells by ex vivo culture, and verifying the engraftment potential in a NOD/SCID mouse model and homing of the expanded cells after brief exposure to the SDF-1 agonist. Our results suggest that CTCE-0214 enhances hematopoietic progenitor cell survival, expansion, and engraftment in NOD/SCID mice.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection and Enrichment of Human Umbilical Cord Blood CD34+ Cells
Cord blood samples were collected from umbilical veins during normal full-term, vaginal deliveries. The samples were stored in preservative-free heparin (10 IU/ml; David Bull Laboratories, Victoria, Australia) at room temperature and processed within 24 hours. Informed consent was obtained from the mother for all cord blood collections, and the study was approved by the Ethics Committee for Clinical Research of The Chinese University of Hong Kong. Mononuclear cells were prepared by density gradient centrifugation (Ficoll Hypaque 1.077 g/ml; Amersham Biosciences, Uppsala, Sweden, http://www.amersham.com). CD34+ cells were enriched using the VarioMACS Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) according to the manufacturer’s instruction. The purity of enriched CD34+ cells, evaluated by flow cytometry, was 91.5% ± 0.01% (mean ± standard error of the mean [SEM]; range, 86.1%–96.3%; n = 25).

CTCE-0214 with Single-Cytokine Thrombopoietin, Stem Cell Factor, or flt-3 Ligand on CD34+ Cells in Culture
CTCE-0214 is a C-terminal amide peptide analogue of SDF-1 belonging to the family of analogues in which the disordered N-terminal region (residue 1–14) is linked to the helical-C-terminal region (residue 55–67) of SDF-1 by a bifunctional molecule (the linker) (Fig. 1Go). This analogue was cyclized between amino acid residue at positions 20 and 24. CTCE-0214 was synthesized by the Fmoc Continuous Flow method, as previously described [22]. Enriched CD34+ cells at 2 x 104/ml were cultured in QBSF-60 serum-free medium (Quality Biological, Gaithersburg, MD, http://www.qualitybiological.com) in a 24-well culture plate (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). The cultures contained various concentrations of CTCE-0214 (0, 0.01, 1, or 10 ng/ml; Chemokine Therapeutics Corporation, Vancouver, BC, Canada, http://www.chemokine.net) with or without thrombopoietin (TPO) (50 ng/ml), stem cell factor (SCF) (50 ng/ml), or flt-3 ligand (FL) (80 ng/ml) and were incubated at 37°C and 5% CO2 in a fully humidified atmosphere. All cytokines were purchased from Peprotech (Rocky Hill, NJ, http://www.peprotech.com), unless specified otherwise. Total nucleated cell (TNC) counts, flow cytometric analysis of progenitor cells, and colony forming unit (CFU) assays were performed on days 0 and 4. Cell viability was determined by the trypan blue (Gibco, Grand Island, NY, http://www.invitrogen.com) dye exclusion assay.


Figure 1
View larger version (6K):
[in this window]
[in a new window]
  		Figure 1. Amino acid sequences of SDF-1 and CTCE-0214. Common sequences are underlined.

 
Ex Vivo Expansion of Cord Blood CD34+ Cells
A pilot study was performed to determine the optimal dose of CTCE-0214 for the expansion of CD34+ cells. Enriched CD34+ cells at 2 x 104/ml were cultured in QBSF-60 medium for 4 days in the presence of TPO (50 ng/ml), SCF (50 ng/ml), and FL (80 ng/ml). At day 4, the cells were split into three wells with fresh medium, cytokines, and various concentrations of CTCE-0214 (0.0001–100 ng/ml) added. At day 8, the cells were harvested and stem and progenitor cells were quantified by flow cytometry and CFU assays.

In subsequent expansion experiments, four treatment groups were studied. In group TSF, CD34+ cells (2 x 104/ml) were cultured in QBSF-60 medium for 4 days in the presence of TPO, SCF, and FL (TSF). At day 4, each culture was split into three portions, with fresh medium and cytokines added. In group CTCE [0.01], in addition to TSF, 0.01 ng/ml CTCE-0214 was added to the cultures at day 4. In groups CTCE [100] and CTCE [500], culture conditions were similar to those of group TSF (without any CTCE-0214), but at day 8, cells in cultures were pulsed with 100 or 500 ng/ml CTCE-0214 for 4 hours before harvesting. At days 0 and 8, TNC counts, flow cytometry analysis of progenitor cells, and CFU assays were performed. Expanded cells were infused into sublethally irradiated NOD/SCID mice for the analysis of SCID-repopulating cells.

Flow Cytometric Analysis of Hematopoietic Progenitor Cells
Enriched CD34+ cells or expanded cells were stained with CD34-fluorescein isothiocyanate (FITC), CD38-phycoerythrin (PE), CXCR4-PE, CD61-FITC (DakoCytomation, Copenhagen, Denmark, http://www.dakocytomation.com), CD41-PE (Dako-Cytomation), and respective isotype controls for 20 minutes in the dark at room temperature. All antibodies and cytometric reagents were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen), unless specified otherwise. The cells were then washed and resuspended in phosphate-buffered saline (PBS) (Gibco) with 0.5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). 7-Amino-actinomycin D (7-AAD) was added to the cells before flow cytometric acquisition. Ten thousand and 60,000 events were acquired for samples at day 0 and after expansion, respectively. These cells were acquired and analyzed using a FACS Calibur flow cytometer and the CellQuest software (BD Pharmingen). Dead cells, which were 7-AAD positive, were gated out during data analysis.

Colony-Forming Unit Assay
Colony-forming unit–granulocyte macrophages (CFU-GMs), burst-forming unit/colony forming unit–erythroids (BFU/CFU-Es), and colony forming unit–mixed (CFU-GEMMs) were assayed in 1% methylcellulose cultures supplemented with 30% fetal calf serum (FCS) and 1% BSA, 0.1 mM ß-mercaptoethanol (ß-ME) (Gibco) in the presence of 3 IU/ml erythropoietin (Cilag, Zug, Switzerland, http://www.janssen-cilag.ch), 10 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF) (Sandoz, Basel, Switzerland, http://www.sandoz.com), 10 ng/ml interleukin-3 (IL-3), and 50 ng/ml SCF. Enriched CD34+ cells or expanded cells at 3 x 103/ml were seeded in triplicate and incubated for 14 days. Colonies were scored in a blinded manner using an inverted light microscope as described previously [24].

Colony forming unit–megakaryocytes (CFU-MKs) were assayed using the plasma clot system. Enriched CD34+ cells or expanded cells at 3 x 103/ml were cultured in duplicate in Iscove’s modified Dulbecco’s medium containing 10% bovine plasma (Sigma-Aldrich), 10% FCS, 1% BSA, 0.1 mM ß-ME, 0.34 mg/ml calcium chloride in the presence of 50 ng/ml TPO, and 20 ng/ml IL-3. After 12 days of culture, the clots were air dried and fixed with 1% paraformaldehyde in PBS. Colonies were labeled with monoclonal antibody CD61-FITC (DakoCytomation). A CFU-MK was identified as a cluster of three or more strongly stained CD61-positive cells examined by fluorescence microscopy.

Engraftment of Human Cells in NOD/SCID Mice
NOD/LtSZ-SCID/SCID mice were purchased from The Walter and Eliza Hall Institute of Medical Research (Melbourne, Victoria, Australia), bred, and maintained in the Laboratory Animal Services Center at The Chinese University of Hong Kong. All procedures were approved by the Animal Research Ethics Committee, The Chinese University of Hong Kong. Mice at 8 to 10 weeks of age (n = 177) were exposed to 280 to 320 cGy of total body irradiation from a 137Cs source (Gammacell-1000 Elite Irradiator; MDS Nordion, Kanata, Ontario, Canada, http://www.mds.nordion.com). In each experiment (n = 18), ex vivo–expanded cells from a cord blood sample were infused into sex-and age-matched mice (progenies of 3 x 104 CD34+ cells at day 0 per mouse). To prevent the loss of data due to animal mortality, two or three mice were assigned to each treatment group and the engraftment parameters were averaged as a single data for analysis, as described previously [25, 26]. These animals were euthanized 6 weeks after transplantation.

For the assessment of human (hu) CD45+ cells and subsets, bone marrow (BM) cells were flushed from both femurs of each mouse. Spleen cells were obtained by mincing and flushing separated cells from the tissue. PB cells were collected by heart puncture. For flow cytometric analysis, red blood cells were lysed with 0.83% ammonium chloride and washed with PBS/0.1% BSA. The cells were resuspended at 5 x 105 cells/100 µl and incubated with mouse immunoglobulin G and 5% human serum (Gibco). They were then incubated with monoclonal antibody specific for huCD45 conjugated to phycoerythrin-cyanine 5-succinimidylester (Immunotech, Marseille, France, http://www.immunotech.com) and propidium iodide (PI) (10 µg/ml; Sigma-Aldrich) for 20 minutes at room temperature. Seventy thousand events were acquired, and for those BM samples that contained more than 1% human cells, we performed additional staining using anti-human antibodies CD34-PE, CD19-PE, CD14-PE, CD33-PE, CD61-PE (DakoCytomation), and their isotypic controls. Nonviable cells (PI positive) were gated out during data analysis. We assayed human CFUs in the BM of NOD/SCID mice that contained more than 1% huCD45+ cells using methylcellulose culture and scored after 14 days. As described previously [25], this culture duration was selective for human CFU assay and did not support murine CFU formation, which normally took 7 days. For CFU-MK assay, the plasma-clot system was performed in duplicate. Analyses of NOD/SCID parameters were performed in a blinded manner.

Statistical Analysis
Treatment groups were compared by analysis of variance and paired-t-test or Wilcoxon signed-rank test, depending on data distribution, using the SigmaStat software (Systat Software, Richmond, CA, http://www.systat.com). A p value of ≤ .05 was considered statistically significant. We compared survival rates of NOD/SCID mice using the Fisher’s exact test. All values were expressed as mean ± SEM.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Synergistic Effects of CTCE-0214 with TPO, SCF, or FL on Survival of Hematopoietic Progenitor Cells
Culturing of enriched CD34+ cells in QBSF-60 for 4 days without any supplementary cytokine (control) resulted in low cell viability (48.4% ± 0.02%, n = 4). The addition of CTCE-0214 at 0.01, 1, or 10 ng/ml did not increase the viability of progenitor cells in these cultures (Fig. 2Go). The presence of TPO, SCF, or FL significantly increased the cell viability to 56.1% ± 0.04%, 64.6% ± 0.02%, and 70.5% ± 0.01%, respectively (p < .05). The addition of CTCE-0214 at 1 ng/ml only slightly increased the viability of the SCF (p = .05) and FL (p = .03) cultures.


Figure 2
View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. Synergistic effects of CTCE-0214 with TPO, SCF, or FL on cell viability and progenitor cell survival in culture. Enriched CD34+ cells were cultured for 4 days in QBSF-60 serum-free medium containing four groups of growth factor combinations: (1) without any growth factor supplement (Cont) or with 0.01, 1, or 10 ng/ml CTCE-0214 (CTCE [0.01], [1], or [10]); (2–4) with 50 ng/ml of single-factor TPO, SCF, or FL and 0, 0.01, 1, or 10 ng/ml CTCT-0214, respectively. *p < .05, **p < .01 when comparing parameters from cultures containing single growth factor with those from the control cultures. #p < .05, ##p < .01 when comparing parameters from cultures containing single growth factor TPO, SCF, or FL with and without CTCE-0214. Data are mean – standard error of mean; n = 4. Abbreviations: FL, flt-3 ligand; SCF, stem cell factor; TPO, thrombopoietin.

 
Of the viable cells, cultures without supplementary cytokines yielded reduced levels of TNCs and all subsets of progenitor cells (Fig. 2Go). The addition of 1 ng/ml CTCE-0214 alone significantly increased TNCs, CD34+ cells, CFU-GMs, BFU/CFU-Es, and CFU-GEMMs (*p < .05), and a trend was demonstrated in the number of CD34+CD38– cells (p = .06) compared with those in cultures without any supplementary factor. CTCE-0214 at the other two concentrations did not change the outcomes of the cultures. TPO significantly increased TNCs, CD34+ cells, CFU-GMs, and BFU/CFU-Es. SCF stimulated all cell types (*p < .05) except that only a trend was observed in the number of CFU-GEMMs (p = .087). FL significantly increased CD34+ cells (*p = .022) but not other subsets.

The synergistic effects of CTCE-0214 with each single cytokine on the culture outcomes were apparent, especially at the dose of 1 ng/ml. Significant increases were observed in CD34+ cells, CFU-GMs, and BFU/CFU-Es when 1 ng/ml of CTCE-0214 was added to TPO-only cultures (p < .05), and a trend was observed in the level of CFU-GEMM (p = .058). The same dose of CTCE-0214 enhanced SCF on TNCs, CD34+ cells, CFU-GMs, and BFU/CFU-Es (p < .05), and similar trends were seen with the CD34+CD38– (p = .09) and CFU-GEMM (p = .07) populations. CTCE-0214 at 1 ng/ml significantly increased all cell parameters compared with those in cultures containing only FL (p < .01).

Effects of CTCE-0214 on Ex Vivo Expansion of CD34+ Cells
In the pilot study using 10 concentrations of CTCE-0214 for the ex vivo expansion of CD34+ cells in the presence of TSF, we observed a dose-dependent effect of CTCE-0214 on the yield of all progenitor cell types, starting from a low concentration of 0.0001 ng/ml (Fig. 3Go). CTCE-0214 at 0.01, 0.05, or 0.1 ng/ml significantly increased the expansion of all cell subsets studied, including early progenitor cells (CD34+ cells, CD34+CD38– cells, and CFU-GEMMs) and committed progenitors of the myeloid (CFU-GMs), erythroid (BFU/CFU-Es), and megakaryocytic lineages (CD61+CD41+ cells, CFU-MKs) (p < .05, n = 3).


Figure 3
View larger version (25K):
[in this window]
[in a new window]
  		Figure 3. Dose effects of CTCE-0214 on CD34+ cell expansion. CD34+ cells at 2 x 104 cells/ml were expanded in QBSF-60 in the presence of TPO, SCF, and FL (each 50 ng/ml). At day 4, fresh medium with growth factors and various concentrations of CTCE-0214 was added. Expanded cells were harvested at day 8, and contents of hematopoietic stem and progenitor cells were analyzed by flow cytometry and CFU assays. *p < .05, **p < .01 when comparing cell products in cultures with and without CTCE-0214. Data are mean ± standard error of mean; n = 3. Abbreviations: FL, flt-3 ligand; SCF, stem cell factor; TPO, thrombopoietin.

 
In the subsequent series of experiments, we compared the effects of 4 days of exposure to CTCE-0214 at 0.01 ng/ml (CTCE [0.01]) with those that resulted from short pulses (4 hours) of high concentrations of 100 ng/ml (CTCE [100]) and 500 ng/ml (CTCE [500]) on the yield of various progenitor cells, particularly those capable of engraftment in NOD/SCID mice. Our results showed that the cell viability remained high in all groups at day 4 (mean values > 99.2%) and day 8 (> 91.3%) of culturing. In the presence of TSF, efficient expansions were observed in TNCs and all subsets of progenitor cells (Fig. 4Go). The addition of 0.01 ng/ml CTCE-0214 at day 4 significantly enhanced the expansion of these subsets (p < .05, n = 25). This was not only contributed to by the higher TNCs but also by the increased proportion of CD34+ cells (Fig. 5Go) and CFUs (Fig. 5Go) in day-8 cultures. Pulsing with CTCE-0214 shortly before harvest did not substantially change the total number or proportions of these subsets (Figs. 4Go, 5Go).


Figure 4
View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. Effects of exposure schedules of CTCE-0214 on ex vivo expansion of CD34+ cells. CTCE-0214 was added to the expansion cultures in various doses and durations: Group TSF: CD34+ cells were cultured in QBSF-60 medium in the presence of TPO, SCF, and FL (TSF; each 50 ng/ml), with medium change at day 4. Group + CTCE [0.01]: In addition to TSF, 0.01 ng/ml CTCE-0214 was added to the cultures at day 4. Groups + CTCE [100] and + CTCE [500]: Culture conditions were similar to those of group TSF, but at day 8, cells in cultures were pulsed with 100 or 500 ng/ml of CTCE-0214 for 4 hours before harvesting. *p < .05, **p < .01 when cell products from cultures with or without CTCE-0214 were compared. The treatment of expanding cells for 4 days with 0.01 ng/ml significantly increased all subsets of progenitor cells, but short pulses of the expansion products for 4 hours with high doses of CTCE-0214 did not increase any cell parameters. Data are mean ± standard error of mean; n = 25. Abbreviations: FL, flt-3 ligand; SCF, stem cell factor; TPO, thrombopoietin.

 

Figure 5
View larger version (21K):
[in this window]
[in a new window]
  		Figure 5. Effects of CTCE-0214 on the proportion of progenitor cells in expansion cultures. Exposure of CD34+ cell cultures to 0.01 ng/ml CTCE-0214 for 4 days significantly increased the percentage of CD34+ cells (p < .001, n = 25) and concentrations of CFUs of the myeloid (CFU-GM), erythroid (BFU/CFU-E), megakaryocytic (CFU-MK), and mixed (CFU-GEMM) lineages (p < .01). Short pulses of the expansion products with high doses of CTCE-0214 did not increase the proportions of these progenitor cells.

 
Six weeks after expanded cells were infused into sublethally irradiated NOD/SCID mice, there were no differences in the mortality rates of the animals in the TSF (4.2%), CTCE [0.01] (6.3%), CTCE [100] (7.7%), or CTCE [500] (7.1%) groups. HuCD45+ cells were detectable in the BM, spleen, and PB of the mice (Fig. 6Go). In BM, there was engraftment of human hematopoietic cells of the early (CD34+), myeloid (CD33+, CD14+), B-lymphoid (CD19+), and megakaryocytic (CD61+) lineages. In animals that received expanded cells treated 4 days with 0.01 ng/ml of CTCE-0214, there were significant increases of engraftment compared with those only exposed to TSF, as demonstrated by the high engraftment of huCD45+ cells in their BM and PB (p < .05). In BM, we also observed increased proportions of huCD34+ cells, CD33+ cells, and CFU-GEMMs (p < .05). Interestingly, animals that received expanded cells pulsed with high concentrations of CTCE-0214 for 4 hours also had increased BM engraftment of huCD45+ cells at 8.32% ± 4.04% in the CTCE [100] group (p = .034) and 7.52% ± 3.27% in the CTCE [500] group (p = .071) compared with those in the TSF group (3.03% ± 0.99%). Consistent increases were also observed in the various CFU subsets in the BM of these mice (Fig. 6Go).


Figure 6
View larger version (19K):
[in this window]
[in a new window]
  		Figure 6. Engraftment of expanded cells in NOD/SCID mice. Expanded cells treated with TSF, TSF + 0.01 ng/ml CTCE-0214 for 4 days (group + CTCE [0.01]), and TSF + 100 ng/ml (group + CTCE [100]) or 500 ng/ml (group + CTCE [500]) for 4 hours were infused in sublethally irradiated NOD/SCID mice. Significant increases in the engraftment of huCD45+ cells (BM and spleen) and their CD34+, CD33+, and CFU-GEMM subsets in BM were observed in animals that received expanded cells from the + CTCE [0.01] cultures compared with those from TSF cultures (p < .05, n = 18). Short pulses of expanded cells with CTCE-0214 also increased engraftment of huCD45+ cells (p < .05, + CTCE [100]) and some CFU subsets in the BM of NOD/SCID mice (+ CTCE [100] and + CTCE [500], p < .05). Abbreviations: BM, bone marrow; NOD/SCID, nonobese diabetic/severe combined immuno-deficient; TSF, thrombopoietin, stem cell factor, and flt-3 ligand.

 
Expression of CXCR4 on CD34+ Cells
A high proportion of enriched CD34+ cells from cord blood expressed surface CXCR4 at day 0 (63.4% ± 11.7%, n = 3) (Fig. 7Go). After 8 days of culture, the proportions of CD34+ cells expressing CXCR4 were decreased to 16.9%–17.5% (mean). There were no differences between the proportions of CD34+CXCR4+ cells in the four groups of expansion cultures.


Figure 7
View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. Expression of CXCR4 on CD34+ cells. Enriched CD34+ cells at day 0 and after expansion for 8 days were stained with CD34-FITC and CXCR4-PE antibodies or their isotypic controls (A). The CD34+ cell population was gated by its CD34-FITC–positive and low side-scatter profile (not shown), and its expressions of CXCR4 were quantified on the CD34-FITC/CXCR4-PE dot blots (B–F). Our data demonstrate that the proportion of CD34+ cells expressing CXCR4 was decreased after 8 days of culture compared with that of day 0 (B). No difference was demonstrated on the percentages of CD34+CXCR4+ cells between treatment groups TSF (C), + CTCE [0.01] (D), + CTCE [100] (E), and + CTCE [500] (F). Data were also plotted as histograms in mean ± standard error of mean, n = 3. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; TSF, thrombopoietin, stem cell factor, and flt-3 ligand.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study attempted to address the possible direct and synergistic effects of the small-peptide analogue of SDF-1 on the survival, expansion, and homing of hematopoietic progenitor cells. Our data demonstrated that CTCE-0214, unlike TPO, SCF, or FL, did not increase the viability of progenitor cells in 4-day cultures without supplementary cytokines. However, at 1 ng/ml, CTCE-0214 enhanced the effects of SCF and FL on the viability of these cells. Of the viable cells, 1 ng/ml of CTCE-0214 significantly supported the survival of all subsets of progenitor cell populations and consistently enhanced the effects of TPO, SCF, and FL. The direct prosurvival/antiapoptosis activity of SDF-1 on hematopoietic progenitor cells has been reported and was possibly attributed to the triggering of the ERK, p90RSK, Akt, and G{alpha}i pathways [9, 27, 28]. Our data are in line with those of Lee et al. [27], who demonstrated a slight effect of SDF-1 alone on suppressing apoptosis in response to growth factor withdrawal but significant synergistic activity of SDF-1 when added together with single-factor GM-CSF, TPO, SCF, or FL on the survival of CFU-GM and CFU-GEMM in a semisolid culture of cord blood CD34+ cells. Using single-cell/single-well cultures, the same group also demonstrated that the survival-enhancing action of SDF-1 was mediated directly on CD34+ cells [28].

Consistent with our previous study [24], the cytokine combination of TPO, SCF, and FL significantly expanded progenitors of the primitive, myeloid, erythroid, and megakaryocytic lineages. The addition of CTCE-0214 at 0.01 ng/ml enhanced the number of all progenitor cell subsets. More significant is that the percentages of CD34+ cells and the various CFU populations were increased, indicating that CTCE-0214 not only stimulated differentiation and expansion but also maintained early progenitor cells. Different optimal dosages of SDF-1 have been reported in various studies, ranging from 0.01 to 500 ng/ml [10, 28, 29]. In our culture system that contained moderate concentrations of TPO, SCF, FL, and the serum-free medium QBSF-60, a very low concentration of CTCE-0214 (0.001 ng/ml) was sufficient to significantly enhance expansion of the panel of progenitor cells. In line with Lataillade et al. [29] on PB CD34+ cells, we demonstrated a slight bell-shaped dose-dependent curve and that 0.01 ng/ml of CTCE-0214 was the optimal dose. Some major binding sites of SDF-1, such as Lys24 and Lys27 to heparin sulfate, are missing in CTCE-0214 [30]. We are unsure whether any deviation of the three-dimensional structure of the small peptide from the native SDF-1 has contributed to the increased efficiency of the peptide.

Cord blood units in an adult transplant setting contain ~105 CD34+ cells/kg [23]. In this study, the cytokine combination of TSF expanded CD34+ by 8.45 ± 1.34-fold and TSF + CTCE-0214 by 14.1 ± 2.12-fold. The theoretical total CD34+ cells after expansion would be greater than 106/kg, approaching the dose from a collection of BM or mobilized PB [31]. As discussed earlier, our result showed that primitive hematopoietic cells (CFU-GEMMs) were efficiently expanded (31.9 ± 5.34-fold with TSF and 69.1 ± 10.2-fold with TSF + CTCE-0214). The increase in primitive cells was confirmed in the NOD/SCID mouse transplantation model, in which the repopulating progenitor cells in cultures containing 0.01 ng/ml of CTCE-0214 were consistently enhanced. Also, the early human progenitor subsets (CD34+ cells and CFU-GEMMs) and myeloid cells (CD33+ cells) were higher in the BM of animals receiving cells expanded in CTCE-0214. Our results are in agreement with those reporting that SDF-1 enhanced NOD/SCID repopulating cells in expansion cultures [10].

Short pulsing of expanding cells with CTCE-0214 significantly increased human hematopoietic cell engraftment in the NOD/SCID mice. Because this effect of CTCE-0214 was not paralleled by any increase in the number of progenitor cells, the likely mechanism of the enhanced repopulating potential could be associated with the upregulation of the homing capacity of these cells. Plett et al. [32] reported that the pretreatment (30-minute pulse) of unmanipulated, mobilized PB CD34+ cells with SDF-1{alpha} (100 ng/ml) or low-dose anti-CXCR4 enhanced their engraftment in NOD/SCID mice. Similar effects were not observed with BM CD34+ cells. However, Peled et al. [3] demonstrated that overnight exposure of cord blood CD34+ cells to high concentrations of SDF-1{alpha} inhibited their engraftment in NOD/SCID mice, possibly due to desensitization and CXCR-4 downregulation. The mechanism of SDF-1/CXCR4 on homing is not fully understood and may depend on the dose and timing as well as the source of progenitor cells. For ex vivo expansion/exposure, additional complications can occur, such as interactions with cytokines as well as binding of SDF-1 to surface CXCR4 on other maturing blood cells.

CXCR4 has been known to play a significant role in human stem and progenitor cell homing and repopulation of NOD/SCID mice [3–6]. We observed a decreased percentage of CD34+ cells expressing CXCR4 in culture, and the proportion of this population was not affected by the presence of CTCE-0214 for 4 days or 4 hours. Overall, there were net increases in cell numbers of this subset due to ~8- to 14-fold expansion of total CD34+ cells in various cultures. Ex vivo culturing of CD34+ cells in many systems led to decreased expression of cell-surface CXCR4 [33], and the addition of SDF-1 might further reduce their expression [3, 34]. The association of cell-surface expression of CXCR4 with their engraftment potentials does not appear straightforward. Kollet et al. [5] demonstrated that CD34+CXCR4– cells engrafted NOD/SCID mice and harbored intracellular CXCR4 that could be upregulated within a few hours upon cytokine stimulation. In our study, changes on 8-day expanding cells within 4 hours of treatment with CTCE-0214 were unlikely to be related to cell number or proapoptosis. We speculate that the homing capacity of these cells might be upregulated, possibly involving CXCR4 or other adhesion receptors. In some expansion culture systems, expressions of ß1 integrin [35], very late antigen-4 (VLA-4), VLA-5, and leukocyte function–association antigen-1 [33] on hematopoietic progenitor cells were differentially regulated and suggested to be associated with their homing capacity. Recent studies also demonstrated the modulation of stem cell homing and engraftment by DPPIV/dipeptidylpeptidase IV (CD26) that codistributes with CXCR4 at the cell surface [36]. Other cell-surface molecules, such as heparin, also regulate such interactions [30]. The expression status of these molecules on CD34+ cells in our culture system deserves further investigation.

Our data demonstrate that CTCE-0214 possesses many activities similar to the native peptide SDF-1{alpha}, such as the direct enhancement and synergism with other cytokines on hematopoietic progenitor cell survival and expansion. Further development of this SDF-1 peptide agonist for clinical expansion as well as for enhancement of homing of transplantable hematopoietic stem and progenitor cells might be possible.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors thank Dr. Anthony Edward James, John Tse, and the staff of the Laboratory Animal Services Centre for their assistance in animal experiments and the nurses of the Labour Ward for their assistance in collecting umbilical cord blood. This study was financially supported by the Hong Kong Paediatrics Bone Marrow Transplant Fund, The Chinese University of Hong Kong, and the Industrial Support Fund AF/203/98, Department of Industry, Hong Kong Government Special Administrative Region.

Chemokine Therapeutics Corporation has provided the SDF-1 peptide CTCE-0214 for this study.

DISCLOSURES
P.L., D.W., H.S., and A.M. are employed by Chemokine Therapeutics Corporation, Vancouver, British Columbia, Canada.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nagasawa T, Hirota S, Tachibana D et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996;393:595–599.

  2. Ma Q, Jones D, Borghesani PR et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci U S A 1998;95:9448–9453.[Abstract/Free Full Text]

  3. Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999; 283:845–848.[Abstract/Free Full Text]

  4. Voermans C, Kooi MLK, Rodenhuis S et al. In vitro migratory capacity of CD34+ cells is related to hematopoietic recovery after autologous stem cell transplantation. Blood 2001;97:799–804.[Abstract/Free Full Text]

  5. Kollet O, Petit I, Kahn J et al. Human CD34+CXCR4– sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation. Blood 2002;100:2778–2786.[Abstract/Free Full Text]

  6. Kahn J, Byk T, Jansson-Sjostrand L et al. Overpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration and NOD/SCID repopulation. Blood 2004;103:2942–2949.[Abstract/Free Full Text]

  7. Hattori K, Heissig B, Tashiro K et al. Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood 2001;97:3354–3360.[Abstract/Free Full Text]

  8. Papayannopoulou T. Current mechanistic scenarios in hematopoietic stem/progenitor cell mobilization. Blood 2004;109:1580–1585.

  9. Lataillade J-J, Clay D, Bourin P et al. Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in CD34+ cells: evidence for an autocrine/paracrine mechanism. Blood 2002;99:1117–1129.[Abstract/Free Full Text]

  10. Glimm H, Tang P, Clark-Lewis I et al. Ex vivo treatment of proliferation human cord blood stem cells with stromal-derived factor-1 enhances their ability to engraft NOD/SCID mice. Blood 2002;99:3454–3457.[Abstract/Free Full Text]

  11. Amara A, Le Gall S, Schwartz O et al. HIV coreceptor downregulation as antiviral principle: SDF-1{alpha}-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J Exp Med 1997;186:139–146.[Abstract/Free Full Text]

  12. Babcock GJ, Farzan M, Sodroski J. Ligand-independent dimerization of CXCR4, a principal HIV-1 coreceptor. J Biol Chem 2003;278:3378–3385.[Abstract/Free Full Text]

  13. Colamussi ML, Secchiero P, Gonelli A et al. Stromal derived factor 1{alpha} (SDF-1{alpha}) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95fL) pathway. J Leukoc Biol 2001;69: 263–270.[Abstract/Free Full Text]

  14. Dunussi-Joannopoulos K, Zuberek K, Runyon K et al. Efficacious immunomodulatory activity of the chemokine stromal cell-derived factor 1 (SDF-1): local secretion of SDF-1 at the tumor site serves as T-cell chemoattractant and mediates T-cell-dependent antitumor responses. Blood 2002;100:1551–1558.[Abstract/Free Full Text]

  15. Okabe S, Fukuda S, Kim Y-J et al. Stromal cell-derived factor-1{alpha}/CXCL12-induced chemotaxis of T cells involves activation of the Ras-GAP-associated docking protein p62Dok-1. Blood 2005;105:474–480.[Abstract/Free Full Text]

  16. Balkwill F. The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol 2004;14:171–179.[CrossRef][Medline]

  17. Gazitt Y. Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies. Leukemia 2004;18:1–10.[CrossRef][Medline]

  18. Liles WC, Broxmeyer HE, Rodger E et al. Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood 2003;102:2728–2730.[Abstract/Free Full Text]

  19. Devine SM, Flomenberg N, Vesole DH et al. Rapid mobilization of CD34+ cells following administration of CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J Clin Oncol 2004;22:1095–1102.[Abstract/Free Full Text]

  20. Tudan C, Willick GE, Chahal S et al. C-terminal cyclization of an SDF-1 small peptide analogue dramatically increases receptor affinity and activation of the CXCR4 receptor. J Med Chem 2002;45:2024–2031.[CrossRef][Medline]

  21. Perez LE, Alpdogan O, Shieh J-H et al. Increased plasma levels of stromal-derived factor-1 (SDF-1/CXCL12) enhance human thrombopoiesis and mobilize human colony-forming cells (CFC) in NOD/SCID mice. Exp Hematol 2004;32:300–307.[CrossRef][Medline]

  22. Zhong R, Law P, Wong D et al. Small peptide analogs to stromal derived factor-1 enhance chemotactic migration of human and mouse hematopoietic cells. Exp Hematol 2004;32:470–475.[CrossRef][Medline]

  23. Wagner JE, Barker JN, DeFor TE et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002;100:1611–1618.[Abstract/Free Full Text]

  24. Lam AC, Li K, Zhang XB et al. Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice. Transfusion 2001;41:1567–1576.[CrossRef][Medline]

  25. Su RJ, Zhang XB, Li K et al. Platelet-derived growth factor promotes ex vivo expansion of CD34+ cells from human cord blood and enhances LTC-IC, NOD/SCID repopulating cells and formation of adherent cells. Br J Haematol 2002;117:735–746.[CrossRef][Medline]

  26. Li K, Lee SM, Su RJ et al. Multipotent neural precursors express neural and hematopoietic factors, and enhance ex vivo expansion of cord blood CD34+ cells, colony forming units and NOD/SCID-repopulating cells in contact and noncontact cultures. Leukemia 2005;19:91–97.[Medline]

  27. Lee Y, Gotoh A, Kwon H-J et al. Enhancement of intracellular signalling association with hematopoietic progenitor cell survival in response to SDF-1/CXCL 12 in synergy with other cytokines. Blood 2002;99:4307–4317.[Abstract/Free Full Text]

  28. Broxmeyer HE, Kohli L, Kim CH et al. Stromal cell-derived factor-1/CXCL 12 directly enhances survival/antiapoptosis of myeloid progenitor cells through CXCR4 and Gai proteins and enhances engraftment of competitive, repopulating stem cells. J Leukoc Biol 2003;73:630–638.[Abstract/Free Full Text]

  29. Lataillade J-J, Clay D, Dupuy C et al. Chemokine SDF-1 enhances circulating CD34+ cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood 2000;95:756–768.[Abstract/Free Full Text]

  30. Sadir R, Imberty A, Baleux F et al. Heparan sulfate/heparin oligosaccharides protect stromal cell-derived factor-1 (SDF-1)/CXCL12 against proteolysis induced by CD26/dipeptidyl peptidase IV. J Biol Chem 2004;42:43854–43860.

  31. Heimfeld S. Bone marrow transplantation: how important is CD34 cell dose in HLA-identical stem cell transplantation? Leukemia 2003;17: 856–858.[CrossRef][Medline]

  32. Plett PA, Frankovitz SM, Wolber FM et al. Treatment of circulating CD34+ cells with SDF-1{alpha} or anti-CXCR4 antibody enhances migration and NOD/SCID repopulating potential. Exp Hematol 2002;30:1061–1069.[CrossRef][Medline]

  33. Denning-Kendall P, Singha S, Bradley B et al. Cytokine expansion culture of cord blood CD34+ cells induces marked and sustained changes in adhesion receptor and CXCR4 expressions. STEM CELLS 2003;21:61–70.[Abstract/Free Full Text]

  34. Rosu-Myles M, Gallacher L, Murdoch B et al. The human hematopoietic stem cell compartment is heterogeneous for CXCR4 expression. Proc Natl Acad Sci U S A 2000;97:14626–14631.[Abstract/Free Full Text]

  35. Szilvassy SJ, Meyerrose TE, Ragland PL et al. Homing and engraftment defects in ex vivo expanded murine hematopoietic cells are associated with downregulation of b1 integrin. Exp Hematol 2001;29:1494–1502.[CrossRef][Medline]

  36. Christopherson KW II, Hangoc G, Mantal CR et al. Modulation of hematopoietic stem cell homing and engraftment by CD26. Science 2004;305:1000–1003.[Abstract/Free Full Text]

Received February 27, 2005; accepted for publication June 14, 2005.



This article has been cited by other articles:


Home page
 Stem CellsHome page
M. Yang, K. Li, P. C. Ng, C. K. Y. Chuen, T. K. Lau, Y. S. Cheng, Y. S. Liu, C. K. Li, P. Man Pan Yuen, A. Edward James, et al.
Promoting Effects of Serotonin on Hematopoiesis: Ex Vivo Expansion of Cord Blood CD34+ Stem/Progenitor Cells, Proliferation of Bone Marrow Stromal Cells, and Antiapoptosis
Stem Cells, July 1, 2007; 25(7): 1800 - 1806.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
S. L. Chan, M. Choi, S. Wnendt, M. Kraus, E. Teng, H. F. Leong, and S. Merchav
Enhanced In Vivo Homing of Uncultured and Selectively Amplified Cord Blood CD34+ Cells by Cotransplantation with Cord Blood-Derived Unrestricted Somatic Stem Cells
Stem Cells, February 1, 2007; 25(2): 529 - 536.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0082v1
24/1/55    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, K.
Right arrow Articles by Yuen, P. M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, K.
Right arrow Articles by Yuen, P. M. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS