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作者：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作者单位：a Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong, Peoples Republic of China;b Chemokine Therapeutics Corporation, Vancouver, British Columbia, Canada " t. i- @7 s( W1 _9 L
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【摘要】
- e% W# z; E3 U' T$ O% } 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¨C cells, colony-forming unit-mixed ), 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.
: I7 @- w( I- R( |- F& V; { 【关键词】 SDF- Cord blood CD cells Ex vivo expansion Engraftment Nonobese diabetic/severe combined immunodeficient mice CXCR7 Z2 q" z+ g; S* A8 b' U( m
INTRODUCTION3 S' r2 E! u+ B0 o$ h
5 o+ O. e2 K7 P7 e0 m/ DStromal cell¨Cderived factor-1 (SDF-1 or CXCL12) is a member of the CXC chemokine family that binds to the G-protein¨Ccoupled 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 .
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The function of SDF-1 can be reproduced by small peptide agonists .6 \4 t3 K+ T% w. o/ e% Q+ M8 h, y
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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 . It is possible that SDF-1 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.% T: {, k9 F. g- }' T2 n( O
4 P/ v1 K% Q. V% W" m6 P: U0 JMATERIALS AND METHODS
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6 K _& [. B( N5 QCollection and Enrichment of Human Umbilical Cord Blood CD34 Cells' [# Q' H% o( ~/ ]
$ O& w- [( r* ?9 S" ICord 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% ¡À 0.01% (mean ¡À standard error of the mean ; range, 86.1%¨C96.3%; n = 25).
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: |& S6 q# b. x1 T& ^4 yCTCE-0214 with Single-Cytokine Thrombopoietin, Stem Cell Factor, or flt-3 Ligand on CD34 Cells in Culture* B' J% _5 {4 ?0 w+ B6 Q% O
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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¨C14) is linked to the helical-C-terminal region (residue 55¨C67) of SDF-1 by a bifunctional molecule (the linker) (Fig. 1). 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 . 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¡ãC 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.
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Figure 1. Amino acid sequences of SDF-1 and CTCE-0214. Common sequences are underlined.
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7 i' o8 D1 x$ I( zEx Vivo Expansion of Cord Blood CD34 Cells4 b9 S7 A. J3 m7 K, W6 X
" R- p0 G- E% L8 J, jA 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¨C100 ng/ml) added. At day 8, the cells were harvested and stem and progenitor cells were quantified by flow cytometry and CFU assays.0 z* Z& ?' {% C2 g4 J" W
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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 , 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.- O2 ]1 ^8 p) E
! G1 _7 r9 X" i8 |$ ^Flow Cytometric Analysis of Hematopoietic Progenitor Cells
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$ M! \2 k& }$ N% s" j+ mEnriched 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.
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Colony-Forming Unit Assay7 Z; x! D7 D$ q7 n2 A" k, z ]
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Colony-forming unit¨Cgranulocyte macrophages (CFU-GMs), burst-forming unit/colony forming unit¨Cerythroids (BFU/CFU-Es), and colony forming unit¨Cmixed (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 .
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- l+ h+ c: Z4 L* W# y5 P2 CColony forming unit¨Cmegakaryocytes (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.2 t! B3 T# @$ d
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Engraftment of Human Cells in NOD/SCID Mice
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9 H& L7 R4 u& r2 a' i3 N# ?8 wNOD/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¨Cexpanded 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 . These animals were euthanized 6 weeks after transplantation.
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. H1 J# p, u3 {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 , 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.$ u6 R# N7 i3 s6 m2 S k
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Statistical Analysis
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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 ¡À SEM.
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RESULTS
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$ ` W# O# O' [9 o1 c% I7 v/ M" NSynergistic Effects of CTCE-0214 with TPO, SCF, or FL on Survival of Hematopoietic Progenitor Cells
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3 J$ g3 _) m3 G" WCulturing of enriched CD34 cells in QBSF-60 for 4 days without any supplementary cytokine (control) resulted in low cell viability (48.4% ¡À 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. 2). The presence of TPO, SCF, or FL significantly increased the cell viability to 56.1% ¡À 0.04%, 64.6% ¡À 0.02%, and 70.5% ¡À 0.01%, respectively (p ! k' n+ E8 ~! @! O1 u, P
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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 ); (2¨C4) with 50 ng/ml of single-factor TPO, SCF, or FL and 0, 0.01, 1, or 10 ng/ml CTCT-0214, respectively. *p
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Of the viable cells, cultures without supplementary cytokines yielded reduced levels of TNCs and all subsets of progenitor cells (Fig. 2). The addition of 1 ng/ml CTCE-0214 alone significantly increased TNCs, CD34 cells, CFU-GMs, BFU/CFU-Es, and CFU-GEMMs (*p W4 W8 A ?3 G7 j% W/ w1 P
" v0 C4 m. T! ?& O s! vThe 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 ) S( R5 q8 y; C
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Effects of CTCE-0214 on Ex Vivo Expansion of CD34 Cells+ s& l, f4 B9 F; l- O( h
; \, t! }! T/ V: cIn 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. 3). 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¨C 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
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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
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In the subsequent series of experiments, we compared the effects of 4 days of exposure to CTCE-0214 at 0.01 ng/ml (CTCE ) 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. 4). The addition of 0.01 ng/ml CTCE-0214 at day 4 significantly enhanced the expansion of these subsets (p ) q$ U, b# }, X% A' d
! T7 o6 y' _- f) T6 @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 : 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 ' w8 [4 k! z# p4 x3 k: G) I' j7 |
& U' F: S+ b$ C# ]2 y9 c6 gFigure 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 + H+ _+ E& c+ ~
5 B# y9 |! |% kSix 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 group (p = .071) compared with those in the TSF group (3.03% ¡À 0.99%). Consistent increases were also observed in the various CFU subsets in the BM of these mice (Fig. 6).
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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 , p
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" |( K; m; R: {/ c* `Expression of CXCR4 on CD34 Cells6 S5 E8 Q/ E0 ]7 u5 T: I' \
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A high proportion of enriched CD34 cells from cord blood expressed surface CXCR4 at day 0 (63.4% ¡À 11.7%, n = 3) (Fig. 7). After 8 days of culture, the proportions of CD34 cells expressing CXCR4 were decreased to 16.9%¨C17.5% (mean). There were no differences between the proportions of CD34 CXCR4 cells in the four groups of expansion cultures.
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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¨Cpositive and low side-scatter profile (not shown), and its expressions of CXCR4 were quantified on the CD34-FITC/CXCR4-PE dot blots (B¨CF). 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 (F). Data were also plotted as histograms in mean ¡À standard error of mean, n = 3. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; TSF, thrombopoietin, stem cell factor, and flt-3 ligand.
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5 d- d8 Y7 q: U/ @0 G# B, `DISCUSSION
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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 Gi pathways .. c- S" V6 P$ K3 L
4 T, ]6 t& r7 J m8 M9 WConsistent with our previous study . 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.* d' `+ [3 {) ^4 _! o8 V
5 {) }2 ^# \/ V/ a: Z: x* tCord blood units in an adult transplant setting contain ~105 CD34 cells/kg ., b' j/ ~; @# p" B
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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. demonstrated that overnight exposure of cord blood CD34 cells to high concentrations of SDF-1 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.
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h0 s& Y1 o" qCXCR4 has been known to play a significant role in human stem and progenitor cell homing and repopulation of NOD/SCID mice . The expression status of these molecules on CD34 cells in our culture system deserves further investigation.
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9 b! c% w" `# j7 U% hOur data demonstrate that CTCE-0214 possesses many activities similar to the native peptide SDF-1, 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.% i* w: h- Q) q8 c9 \. W
* {8 ]5 s1 j) j. P, xACKNOWLEDGMENTS8 E2 ]* H3 z2 Q% b4 ]' D$ \7 d
" d* |- T* ^! }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.
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Chemokine Therapeutics Corporation has provided the SDF-1 peptide CTCE-0214 for this study.
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DISCLOSURES, a. K1 E8 H: a2 A; D8 n7 J! e8 l+ {
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P.L., D.W., H.S., and A.M. are employed by Chemokine Therapeutics Corporation, Vancouver, British Columbia, Canada.9 u& B- @/ i ?8 j \ B
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" w5 ] @! Y6 fMa 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¨C9453." [/ W/ G9 W$ W# l4 S1 X2 q- {
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% P5 j; o( y- {# cVoermans 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¨C804.
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Kollet O, Petit I, Kahn J et al. Human CD34 CXCR4¨C sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation. Blood 2002;100:2778¨C2786.' |1 s! ~2 J, B! N
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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¨C2949.2 ]4 @, Z$ [% V" M' Q# b
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发表于 2015-6-2 11:18
