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2009 114: 2530-2541 Prepublished online July 14, 2009; doi:10.1182/blood-2009-04-214403
Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize different CD34 + cell populations based on global gene and microRNA expression signatures Robert E. Donahue, Ping Jin, Aylin C. Bonifacino, Mark E. Metzger, Jiaqiang Ren, Ena Wang and David F. Stroncek
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Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize different CD34⫹ cell populations based on global gene and microRNA expression signatures Robert E. Donahue,1 Ping Jin,2 Aylin C. Bonifacino,1 Mark E. Metzger,1 Jiaqiang Ren,2 Ena Wang,2 and David F. Stroncek2 1Hematology Branch, National Heart, Lung, and Blood Institute; and 2Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD
Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize peripheral blood stem cells by different mechanisms. A rhesus macaque model was used to compare plerixafor and G-CSF–mobilized CD34ⴙ cells. Three peripheral blood stem cell concentrates were collected from 3 macaques treated with G-CSF, plerixafor, or plerixafor plus G-CSF. CD34ⴙ cells were isolated by immunoselection and were analyzed by global gene and microRNA (miR) expression microarrays. Unsupervised hierarchi-
cal clustering of the gene expression data separated the CD34ⴙ cells into 3 groups based on mobilization regimen. Plerixaformobilized cells were enriched for B cells, T cells, and mast cell genes, and G-CSF– mobilized cells were enriched for neutrophils and mononuclear phagocyte genes. Genes up-regulated in plerixafor plus G-CSF–mobilized CD34ⴙ cells included many that were not up-regulated by either agent alone. Two hematopoietic progenitor cell miR, miR-10 and miR-126, and a dendritic cell miR, miR-155, were up-
regulated in G-CSF–mobilized CD34ⴙ cells. A pre-B-cell acute lymphocytic leukemia miR, miR-143-3p, and a T-cell miR, miR-143-5p, were up-regulated in plerixafor plus G-CSF–mobilized cells. The composition of CD34ⴙ cells is dependent on the mobilization protocol. Plerixaformobilized CD34ⴙ cells include more B-, T-, and mast cell precursors, whereas G-CSF–mobilized cells have more neutrophil and mononuclear phagocyte precursors. (Blood. 2009;114:2530-2541)
Introduction Granulocyte colony-stimulating factor (G-CSF) has been the standard hematopoietic growth factor for increasing the levels for circulating hematopoietic stem cells (HSCs) in both healthy subjects donating cells for allogeneic transplantation and patients donating cells for autologous transplantation. G-CSF–mobilized HSCs collected by apheresis have been used for transplantation, immune therapy, and the treatment of cardiac ischemia. Another HSC-mobilizing agent, plerixafor (AMD3100), has been used in large animal models,1,2 with G-CSF to mobilize stem cells for autologous transplants3 and is currently being evaluated as a single agent to mobilize HSCs for allogeneic donor transplants.4 The mechanisms by which plerixafor and G-CSF alter HSC trafficking and mobilize HSCs are different, suggesting that HSCs with different intrinsic properties may be mobilized by these agents. Plerixafor, as a CXC chemokine receptor 4 (CXCR4) antagonist, mobilizes HSCs within 6 hours5-7 by disrupting the engagement of stem cell surface CXCR48,9 with its ligand SDF-1 (CXCL12), which is expressed on marrow osteoblasts.10,11 In contrast, G-CSF mobilizes stem cells indirectly by downregulating the expression of CXCL12 on marrow osteoblasts and by releasing neutrophil and monocyte proteolytic enzymes, including neutrophil elastase, cathepsin G, and matrix metalloproteinase-9, which in turn degrade important HSC-trafficking and adhesion molecules c-kit, VCAM-1, and CXCR4.12 Animal models suggest that plerixafor mobilizes a CD34⫹ cell population different from that mobilized by G-CSF,2,13,14 due to differences in mechanisms of mobilization.
To explore differences between plerixafor and G-CSF mobilization, we used a rhesus macaque HSC-mobilization model to compare CD34⫹ cells mobilized by G-CSF and plerixafor. We also compared CD34⫹ cells mobilized with G-CSF and plerixafor in combination. The combination of G-CSF plus plerixafor was tested because it is being used clinically, and when these agents are used together, more CD34⫹ cells are mobilized than when either agent is used alone.3,15 The CD34⫹ cells were compared using global gene and microRNA expression analysis.
Submitted April 13, 2009; accepted June 24, 2009. Prepublished online as Blood First Edition paper, July 14, 2009; DOI 10.1182/blood-2009-04-214403.
payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
Methods Study design Young rhesus macaques (Macaca mulatta) were studied before and after the completion of 3 different mobilization protocols. The protocols involved the administration of 5 days of G-CSF, one dose of plerixafor, and 5 days of G-CSF plus one dose of plerixafor. Peripheral blood stem cell (PBSC) concentrates were collected by apheresis after the mobilization protocol was complete. Three rhesus macaques were given all 3 mobilization protocols, and PBSCs were collected from each of these 3 animals. Each course of PBSC mobilization was separated by at least 6 weeks. Two monkeys were given plerixafor first, one was given G-CSF first, and all 3 were given plerixafor plus G-CSF last (Table 1). The CD34⫹ cells were isolated from the PBSCs by using CD34 monoclonal antibodies and magnetic particles. The CD34⫹ cells were analyzed using cDNA gene and miRNA expression microarrays.
The publication costs of this article were defrayed in part by page charge
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Table 1. Composition of PBSC products collected from 3 rhesus macaque and the yield of CD34ⴙ cells isolated from each product Isolated CD34ⴙ cells
PBSC products Mobilizing agents
Mononuclear cells/mL, ⴛ109
Granulocytes/mL, ⴛ109
Animal ID
Collection date
RQ4766
3/28/2008
49
163
1.5 ⫻ 107
86.5
2RC104
1/22/2008
90
81
2.5 ⫻ 107
98.8
RQ4963
1/23/2008
30
184
2.0 ⫻ 107
96.4
56 ⫾ 31
143 ⫾ 54
2.0 ⫾ 0.5 ⫻ 107
93.9 ⫾ 6.5
Total no.
Purity (%)
G-CSF
Mean ⫾ SD Plerixafor RQ4766
11/14/2007
103
30
1.5 ⫻ 107
82.0
2RC104
3/11/2008
99
28
2.6 ⫻ 107
95.1
RQ4963
10/31/2007
107
61
2.0 ⫻ 107
82.0
103 ⫾ 4
40 ⫾ 18
2.0 ⫾ 0.6 ⫻ 107
86.4 ⫾ 7.6
Mean ⫾ SD Plerixafor ⴙ G-CSF RQ4766
7/14/2008
63
215
8.9 ⫻ 107
78.8
2RC104
7/12/2008
56
215
12.5 ⫻ 107
99.5
RQ4963
7/16/2008
55
266
6.4 ⫻ 107
85.5
58 ⫾ 4
233 ⫾ 29
9.3 ⫾ 3.1 ⫻ 107
88.0 ⫾ 10.6
Mean ⫾ SD
Mobilization and collection of PBSC concentrates CD34⫹ cells were mobilized, and PBSC concentrates were collected from rhesus macaques that were housed and handled in accordance with the guidelines set by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHHS publication no. NIH 85-23). The protocol was approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute, National Institutes of Health. G-CSF mobilization involved the administration of 10 g/kg G-CSF (Filgrastim; Amgen) subcutaneously daily for 5 days. When plerixafor
CD34+
(Genzyme) was used to mobilize peripheral blood mononuclear cells (PBMCs), a single dose of 1 mg/kg plerixafor was given subcutaneously, and when both G-CSF and plerixafor were given, 10 g/kg G-CSF was given for 5 days and 1 mg/kg plerixafor was given on the fifth day. PBSC concentrates were collected by apheresis approximately 2 hours after the last dose of G-CSF or plerixafor. The PBSCs were collected using a CS3000 plus blood cell separator (Baxter Healthcare Corp, Fenwal Division).16 The concentrates were processed to remove contaminating red blood cells and granulocytes by using Ficoll gradient separation. CD34⫹ cells were isolated by immunomagnetic separation as previously described.16 Purification was determined following immunoselection using phycoerythrin-conjugated murine anti-rhesus/human CD34 antibody (clone 563; BD Biosciences), which recognizes a different epitope from that used in the immunoselection procedure. The processed CD34⫹ cells were frozen and stored at ⫺80°C until analysis.
Plerixafor + G-CSF G-CSF
CD34-
Figure 1. Comparison of gene expression profiles of CD34ⴙ cells mobilized with plerixafor, G-CSF, and plerixafor plus G-CSF with PBMCs. CD34⫹ cells were isolated by immunomagnetic selection from plerixafor, G-CSF, and plerixafor plus G-CSF– mobilized PBSCs collected by apheresis. The CD34⫹ cells and unabsorbed PBMCs were analyzed by gene expression profiling using a cDNA microarray with more than 17 000 genes. The 5378 genes that were expressed in at least 80% of the samples and were increased at least 2-fold in one sample were analyzed by hierarchical cluster of Eisen. G indicates G-CSF; A, plerixafor; A⫹G, plerixafor plus G-CSF.
Plerixafor
Figure 2. Unsupervised hierarchical clustering analysis of gene differentially expressed among CD34ⴙ cells mobilized plerixafor, G-CSF, and plerixafor plus G-CSF in 3 rhesus macaques. The CD34⫹ cells were analyzed by gene expression profiling using a cDNA microarray with more than 17 000 genes. Among 5378 genes that were expressed in at least 80% of the samples and were increased at least 2-fold, 1097 were found to be differentially expressed in 3 clusters of CD34⫹ cells (F test, P ⱕ .005). The differentially expressed genes were analyzed by hierarchical cluster of Eisen. G indicates G-CSF; A, plerixafor; A⫹G, plerixafor plus G-CSF.
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Table 2. KEGG pathways with the most genes up-regulated in G-CSF–mobilized CD34ⴙ cells and plerixafor-mobilized CD34ⴙ cells Pathways with genes up-regulated by G-CSF Pathway
Pathways with genes up-regulated by plerixafor
No. of genes in pathway
Pathway
No. of genes in pathway
Cytokine-cytokine receptor interaction
30
Cytokine-cytokine receptor interaction
Jak-STAT signaling pathway
25
Calcium signaling pathway
29 27
MAPK signaling pathway
24
T-cell receptor signaling pathway
27
Focal adhesion
22
Hematopoietic cell lineage
24
Insulin signaling pathway
21
Wnt signaling pathway
22
Hematopoietic cell lineage
20
Primary immunodeficiency
21
Natural killer cell–mediated cytotoxicity
19
Cell cycle
20
Purine metabolism
19
MAPK signaling pathway
20
ErbB signaling pathway
16
Focal adhesion
19 19
VEGF signaling pathway
15
Natural killer cell mediated cytotoxicity
Regulation of actin cytoskeleton
14
TGF-beta signaling pathway
19
Toll-like receptor signaling pathway
14
Fc epsilon RI signaling pathway
18 18
Tyrosine metabolism
14
Jak-STAT signaling pathway
Acute myeloid leukemia
13
GnRH signaling pathway
15
Fc epsilon RI signaling pathway
13
Cell adhesion molecules
14
GnRH signaling pathway
13
Tight junction
13
Neuroactive ligand-receptor interaction
13
Adherens junction
12
Wnt signaling pathway
13
Antigen processing and presentation
12
Calcium signaling pathway
12
B-cell receptor signaling pathway
12
Complement and coagulation cascades
12
Neuroactive ligand-receptor interaction
12
Fructose and mannose metabolism
12
Gap junction
11
Glycine, serine, and threonine metabolism
12
Long-term depression
11
Leukocyte transendothelial migration
12
Melanogenesis
11
Melanogenesis
12
Regulation of actin cytoskeleton
11
Analysis of gene expression with a 17 500 gene cDNA microarray Total RNA was extracted from the rhesus samples and amplified into antisense RNA (aRNA). In addition, total RNA from human PBMCs pooled from 6 healthy subjects was extracted and amplified into aRNA
to serve as a constant reference. Test and reference RNA were labeled with Cy5 (red) and Cy3 (green), respectively, and cohybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. The microarrays were printed in the Immunogenetics Section, Department of Transfusion Medicine (DTM), Clinical Center (CC), National Institutes
Table 3. Genes whose expression was increased the greatest in G-CSF–mobilized CD34ⴙ cells compared with plerixafor-mobilized CD34ⴙ cells Gene
Fold increase*
P
9.04
5.30 ⫻ 10⫺6
NCF4
Neutrophil cytosolic factor 4, 40 kDa
VNN2
Vanin 2
7.57
7.90 ⫻ 10⫺6
PRTN3
Proteinase 3 (serine proteinase, neutrophil, Wegener granulomatosis autoantigen)
7.25
4.49 ⫻ 10⫺4
LTBR
Lymphotoxin-beta receptor precursor ⫽ tumor necrosis factor receptor 2 related protein ⫽ tumor necrosis
7.23
1.33 ⫻ 10⫺4
factor C receptor CST7
Cystatin F (leukocystatin) (inhibits cathepsins)
7.05
9.00 ⫻ 10⫺6
FCGR3A
CD16 ⫽ Fc-gamma receptor IIIa
7.00
2.60 ⫻ 10⫺5
CTSL
Cathepsin L
6.50
8.00 ⫻ 10⫺7
ANXA3
Annexin A3
6.17
1.17 ⫻ 10⫺4
CST7
Cystatin F (leukocystatin)
5.83
5.80 ⫻ 10⫺6
TSPYL2
TSPY-like 2
4.73
8.58 ⫻ 10⫺4
HSPA1B
Heat shock 70-kDa protein 1B
4.67
.012
ANXA3
Annexin A3
4.64
8.42 ⫻ 10⫺5
DYSF
Dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive)
4.65
1.47 ⫻ 10⫺3
CLU
Clusterin
4.51
8.50 ⫻ 10⫺6
MYCO
Myocilin, trabecular meshwork inducible glucocorticoid response
4.23
1.20 ⫻ 10⫺3
PPBP
CTAP-III ⫽ NAP2 ⫽ connective tissue activation peptide III ⫽ pro-platelet basic protein
4.09
6.47 ⫻ 10⫺3
C1QB
Complement component 1, q subcomponent, B chain
4.00
4.14 ⫻ 10⫺3
RNASE2
Ribonuclease, RNase A family, 2 (liver, eosinophil-derived neurotoxin)
3.99
7.57 ⫻ 10⫺4
GOS2
putative lymphocyte G0/G1 switch gene
3.96
8.50 ⫻ 10⫺5
TNFRSF1
Tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator)
3.84
3.72 ⫻ 10⫺4
CXCL2
Chemokine (C-X-C motif) ligand
3.65
6.73 ⫻ 10⫺4
FCGR2B
Low-affinity IgG Fc receptor II-B and C isoforms
3.61
1.98 ⫻ 10⫺4
IL21R
Interleukin-21 receptor
3.55
3.12 ⫻ 10⫺4
CX3CR1
Chemokine (C-X3-C motif) receptor 1
3.52
1.20 ⫻ 10⫺4
SLCIA5
Solute carrier family 1 (neutral amino acid transporter), member 5
3.35
2.43 ⫻ 10⫺4
PTRF
Polymerase I and transcript release factor
3.28
3.92 ⫻ 10⫺5
*The fold increase for each gene is the average expression of each gene in G-CSF–mobilized CD34⫹ cells from 3 rhesus macaque divided by the average expression of the same gene in plerixafor-mobilized CD34⫹ cells from 3 monkeys.
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Figure 3. Analysis of differentially expressed mobilized CD34ⴙ cells genes by qRT-PCR. The expression of 5 genes (FLT3, HOXA9, PTGS2, CXCR4, and CTSL) in CD34⫹ cells from the 3 rhesus macaque mobilized by plerixafor (A), G-CSF (G), or plerixafor plus G-CSF (A⫹G) were analyzed by qRT-PCR. The expression of CXCR4 was greater in plerixafor and plerixafor plus G-CSF–mobilized CD34⫹ cells. The expression of CTSL was greater on G-CSF– and G-CSF plus plerixafor-mobilized CD34⫹ cells. FLT3 expression was greatest on plerixafor-mobilized CD34⫹ cells, and the expression of PTGS2 was greatest on plerixafor plus G-CSF–mobilized cells. The level of expression of HOXA9 by the 3 types of CD34⫹ cells was similar but much greater than peripheral blood leukocytes. The expression of the 5 genes in leukocytes that were not absorbed by anti-CD34 from the 9 mobilized PBSC concentrates (CD34⫺) are shown as a control.
of Health with a configuration of 32 ⫻ 24 ⫻ 23 and contained 17 500 elements. The clones used for printing the arrays included a combination of the Research Genetics RG_HsKG_031901 8k clone set and 9000 clones selected from the RG_Hs_seq_ver_070700 40k clone set. The 17 500 spots included 12 072 uniquely named genes, 875 duplicated genes, and approximately 4000 expression sequence tags. A complete list of genes included in the Hs-CCDTM-17.5k-1px printing is available at http://nciarray.nci.nih.gov/gal_files/index.shtml.
Analysis of miRNA expression with a microarray Two g total RNA from each sample were labeled with Hy5 and the reference sample (total RNA from Epstein-Barr virus-transformed lymphoblastoid cell lines) with Hy3 by using a miRCURY LNA Array Power Labeling Kit (Exiqon) following the recommended protocol by the manufacturer’s instruction. A commercially available miRNA probe set with 827 human, mouse, and Epstein-Barr virus miRNA was purchased from Ambion and printed onto the glass slides in the Immunogenetics Section of DTM. A complete list of miR included in our microRNA assay is available at http://nciarray.nci.nih.gov/gal_files/CCDTM-miRNA700-V3px-
A.gal. After labeling, the sample and reference were cohybridized to the miRNA array at room temperature overnight. Both the processed cDNA and the miRNA array slides were scanned by using GenePix Pro 4.0 (Axon). Microarray data have been deposited with Gene Expression Omnibus under accession number GSE16936.
Data and statistical analysis For analysis of the cDNA and miRNA array data, the raw dataset was filtered according to a standard procedure to exclude spots with minimum intensity. This filtering was arbitrarily set to an intensity parameter of 300 for cDNA expression data and 100 for miRNA array data in both fluorescence channels. Spots flagged by the analysis software and/or spots with diameters less than 25 m for cDNA expression array and less than 10 m for the miRNA array were excluded from the analysis. The filtered data were normalized using Lowess Smoother and were retrieved by the BRB ArrayTool (http://linus.nci.nih.gov/BRB-ArrayTools. html) developed at the National Cancer Institute, Biometric Research Branch, Division of Cancer Treatment and Diagnosis. Hierarchical cluster
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Table 4. Genes whose expression was increased the greatest in plerixafor-mobilized CD34ⴙ cells compared with G-CSF–mobilized CD34ⴙ cells Gene
Fold increase*
P
ICSBP1
Interferon consensus sequence binding protein 1
6.14
9.20 ⫻ 10⫺6
BLNK
B-cell linker
5.99
1.30 ⫻ 10⫺6
CXCR4
CXC chemokine receptor 4
5.17
1.17 ⫻ 10⫺5
CXCR4
CXC chemokine receptor 4
5.07
4.80 ⫻ 10⫺6
CDK5R1
Cyclin-dependent kinase 5, regulatory subunit 1 (p35)
4.62
3.80 ⫻ 10⫺6
TP53I11
Tumor protein p53 inducible protein 11
4.35
8.40 ⫻ 10⫺6
SMAD7
Homologue of Mothers Against Decapentaplegic (MAD) ⫽ negative regulator of TGF-beta signaling
4.19
8.90 ⫻ 10⫺6
FLT3
Fms-related tyrosine kinase 3
3.44
2.05 ⫻ 10⫺6
CXCR4
CXC chemokine receptor 4
3.32
2.49 ⫻ 10⫺4
LDB2
LIM domain binding 2
3.23
3.24 ⫻ 10⫺4
COG1
Component of oligomeric golgi complex 1
3.12
3.15 ⫻ 10⫺5
DCN
Decorin
3.04
5.96 ⫻ 10⫺5
IL23A
Enhancer of polycomb homolog 1 (Drosophila)
2.95
3.58 ⫻ 10⫺3
AGTRL1
Angiotensin II receptor-like 1
2.92
4.68 ⫻ 10⫺4
SPIB
Spi-B transcription factor
2.91
5.17 ⫻ 10⫺4
EBI2
Epstein-Barr virus–induced gene 2 (lymphocyte-specific G protein–coupled receptor)
2.90
1.81 ⫻ 10⫺4
IL7R
Interleukin-7 receptor
2.86
1.81 ⫻ 10⫺4
TRAT1
T-cell receptor–associated transmembrane adaptor 1
2.83
.014
GAP43
Growth associated protein 43
2.82
7.42 ⫻ 10⫺4
CD79A
CD79A, BCR alpha chain ⫽ mb-1
2.95
3.58 ⫻ 10⫺3
TIEG
TGF-beta inducible early protein
2.75
1.84 ⫻ 10⫺4
IL18R1
Interleukin-18 receptor 1
2.73
8.62 ⫻ 10⫺3
SMARCD
SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 2
2.73
2.73 ⫻ 10⫺3
BTK
Btk ⫽ Bruton agammaglobulinemia tyrosine kinase
2.72
2.67 ⫻ 10⫺3
FCER1A
Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide
2.67
.037
KHDRBS3
KH domain containing, RNA binding, signal transduction associated 3
2.63
3.46 ⫻ 10⫺3
*The fold increase for each gene is the average expression of each gene in plerixafor-mobilized CD34⫹ cells from 3 rhesus macaque divided by the average expression of the same gene in G-CSF–mobilized CD34⫹ cells from 3 monkeys.
analysis was conducted on the genes or miRNA by using Cluster and TreeView software.17 The relationships between the different groups of mobilized CD34⫹ cells were tested by applying unsupervised the Eisen hierarchical clustering method.17 For annotation of genes and functional pathways, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) 2007 software (http://david.abcc.ncifcrf.gov/)18 and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway (http://www.genome.ad.jp/kegg) were used. All target prediction analysis used BRB ArrayTool microRNA targets program (http://linus.nci.nih.gov/BRB-ArrayTools.html), TargetScan (http://www.targetscan.org), and miRBase Targets (http://microrna.sanger.ac.uk).
Results PBSC mobilization and collection
The greatest quantities of CD34⫹ cells were isolated from plerixafor plus G-CSF–mobilized PBMC concentrates (Table 1). Similar quantities of CD34⫹ cells were isolated from G-CSF– mobilized and plerixafor-mobilized PBSC concentrates. Interestingly, the composition of the products differed with more mononuclear cells appearing in the plerixafor-mobilized products than either the G-CSF–mobilized or plerixafor plus G-CSF– mobilized products. Gene expression analysis
Mobilized CD34⫹ cells and the peripheral blood leukocytes that were not absorbed with anti-CD34 were analyzed by gene expression profiling using a microarray with 17 500 cDNA probes. The 5378 genes that were expressed in 80% of these samples and whose expression was increased 2-fold or more in
at least one sample were analyzed by unsupervised hierarchical clustering of Eisen (Figure 1). The samples clustered into 2 major groups: the CD34⫹ group, which included all 9 CD34⫹ cell samples, and the peripheral blood leukocyte group, which included all 9 unabsorbed leukocyte samples. Within the CD34⫹ cell group, a large cluster of genes were expressed at similar levels among all CD34⫹ cell samples. The expression of a second cluster of genes, however, differed among CD34⫹ cells. In fact, the CD34⫹ group was made up of 3 separate clusters: one group that contained all 3 G-CSF–mobilized CD34⫹ cell samples and one plerixafor-mobilized CD34⫹ cell sample, one group that contained 2 plerixafor-mobilized CD34⫹ cell samples, and one that contained all 3 plerixafor plus G-CSF–mobilized CD34⫹ cells samples. These results suggest that CD34⫹ cells mobilized by G-CSF alone and plerixafor alone differ and CD34⫹ cells mobilized by the combination of plerixafor plus G-CSF differed from those mobilized by either G-CSF alone or by plerixafor alone. To further explore the differences among the 3 types of CD34⫹ cells, we identify 1097 genes that were differentially expressed among the 3 types of CD34⫹ cells (F tests, P ⬍ .005). Hierarchical clustering analysis of the differentially expressedgenes separated the CD34⫹ cells into 3 clusters: one with all 3 G-CSF–mobilized samples, one with all 3 plerixaformobilized samples, and one with all 3 plerixafor plus G-CSF– mobilized samples (Figure 2). Although the CD34⫹ cells samples were clustered into 3 separate groups, according to mobilization protocol, the transcription profiles of the plerixafor plus G-CSF–mobilized CD34⫹ cells were not simply a mixture of genes differently expressed by G-CSF–mobilized and plerixafor-mobilized cells. Although some genes that were upregulated in plerixafor plus G-CSF–mobilized CD34⫹ cells
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Table 5. KEGG pathways with the most genes up-regulated and down-regulated in CD34ⴙ cells mobilized by plerixafor plus G-CSF compared with those mobilized by plerixafor alone and G-CSF alone Pathways with genes up-regulated by plerixafor plus G-CSF Pathway
Pathways with genes down-regulated by plerixafor plus G-CSF
No. of up-regulated genes in pathway
Pathway
No. of down-regulated genes in pathway
Cytokine-cytokine receptor interaction
68
MAPK signaling pathway
MAPK signaling pathway
63
Focal adhesion
51 46
Natural killer cell–mediated cytotoxicity
41
Oxidative phosphorylation
40
Jak-STAT signaling pathway
39
Calcium signaling pathway
37
Focal adhesion
33
Ribosome
36
Leukocyte transendothelial migration
33
Cytokine-cytokine receptor interaction
33
Regulation of actin cytoskeleton
32
Wnt signaling pathway
33
Insulin signaling pathway
29
Tight junction
31
Cell cycle
28
Jak-STAT signaling pathway
29
Gap junction
28
Cell adhesion molecules
28
Calcium signaling pathway
25
Natural killer cell–mediated cytotoxicity
28
Fc epsilon RI signaling pathway
25
Insulin signaling pathway
27
Oxidative phosphorylation
25
Axon guidance
26
Ubiquitin mediated proteolysis
25
ErbB signaling pathway
25
T-cell receptor signaling pathway
24
Small cell lung cancer
25
B-cell receptor signaling pathway
22
Antigen processing and presentation
23
Cell adhesion molecules
22
Leukocyte transendothelial migration
23
Toll-like receptor signaling pathway
22
Gap junction
22
Wnt signaling pathway
22
Hematopoietic cell lineage
22
GnRH signaling pathway
21
Long-term depression
22
Melanogenesis
21
Regulation of actin cytoskeleton
22 22
Tight junction
21
VEGF signaling pathway
Apoptosis
20
Colorectal cancer
21
Hematopoietic cell lineage
20
T-cell receptor signaling pathway
21
Pathogenic Escherichia coli infection—EHEC
20
Ubiquitin mediated proteolysis
21
were also up-regulated in plerixafor-mobilized or G-CSF– mobilized CD34⫹ cells, many genes were only up-regulated in plerixafor plus G-CSF–mobilized CD34⫹ cells (Figure 2). Similarly, many genes were only down-regulated in plerixafor plus G-CSF–mobilized CD34⫹ cells (Figure 2). These results suggest that G-CSF, plerixafor, and the combination of plerixafor plus G-CSF mobilized 3 biologically distinct types of HSCs. Comparison of gene expression in G-CSF– and plerixafor-mobilized CD34ⴙ cells
Student t tests were used to identify genes that were differentially expressed between G-CSF–mobilized CD34⫹ cells and plerixafor-mobilized CD34⫹ cells. The expression of 593 genes was greater in G-CSF–mobilized CD34⫹ cells than plerixaformobilized CD34⫹ cells, and the expression of 618 was greater in plerixafor-mobilized CD34⫹ cells (P ⬍ .05). Genes in the complement and coagulation cascades, Toll-like receptor signaling, ErbB signaling, acute myeloid leukemia, vascular endothelial growth factor signaling, and several metabolism and biosynthesis pathways were more likely to be up-regulated in G-CSF– mobilized CD34⫹ cells (Table 2). Up-regulated genes in plerixafor-mobilized CD34⫹ cells were more likely to include those in the T-cell receptor, primary immunodeficiency, TGFbeta signaling pathway, cell cycle, B-cell receptor, antigen presentation, and cell adhesion molecules signaling pathways. Genes in several pathways were up-regulated in both G-CSF– and plerixafor-mobilized CD34⫹ cells, including cytokinecytokine interaction, Jak-STAT signaling pathway, MAPK signaling pathway, hematopoietic cell lineage, natural killer cell (NK)–mediated cytotoxicity, Fc epsilon RI signaling, Wnt signaling, focal adhesion, and neuroactive ligand-receptor interaction (Table 2).
Comparison of the specific genes up-regulated in G-CSF– mobilized and plerixafor-mobilized CD34⫹ cells revealed that the genes maximally up-regulated by G-CSF included the neutrophil genes, neutrophil cytosolic factor 4 (NCF4) and proteinase 3 (PR3), and the NK-cell gene Fc-gamma receptor IIIa (CD16a; Table 3). G-CSF–mobilized CD34⫹ cells were also more likely to express mononuclear phagocyte genes CX3CR1, cathepsin L (CTSL), and lymphotoxin B receptor precursor. Genes expressed by several types of leukocytes that were up-regulated in G-CSF– mobilized CD34⫹ cells included Fc-gamma receptor IIb, which is expressed by neutrophils and B cells; cystatin F, which is expressed by NK cells and mononuclear phagocytes; and IL-21 receptor, which is expressed by T cells, B cells, NK cells, and mononuclear phagocytes. Analysis of the expression of CTSL by quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that CTSL expression was greater in G-CSF–mobilized CD34⫹ cells than in plerixafor-mobilized CD34⫹ cells (Figure 3). The level of CTSL expressed by plerixafor plus G-CSF– and G-CSF–mobilized CD34⫹ cells were similar. The genes differentially up-regulated most by plerixafor included several copies of CXCR4, which is expressed by hematopoietic stem cells, T cells, B cells, monocytes, and neutrophils. Other plerixafor up-regulated genes included the B-cell genes CD79A, interleukin-7 receptor, Epstein-Barr virus–induced gene 2, and Bruton agammaglobulinemia tyrosine kinase; a T-cell gene, T-cell receptor–associated transmembrane adaptor 1; and a mast cell gene, Fc receptor of IgE 1a (Table 4). In addition, the expression of the HSC marker Fms-related tyrosine kinase 3 (FLT3) was up-regulated in plerixafor-mobilized CD34⫹ cells. Analysis of CXCR4 and FLT3 expression by qRT-PCR confirmed that the expression of both was greater in plerixafor-mobilized CD34⫹ cells than G-CSF– mobilized CD34⫹ cells (Figure 3). These results suggest that G-CSF–
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Table 6. Genes whose expression was increased the greatest in plerixafor plus G-CSF–mobilized CD34ⴙ cells compared with those mobilized by plerixafor alone and G-CSF alone Gene
Description
Fold increase*
Parametric P 2.45 ⫻ 10⫺4
MGC27165
Immunoglobulin alpha (1 or 2) heavy chain constant region
9.04
BTK
btk ⫽ Bruton agammaglobulinemia tyrosine kinase
5.13
⬍ 1 ⫻ 10⫺7
PILRB
Similar to hCG2024106
5.07
2.70 ⫻ 10⫺6
IGHG3
Immunoglobulin gamma 3 heavy chain constant region
5.04
3.26 ⫻ 10⫺3
IL1R2
Interleukin 1 receptor, type II
4.82
2.69 ⫻ 10⫺4
GNA14
G alpha 14 ⫽ guanine nucleotide binding protein alpha 14
4.72
⬍1 ⫻ 10⫺7
ULK2
Unc-51-like kinase 2 (C. elegans)
4.26
1.27 ⫻ 10⫺3
BAI3
Brain-specific angiogenesis inhibitor 3
4.16
3.92 ⫻ 10⫺4
IL8
Interleukin 8
3.98
2.28 ⫻ 10⫺4
HRASLS3
HRAS-like suppressor 3
3.68
2.24 ⫻ 10⫺4
PTGS2
Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)
3.67
1.61 ⫻ 10⫺4
PPM1A
Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform
3.61
1.30 ⫻ 10⫺6
CYP3A7
Cytochrome P450, family 3, subfamily A, polypeptide 7
3.59
1.45 ⫻ 10⫺5
CCR1
Chemokine (C-C motif) receptor 1
3.58
1.31 ⫻ 10⫺3
CX3CR1
Chemokine (C-X3-C motif) receptor 1
3.58
1.39 ⫻ 10⫺3
UBE2K
Ubiquitin-conjugating enzyme E2K (UBC1 homolog, yeast)
3.43
3.00 ⫻ 10⫺7
RPL3L
Ribosomal protein L3-like
3.42
3.00 ⫻ 10⫺7
HOXA9
Homeobox A9
3.40
2.10 ⫻ 10⫺4
TMSB10
Thymosin beta 10
3.38
1.34 ⫻ 10⫺3
ZNF644
Zinc finger protein 644
3.34
1.74 ⫻ 10⫺3
HIRA
HIR histone cell-cycle regulation defective homolog A (S cerevisiae)
3.31
2.33 ⫻ 10⫺5
LLT1
Lectin-like NK-cell receptor
3.30
4.62 ⫻ 10⫺5
PRDM15
PR domain containing 15
3.23
2.82 ⫻ 10⫺4
TIMP3
Tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory)
3.19
2.26 ⫻ 10⫺4
ACACA
Acetyl-coenzyme A carboxylase alpha
3.14
2.70 ⫻ 10⫺6
GZMA
Granzyme A (granzyme 1, cytotoxic T-lymphocyte–associated serine esterase 3)
3.12
1.19 ⫻ 10⫺3
ICOS
Inducible T-cell co-stimulator
3.09
2.63 ⫻ 10⫺4
CYP1B1
Cytochrome P450, family 1, subfamily B, polypeptide 1
3.05
3.64 ⫻ 10⫺5
LYN
Lyn ⫽ tyrosine kinase
3.02
8.78 ⫻ 10⫺5
GPA33
Glycoprotein A33 (transmembrane)
3.02
1.74 ⫻ 10⫺4
*The fold increase for each gene is the average expression in the plerixafor plus G-CSF–mobilized CD34⫹ cells from 3 rhesus divided by the average expression of the same gene in the CD34⫹ cells mobilized by plerixafor from 3 monkeys and CD34⫹ cells mobilized by G-CSF from 3 monkeys.
mobilized CD34⫹ cells are more likely to include neutrophil and macrophage precursors, and plerixafor-mobilized CD34⫹ cells are more likely to contain T-cell, B-cell, and mast cell precursors. Gene expression analysis of CD34ⴙ cells mobilized by plerixafor plus G-CSF
To assess the nature of CD34⫹ cells mobilized by the combination of plerixafor plus G-CSF, we combined the gene expression results from CD34⫹ cells mobilized with G-CSF alone and with plerixafor alone, and compared this dataset with CD34⫹ cells mobilized with plerixafor plus G-CSF. A total of 1375 genes were up-regulated in plerixafor plus G-CSF–mobilized CD34⫹ cells compared with those mobilized with G-CSF alone and plerixafor alone, and 1449 were up-regulated in CD34⫹ cells regulated with G-CSF alone and plerixafor alone (t tests, P ⬍ .05). Analysis of the pathways that contained the most genes up-regulated in CD34⫹ cells found that the genes up-regulated in the plerixafor plus G-CSF–mobilized CD34⫹ cells were more likely to belong to B-cell signaling, Fc epsilon RI signaling, Toll-like receptor, and cell cycle (Table 5). Those down-regulated in the plerixafor plus G-CSF–mobilized CD34⫹ cells were more likely to belong to ribosome, antigen processing and presentation, and vascular endothelial growth factor signaling pathways. Analysis of the specific genes up-regulated in plerixafor plus G-CSF–mobilized CD34⫹ cells found that, when compared with G-CSF alone and plerixafor alone mobilized CD34⫹ cells, plerixafor plus G-CSF–mobilized CD34⫹ cells expressed increased levels of B-cell genes: immunoglobulin alpha heavy chain constant
region, Bruton agammaglobulinemia tyrosine kinase, and immunoglobulin gamma 3 heavy chain constant region (Table 6). In addition, the T-cell gene granzyme A; the HSC gene HOXA9; an NK cell gene, lectin-like NK-cell receptor (LLT1); and a gene encoding a protein found in the extracellular matrix and macrophages tissue inhibitor of metalloproteinase (TIMP3) were upregulated (Table 6). The CD34⫹ cell genes up-regulated most by plerixafor plus G-CSF also included prostaglandin-endoperoxide synthase 2 (PTGS2) or cyclooxygenase 2 (COX2), an important enzyme in arachidonic acid metabolism and prostaglandin production. Analysis of PTGS2 and HOXA9 expression by qRT-PCR confirmed that the expression of PTGS2 was greater in plerixafor plus G-CSF–mobilized CD34⫹ cells than in those mobilized by plerixafor alone or G-CSF alone (Figure 3). The expression of HOXA9 was similar among CD34⫹ cells mobilized by each of the 3 regimens but was much greater than that for peripheral blood leukocytes (Figure 3). We also identified the genes that were down-regulated most in plerixafor plus G-CSF–mobilized CD34⫹ cells. Among the genes down-regulated most in plerixafor plus G-CSF–mobilized CD34⫹ cells were a number of ribosomal protein genes, cathepsin S (an enzyme important for antigen processing), neutrophil receptor formyl peptide receptor 1, bone morphogenic protein 2, laminin, and gamma 3 (Table 7). MIR expression analysis
A comparison of differentially expressed miR between plerixaforand G-CSF–mobilized CD34⫹ cells found that 2 miR were
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Table 7. Genes whose expression was decreased the greatest in plerixafor plus G-CSF–mobilized CD34ⴙ cells compared with those mobilized by plerixafor alone and G-CSF alone Gene symbol
Description
Fold decrease*
Parametric P
CTSS
Cathepsin S
13.95
⬍ 1 ⫻ 10⫺7
C9orf3
Chromosome 9 open reading frame 3
7.64
2.16 ⫻ 10⫺4
NTRK2
Neurotrophic tyrosine kinase, receptor, type 2
5.82
⬍ 1 ⫻ 10⫺7
NMT1
N-myristoyltransferase 1
5.64
2.00 ⫻ 10⫺7
SCN1B
Sodium channel, voltage-gated, type I, beta
5.34
⬍ 1 ⫻ 10⫺7
IL16
Interleukin 16 (lymphocyte chemoattractant factor)
4.98
1.20 ⫻ 10⫺6
CYP27B1
Cytochrome P450, family 27, subfamily B, polypeptide 1
4.56
2.35 ⫻ 10⫺4
KNS2
kinesin 2 (60-70 kDa)
4.45
1.18 ⫻ 10⫺4
SMARCD2
SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d,
4.373
6.81 ⫻ 10⫺4 8.89 ⫻ 10⫺5
member 2 CCL25
TECK chemokine (CCL25)
4.27
PFDN6
Prefoldin subunit 6
4.26
2.53 ⫻ 10⫺3
EIF5A
Eukaryotic translation initiation factor 5A
4.24
6.40 ⫻ 10⫺5
TYMS
Thymidylate synthetase
4.23
2.20 ⫻ 10⫺6
OCLN
Occludin ⫽ tight junction protein
4.21
2.35 ⫻ 10⫺4
NAT2
N-acetyltransferase 2 (arylamine N-acetyltransferase)
4.09
8.14 ⫻ 10⫺4
RPL23
Ribosomal protein L23
4.04
6.20 ⫻ 10⫺6
WISP2
WNT1 inducible signaling pathway protein 2
4.01
1.20 ⫻ 10⫺6
MVD
Mevalonate (diphospho) decarboxylase
3.84
2.33 ⫻ 10⫺5
BMP2
Bone morphogenetic protein 2
3.79
3.03 ⫻ 10⫺4
UXT
Ubiquitously expressed transcript
3.78
2.80 ⫻ 10⫺6
IQGAP1
IQ motif containing GTPase activating protein 1
3.67
4.00 ⫻ 10⫺6
KCNA5
Potassium voltage-gated channel, shaker-related subfamily, member 5
3.67
1.80 ⫻ 10⫺3
RPS27A
Ribosomal protein S27a ⫽ fused to ubiquitin
3.60
1.00 ⫻ 10⫺5
ECH1
Enoyl coenzyme A hydratase 1, peroxisomal
3.52
2.10 ⫻ 10⫺6
FPR1
Formyl peptide receptor 1
3.41
8.60 ⫻ 10⫺6
STAP2
Signal transducing adaptor family member 2
3.36
3.15 ⫻ 10⫺4
ZNF525
Zinc finger protein 525
3.27
8.30 ⫻ 10⫺5
LAMC3
Laminin, gamma 3
3.23
1.03 ⫻ 10⫺3
DES
Desmin
3.18
1.78 ⫻ 10⫺5
RPS15
Ribosomal protein S15
3.17
2.80 ⫻ 10⫺6
*The fold decrease for each gene is the average expression in the plerixafor plus G-CSF–mobilized CD34⫹ cells from 3 rhesus divided by the average expression of the same gene in the CD34⫹ cells mobilized by plerixafor from 3 monkeys and CD34⫹ cells mobilized by G-CSF from 3 monkeys.
up-regulated in plerixafor-mobilized CD34⫹ cells, miR-34c and miR-432 (Table 8). A miR, miR-126, that was previously found to be up-regulated in G-CSF–mobilized peripheral blood CD34⫹ cells compared with peripheral blood leukocytes19 was up-regulated in G-CSF–mobilized CD34⫹ cells compared with plerixaformobilized CD34⫹ cells (Table 8). The expression of miR-155, which is up-regulated during macrophage activation,20 was also greater in G-CSF–mobilized CD34⫹ cells. In addition, among the
miR up-regulated in G-CSF–mobilized CD34⫹ cells were miR-21, miR-129-3p, miR-146b, miR-99a, miR-126*, and miR-10a*. We also identified miRs differentially expressed between CD34⫹ cells mobilized by plerixafor plus G-CSF and those mobilized by G-CSF alone and plerixafor alone. The levels of miR-126* and miR-142-3p were greater in plerixafor plus G-CSF–mobilized CD34⫹ cells than in those mobilized by either G-CSF alone (Table 9) or plerixafor alone (Table 10). The
Table 8. miRs that were differently expressed between plerixafor-mobilized CD34ⴙ cells and G-CSF–mobilized CD34ⴙ cells Increased in plerixafor-mobilized CD34ⴙ cells miR
Increased in G-CSF–mobilized CD34ⴙ cells
Fold increase*
P
miR
hsa-miR-34c
1.69
.031
hsa-miR-21
3.67
1.72 ⫻ 10⫺4
hsa-miR-432*
1.45
.032
hsa-miR-126*
2.80
1.21 ⫻ 10⫺3
mmu-miR-129-3p
2.62
3.67 ⫻ 10⫺3
hsa-miR-146b
2.31
1.35 ⫻ 10⫺3
hsa-miR-99a
2.13
.011
hsa-miR-126*
2.08
9.23 ⫻ 10⫺3
hsa-miR-10a*
1.98
.019
hsa-miR-424
1.90
8.57 ⫻ 10⫺3
hsa-miR-126
1.70
.041
hsa-miR-155
1.65
.033
hsa-miR-335
1.55
.049
CD34⫹
Fold increase†
P
*The fold increase is the average expression of each microRNA in cells from 3 rhesus macaque given plerixafor divided by the average expression of the same microRNA in 3 monkeys given G-CSF. †The fold increase is the average expression of each microRNA in CD34⫹ cells from 3 rhesus macaque given G-CSF divided by the average expression of the same microRNA in 3 monkeys given plerixafor.
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Table 9. miRs that were differently expressed between plerixafor plus G-CSF–mobilized CD34ⴙ cells and those mobilized with G-CSF alone Increased in plerixafor ⴙ G-CSF–mobilized CD34ⴙ cells miR
Fold increase*
Increased in G-CSF–mobilized CD34ⴙ cells P
miR 10⫺3
Fold increase†
P
hsa-miR-142-3p
3.27
3.34 ⫻
sv40-miR-S1-5p
2.56
hsa-miR-142-3p
3.15
3.04 ⫻ 10⫺3
hsa-miR-335
2.17
.045
hsa-miR-142-5p
2.91
3.95 ⫻ 10⫺3
hsa-mir-615
2.00
.03
hsa-miR-29b
2.36
7.71 ⫻ 10⫺3
hsa-miR-363*
1.94
.012
hsa-miR-101
2.34
8.77 ⫻ 10⫺3
mmu-miR-10b
1.81
.012
hsa-miR-525
2.18
8.19 ⫻ 10⫺3
hsa-mir-622
2.16
.019
3.17 ⫻ 10
⫺3
mmu-mir-691
1.80
.01
hsa-miR-299-3p
1.77
.018 8.92 ⫻ 10⫺3
hsa-miR-487b
2.15
9.93 ⫻ 10⫺3
mmu-miR-346
1.72
hsa-miR-126*
2.11
9.92 ⫻ 10⫺3
mmu-mir-615
1.71
.01
hsa-miR-199b
2.10
.012
hsa-miR-320
1.61
.031
mmu-mir-804
2.09
0.016
hcmv-miR-UL70-3p
1.56
.03
hsa-miR-486
2.08
3.63 ⫻ 10⫺3
kshv-miR-K12-10a
1.54
.038
hsa-miR-197
1.99
6.74 ⫻ 10⫺3
hsa-miR-518f*
1.54
.029
hsa-miR-19a
1.98
.01
mmu-mir-423
1.51
.044
rno-miR-343
1.97
.015
hsa-mir-765
1.47
.048
hsa-miR-15a
1.97
.011
hsa-miR-19b
1.96
.012
hsa-miR-29c
1.87
.026
hsa-miR-27a
1.74
.017
hsa-miR-374
1.71
.027
hsa-miR-30e-5p
1.70
.044
hsa-miR-29a
1.68
.029
hsa-mir-662
1.66
.02
mmu-miR-489
1.63
.025
hsa-mir-421
1.60
.03
hsa-miR-20a
1.60
.025
hsa-miR-106a
1.59
.022
hsa-miR-203
1.58
.032
hsa-miR-20b
1.58
.023
ebv-mir-BART3
1.57
.022
hsa-miR-17-5p
1.55
.034
*The fold increase is the average expression of each microRNA in CD34⫹ cells from 3 rhesus macaque given plerixafor plus G-CSF divided by the average expression of the same microRNA in CD34⫹ cells from 3 monkeys given G-CSF. †The fold increase is the average expression of each microRNA CD34⫹ cells from 3 rhesus given G-CSF divided by the average expression of the same microRNA in CD34⫹ cells from 3 monkeys given plerixafor plus G-CSF.
expression of miR-21 was also increased in plerixafor plus G-CSF–mobilized CD34⫹ cells compared with plerixaformobilized CD34⫹ cells (Table 10). The expression of miR-126*, miR-155, miR-142-3p, miR-1293p, and miR-99a were also measured by qRT-RCR (Figure 4). This analysis confirmed that the expression of miR-126* and miR142-3p was greater in plerixafor plus G-CSF–mobilized CD34⫹ cells than in the other 2 types of CD34⫹ cells. In addition, the analysis confirmed that the expression of miR-99a, miR-129-3p, and miR-155 was greater in G-CSF–mobilized CD34⫹ cells than plerixafor-mobilized CD34⫹ cells.
Discussion Plerixafor has recently been approved by the US Food and Drug Administration for use with G-CSF to mobilize PBSCs for autologous transplantation. Because plerixafor is now clinically available, it will also likely be used to mobilize stem cells in the 1% to 5% of allogeneic donors who mobilize HSCs poorly in response to G-CSF.21,22 The number of CD34⫹ cells mobilized by the combination of plerixafor and G-CSF is significantly greater than either agent alone. Because the success of allogeneic transplants is highly dependent on rapid myeloid engraftment and immune reconstitution in the transplant recipient, it is important to deter-
mine whether the critical in vivo biological activity or potency of HSCs mobilized by plerixafor and plerixafor plus G-CSF is similar to those mobilized by G-CSF. We used a surrogate assay for potency testing, global gene and microRNA expression analysis, to compare CD34⫹ cells mobilized by plerixafor, G-CSF, and the combination of the 2 agents. Although many of the same genes were up-regulated in CD34⫹ cells mobilized by either plerixafor or G-CSF, the expression of a large number of genes also differed between CD34⫹ cells mobilized by these 2 agents. G-CSF–mobilized CD34⫹ cells were more likely to express neutrophil and mononuclear phagocyte genes than those mobilized by plerixafor. In addition, G-CSF–mobilized CD34⫹ cells also expressed high levels of miR-155, which is expressed in maturing dendritic cells.20 In contrast, plerixaformobilized CD34⫹ cells were more likely to express B-, T-, and mast cell genes. These results suggest that G-CSF mobilizes a greater proportion of HSCs that are committed toward myeloid differentiation, whereas those mobilized by plerixafor are more likely to be committed toward B, T, and mast cells. Surprisingly, the combination of plerixafor plus G-CSF mobilized a population of CD34⫹ cells that were distinct from CD34⫹ cells mobilized by either agent alone. CD34⫹ cells mobilized by plerixafor plus G-CSF were more likely to express B-cell and T-cell genes than G-CSF–mobilized and plerixafor-mobilized CD34⫹ cells. In addition, plerixafor plus G-CSF–mobilized CD34⫹ cells
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Table 10. miRs that were differently expressed between plerixafor plus G-CSF–mobilized CD34ⴙ cells and those mobilized with plerixafor alone Increased in plerixafor ⴙ G-CSF–mobilized CD34ⴙ cells miR
Increased in plerixafor-mobilized CD34ⴙ cells
Fold increase*
P
hsa-miR-21
5.98
4.29 ⫻ 10⫺5
Fold increase†
P
hsa-mir-615
miR
2.06
.0305
hsa-miR-126*
3.91
1.6 ⫻ 10
hsa-miR-126
3.34
2.90 ⫻ 10⫺4
mmu-miR-329
2.01
.0210
sv40-miR-S1-5p
1.88
hsa-miR-142-3p
2.88
.0129
8.98 ⫻ 10⫺4
kshv-miR-K12-1
1.79
hsa-miR-142-3p
.0143
2.75
1.03 ⫻ 10⫺3
kshv-miR-K12-10a
1.78
.0103
hsa-miR-29b
2.57
2.92 ⫻ 10⫺3
mmu-mir-709
1.69
.0235
hsa-miR-142-5p
2.56
1.22 ⫻ 10⫺3
rno-miR-327
1.68
.0155
hsa-miR-101
2.54
3.01 ⫻ 10⫺3
hsa-miR-518f*
1.63
.0312
hsa-miR-29c
2.44
8.59 ⫻ 10⫺3
hsa-mir-612
1.62
.0353
hsa-miR-345
2.39
8.68 ⫻ 10⫺3
hcmv-miR-UL70-3p
1.58
.0457
mmu-mir-705
1.53
.0365
ebv-mir-BART6
1.51
.0475
hsa-mir-557
1.51
.0404
hsa-miR-363*
1.50
.0440
⫺3
hsa-miR-424
2.34
.011
hsa-miR-27a
2.21
9.84 ⫻ 10⫺3
hsa-miR-148a
2.21
.044
hsa-miR-29a
2.15
5.57 ⫻ 10⫺3
mmu-mir-804
2.09
.01
hsa-miR-146b
2.07
5.72 ⫻ 10⫺3
hsa-miR-19a
2.07
3.23 ⫻ 10⫺3
hsa-miR-24
2.03
.014
hsa-miR-19b
1.93
4.96 ⫻ 10⫺3
hsa-miR-146a
1.90
.021
hsa-miR-374
1.72
.025
hsa-miR-487b
1.71
.043
hsa-miR-486
1.71
.034
hsa-miR-15a
1.71
.029
hsa-miR-18a
1.69
.025
rno-miR-374
1.62
.03
hsa-miR-30e-5p
1.62
.04
hsa-miR-20a
1.59
.04
hsa-miR-106a
1.53
.043
*The fold increase in the average expression of each microRNA in CD34⫹ cells from 3 rhesus macaque given plerixafor plus G-CSF divided by the average expression of the same microRNA in CD34⫹ cells from 3 monkeys given plerixafor. †The fold increase in the average expression of each microRNA in CD34⫹ cells from in 3 rhesus macaque given plerixafor divided by the average expression of the same microRNA in CD34⫹ cells from monkeys given plerixafor plus G-CSF.
were also remarkable for their increased expression of miR142-3p and miR-142-5p. miR-142-3p and miR-142-5p are highly expressed in T cells,23 and miR-142-3p has been found to be increased in childhood B-cell precursor acute lymphoblastic leukemia.24 The results suggest plerixafor plus G-CSF mobilizes a greater proportion of B- and T-cell precursors than either G-CSF or plerixafor alone. It is not clear if plerixafor, G-CSF, or their combination mobilizes a more primitive HSC. CD34⫹ cells mobilized by plerixafor expressed greater levels of CXCR4 and FLT3 than G-CSF–mobilized CD34⫹ cells. Increased expression of CXCR4 in the plerixafor-mobilized population is not surprising and is consistent with the nature of the mobilization protocol, that is, interfering with the binding of CXCR4 and CXCL12. Thus, CD34⫹ cells mobilized with plerixafor alone or in combination with G-CSF should be expressing CXCR4. FLT3 is a member of the receptor tyrosine kinase family and plays an important role in hematopoietic homeostasis.25 It is expressed on both common lymphoid and multipotent progenitors. Higher FLT3 expression was observed in those CD34⫹ cells mobilized with plerixafor alone or with the combination plerixafor plus G-CSF than with G-CSF alone. These increases in expression were confirmed by qRT-PCR analysis. We also found that the expression of COX2 was up-regulated in plerixafor plus G-CSF–mobilized CD34⫹ cells. COX2 is an inducible enzyme that metabolizes arachidonic acid to a number of prostaglandins, including prostaglandin E2 (PGE2). PGE2 has been found to be important in hematopoiesis. In zebrafish PGE2
increases HSC numbers, and in mice it enhances spleen colonyforming units and the frequency of long-term repopulating cells.26 PEG2 also has been found to enhance the expression of CXCR4 on mouse CD34⫹ cells.27 This suggests that greater quantities of these progenitors are among plerixafor-mobilized and plerixafor plus G-CSF–mobilized CD34⫹ cells than G-CSF–mobilized CD34⫹ cells. G-CSF–mobilized CD34⫹ cells, however, have increased levels of miR-126, miR-126*, and miR-10a* compared with plerixaformobilized CD34⫹ cells. miR-126 and miR-10a have previously been found to be expressed by CD34⫹ cells.19 In addition to being expressed by HSCs, miR-126 is also expressed by megakaryocytes and endothelial cells and is important in angiogenesis.28,29 miR126* and miR-10a* are mirror images of miR-126 and miR-10a, respectively, and they are derived from the same pre-miR stem loop, so it is not surprising that both miR-126 and miR-126* were expressed in G-CSF–mobilized CD34⫹ cells. These 2 miR, however, target different genes, and they may play a different role in HSC function. We found that the levels of miR-126 and miR-126* were greater in plerixafor plus G-CSF–mobilized CD34⫹ cells than in the other types of CD34⫹ cells. Another miR whose expression was increased in G-CSF–mobilized CD34⫹ cells was miR-99a, which is also expressed by megakaryocytes.29 These results suggest that G-CSF–mobilized CD34⫹ cells may contain a wider range of lineage progenitors. Transplantation studies will be required to determine whether plerixafor, G-CSF, or the combination of these agents mobilizes a more primitive HSC.
From bloodjournal.hematologylibrary.org by guest on May 18, 2011. For personal use only. 2540
DONAHUE et al
BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
Figure 4. Analysis of differentially expressed mobilized CD34ⴙ cell miRs by qRT-PCR. The expression of miR-126*, miR-129-3p, miR-142-3p, miR-99, and miR-155 was analyzed by qRT-PCR in CD34⫹ cells from 3 rhesus macaque mobilized by plerixafor (A), G-CSF (G), or plerixafor plus G-CSF (A⫹G). The expression of the 5 microRNAs in leukocytes that were not absorbed by anti-CD34 from the 9 mobilized PBSC concentrates (CD34⫺) are shown as a control.
The finding that plerixafor, G-CSF, and plerixafor plus G-CSF mobilize different types of CD34⫹ cells is consistent with other studies that found that different agents mobilized different populations of HSCs. In a mouse model, Pitchford and colleagues have found that G-CSF mobilized greater quantities of hematopoietic progenitor cells than plerixafor but plerixafor mobilized greater quantities of endothelial progenitor cells than G-CSF.30 They also found that the combination of G-CSF plus plerixafor mobilized greater quantities of hematopoietic progenitors than either agent alone, but not endothelial progenitors. The results of this study have several implications for clinical HSC transplantation with mobilized PBSCs. Because plerixafor, G-CSF, and plerixafor plus G-CSF mobilize different subpopulations of CD34⫹ cells, the potency of PBSCs mobilized by these agents may differ. The rate of engraftment and profile of transplantassociated comorbidities may differ between recipients of allogeneic transplants with plerixafor-mobilized PBSCs and those who receive G-CSF–mobilized PBSCs. In addition, the dose of CD34⫹ cells required for successful engraftment may be different for plerixafor- and plerixafor plus G-CSF–mobilized PBSC concentrates. A preliminary study of allogeneic transplantations involving 20 HLA-compatible siblings has shown that plerixafor-mobilized
PBSCs resulted in prompt neutrophil and platelet engraftment although a relatively low median dose of CD34⫹ cells was given, 2.9 ⫻ 106 per kilogram of recipient weight.4 This same study found that plerixafor-mobilized PBSCs contained greater quantities of CD3⫹, CD4⫹, and CD8⫹ cells than G-CSF–mobilized PBSCs, but the rates of acute and chronic graft versus host disease that were similar to those of historic controls who received G-CSF– mobilized PBSCs at the same institution.4 Our data suggest that if similar quantities of CD34⫹ cell are administered, those mobilized with G-CSF may result in faster neutrophil recovery and those mobilized with plerixafor or plerixafor plus G-CSF may have more rapid B-cell and T-cell recovery. Larger controlled studies will be required to assess similarities and differences in rates of engraftment, acute and chronic graft-versus-host disease, and immune reconstitution between plerixafor- and G-CSF–mobilized PBSCs. Clinical correlates will be required to determine the minimum and optimal CD34⫹ cell doses of plerixafor-mobilized and plerixafor plus G-CSF–mobilized PBSCs required for successful transplantations and to determine whether the rate and sequence of immune recovery differs among the mobilization regimens. In conclusion, the composition of CD34⫹ cells in PBSC concentrates is dependent on the mobilization regimen, and the
From bloodjournal.hematologylibrary.org by guest on May 18, 2011. For personal use only. BLOOD, 17 SEPTEMBER 2009 䡠 VOLUME 114, NUMBER 12
composition of CD34⫹ cells mobilized by a combination of agents is not a simple mixture of the cells mobilized by single agents. Unique expression profile signatures appear between populations. G-CSF, plerixafor, and plerixafor plus G-CSF mobilized different populations of CD34⫹ cells, but further in vitro and clinical studies are needed to compare the potency of those cells mobilized by these agents.
Acknowledgments This study was funded by the Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, and the Department of Transfusion Medicine, Clinical Center, National Institutes of Health.
PLERIXAFOR- AND G-CSF–MOBILIZED CD34⫹ CELLS
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Authorship Contribution: R.E.D. designed the study, analyzed data, and wrote the manuscript; P.J. helped design the study, performed studies, analyzed data, and wrote the manuscript; A.C.B., M.E.M., J.R., and E.W. performed studies and analyzed data; and D.F.S. helped design the study, analyzed data, and wrote the manuscript. Conflict-of-interest disclosure: A drug used in this study, plerixafor, was provided by Genzyme. The authors declare no competing financial interests. Correspondence: David Stroncek, MD, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, 10 Center Dr, Bldg 10, Rm 1C711, Bethesda, MD 20892-1184; e-mail:
[email protected].
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