Characterization of Chemokine Receptors Expressed in Primitive Blood Cells During Human Hematopoietic Ontogeny

Authors

  • M. Rosu-Myles,

    1. The John P. Robarts Research Institute, Developmental Stem Cell Biology, London, Ontario; Department of Microbiology and Immunology, University of Western Ontario, London, Ontario
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  • M. Khandaker,

    1. The John P. Robarts Research Institute, Developmental Stem Cell Biology, London, Ontario; Department of Microbiology and Immunology, University of Western Ontario, London, Ontario
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  • D. M. Wu,

    1. The John P. Robarts Research Institute, Developmental Stem Cell Biology, London, Ontario; Department of Microbiology and Immunology, University of Western Ontario, London, Ontario
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  • M. Keeney,

    1. London Health Sciences, Department of Medicine, London, Ontario
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  • S. R. Foley,

    1. Henderson Hospital, Hamilton, Ontario
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  • K. Howson-Jan,

    1. London Health Sciences, Department of Medicine, London, Ontario
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  • I. Chin Yee,

    1. London Health Sciences, Department of Medicine, London, Ontario
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  • F. Fellows,

    1. St. Joseph's Hospital, London, Ontario
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  • D. Kelvin,

    1. The John P. Robarts Research Institute, Developmental Stem Cell Biology, London, Ontario; Department of Microbiology and Immunology, University of Western Ontario, London, Ontario
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  • M. Bhatia M.D.

    Corresponding author
    1. The John P. Robarts Research Institute, Developmental Stem Cell Biology, London, Ontario; Department of Microbiology and Immunology, University of Western Ontario, London, Ontario
    • The John P. Robarts Research Institute, Developmental Stem Cell Biology, 100 Perth Drive, London, Ontario, N6A 5K8, Canada, and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada. Telephone: 519-663-5777, ext. 34166; Fax: 519-663-3789
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Abstract

Chemokines are capable of regulating a variety of fundamental processes of hematopoietic cells that include proliferation, differentiation, and migration. To evaluate potential chemokine signaling pathways important to the regulation of primitive human hematopoietic cells, we examined chemokine receptor expression of highly purified subpopulations of uncommitted human blood cells. CXCR1-, CXCR2-, CXCR4-, and CCR5-expressing cells were detected by flow cytometry among human blood subsets depleted of lineage-restricted cells (Lin) derived from adult bone marrow, mobilized peripheral blood, cord blood (CB), and circulating fetal blood. Although these chemokine receptors could be detected on Lin cells throughout human development, only CXCR4 could be detected in CD34CD38Lin and CD34+CD38Lin subfractions enriched for stem cell function, suggesting that independent of ontogeny, CXCR4-mediated signals are critical to primitive hematopoiesis. Distinct to other stages of human hematopoietic development, primitive CB cells expressed higher levels of CXCR1, CXCR2, CCR5, and CXCR4 on both CD34CD38Lin and CD34+CD38Lin subsets. Isolation of these fractions revealed expression of additional chemokine receptors CCR7, CCR8, and Bonzo (STRL133), whereas BOB (GPR15) could not be detected. Our study illustrates that rare uncommitted hematopoietic cells express chemokine receptors not previously associated with primitive human blood cells. Based on these results, we suggest that signaling pathways mediated by chemokine receptors identified here may play a fundamental role in hematopoietic stem cell regulation and provide alternative receptor targets for retroviral pseudotyping for genetic modification of repopulating cells.

Introduction

Reconstituting function of human hematopoietic stem cells is dependent on their ability to home to the bone marrow (BM) of transplanted recipients, and subsequently proliferate and differentiate along multiple lineages [1]. This reconstituting ability makes human stem cells an important target for cellular and genetic-based therapies for the treatment of hematopoietic disorders. Accordingly, identifying factors that govern the migration, proliferation, and differentiation of human blood stem cells is essential to optimizing the clinical utility of these rare repopulating cells. The dual function of chemokines as mediators of chemotaxis and growth factors of progenitor blood cells suggests that chemokines are candidate stem cell-regulating molecules [2-4]. Phenotypic analysis of chemokine or chemokine receptor knockout mice indicates that chemokine-mediated signaling is capable of regulating the migration and growth of primitive hematopoietic cells in vivo [5-8]. While these studies have established an important role for chemokines and their receptors in hematopoiesis, the specific chemokine signals actively regulating the homing and proliferation of processes of hematopoietic repopulating cells isolated from humans remain unknown.

In addition to providing migratory and proliferative signals, a proportion of the chemokine receptor family has been shown to function as a mediator for retroviral entry [4, 9-11]. Since retroviruses are capable of stable integration into the host cell genome, retroviral vectors are predominantly used for sustained gene transfer into human blood stem cells [12]. Over the past several years, the efficiency of gene-marking human stem cells with gibbon ape leukemia virus (GALV)-pseudotyped retrovirus has been poor and has only recently shown improvements in providing therapeutic benefits in specific cases [13-15]. Since our current knowledge of retroviral receptors expressed on the surface of primitive human blood cell targets is limited, low gene transfer efficiency into human repopulating cells may partially be due to the use of inappropriate retroviral pseudotypes [16]. Since retroviral pseudotypes can be altered by replacing the gene encoding the envelope glycoprotein [17], specialized retroviral pseudotyping based on chemokine receptor expression of candidate human stem cell populations may prove invaluable for optimizing gene transfer efficiencies.

The ability to survey the large family of chemokines for potential regulation of human blood stem cells is limited due to the extensive and laborious efforts required in using surrogate in vitro or in vivo xenotransplantation assays for individual chemokine receptors [18-20]. Alternatively, a profile of chemokine receptors expressed by highly purified primitive hematopoietic populations would establish a narrow panel of chemokines with the potential to regulate and/or enhance gene transfer efficiencies of human hematopoietic reconstituting cells. While chemokine receptor expression has been well defined on mature human blood cells, the receptor profile of rare stem cell subsets in the human has not been reported. Here, we examined the chemokine receptor expression of primitive human hematopoietic subpopulations throughout human development. Adult sources of mobilized peripheral blood (MPB) and BM, and prenatal sources of full-term cord blood (CB) and circulating fetal blood (FB) were examined for the expression of chemokine receptors CXCR1, CXCR2, CXCR4, and CCR5 using flow cytometry. In addition, reverse-transcriptase-polymerase chain reaction (RT-PCR) was used to determine the chemokine receptor profile of CCR7, CCR8, Bonzo (STRL133), and BOB (GPR15). Our results demonstrate that CXCR4, CCR7, CCR8, and Bonzo (STRL133) are expressed among novel human subpopulations enriched for repopulating ability, and that purified subsets derived from full-term CB express a chemokine receptor profile that is unique from all other hematopoietic sources examined during human ontogeny. Taken together our study suggests potential alternatives for retroviral pseudotypes to target human hematopoietic stem cells and provides insights into the chemokine signaling pathways critical to human stem cell function.

Materials and Methods

Human Cells

Samples of adult human BM, full-term human umbilical CB and human FB from 16-22-week abortus were obtained in conjunction with local ethical and biohazard authorities of the University of Western Ontario and London Health Sciences Centre. Mononuclear cells from PB were obtained by leukapheresis on day 5 following mobilization with human G-CSF. Whole blood was diluted (1 to 3) in alpha-minimal essential medium (GIBCO BRL; Burlington, Ontario) or phosphate-buffered saline (PBS), and mononuclear cells were collected by centrifugation on Ficoll-paque (Pharmacia; Piscataway, NJ; http://www.pnu.com) as shown previously [15].

Purification of Cell Populations

Human mononuclear cells depleted of mature lineage markers (Lin) were purified from various human hematopoietic sources using standard protocols [21, 22]. Briefly, cells were enriched by negative selection using a cocktail of lineage antibodies and the StemSep device as described by the manufacturer (Stem Cell Technologies; Vancouver, BC; http://www.stemcell.com). Purification of Lin subsets was accomplished by fluorescence-activated cell sorting (FACS) following staining with antihuman CD34 conjugated to allopycocyanin (APC), antihuman CD38 conjugated to phycoerythrin (PE [Becton Dickinson; San Jose, CA http://www.bd.com]) and antihuman CD7 conjugated to fluorescein isothiocyanate (FITC) (Immunotech; Marseille, France). Sort gates were set based on isotype controls. Sorted populations were reanalyzed and greater than 98% purity was consistently achieved in all experiments (data not shown).

Phenotypic Analysis

Purified Lin cells (1 × 105) were resuspended in 50 ul of PBS/5% fetal calf serum and incubated for 30 min at 4°C with human CD34-APC (Becton Dickinson), CD38-PeCy5, CD7-FITC (Immunotech) and either antihuman CXCR1-PE (5A12), CXCR2-PE (6C6), CXCR4-PE (12G5) (Pharmingen; Mississauga, Ontario; http://www.pharmingen.com), or CCR5-FITC (45531.111) (R&D Systems; Minneapolis, MN; http://www.rndsystems.com) monoclonal antibodies (mAbs). Flow cytometry was performed on a FACSCalibur (Becton Dickinson).

RT-PCR Analysis

Messenger RNA was extracted from purified CD34+CD38CD7Lin and CD34CD38CD7Lin populations and reverse-transcribed into first strand cDNA using Mo-MuLV Superscript II (GIBCO BRL; Grand View, NY) reverse-transcriptase enzyme. RT-PCR analysis of β-glucuronidase, a housekeeping gene constitutively expressed at a single transcript per cell, was used to define the lower limit of detection for the assay and provided a control for cDNA fidelity [23]. PCR for different chemokine and retroviral receptors was performed using a Perkin-Elmer 9700 thermocycler with the following specific primer sequences: CCR7F, 5′-GGCTGGTCGTGTTGACCTA T-3′; CCR7R, 5′-CCATTGTAGGGCAGCTGGAA-3′; CCR8F, 5′-ATGC CGTGTATGCCCTAAAG-3′; CCR8R, 5′-ACTTTTCA CAGCTCTCCCTA-3′; BonzoF, 5′-CAGGCAT CCATGA ATGGGTGT-3′; BonzoR, 5′-CAAGGCCTATAACTG GAACATGCTG-3′; BOBF, 5′-CATCTGCTCTTTGGT GATG-3′; BOBR, 5′-GTATGGCTTATCATCAATCAGC-3′; GALVR1F, 5′-CCTGCTCCCCTGAAGAATGA-3′; GALVR1R, 5′-TTGCCCAAAGTTCACATTCC-3′; GAL VR2F, 5′-TAAAGGGATGGTCTGGTGTT-3′; GALVR2R, 5′-GAGGGTGGGAGACAGACAAT-3′. Primers were used at a final concentration of 0.5 uM and amplified using 40 cycles of PCR. In all cases cDNA derived from whole fetal tissue was used as a positive control. All amplified products were gel-purified, sequenced and then compared with published sequences (data not shown) to verify specificity of PCR product.

Results

Expression of Chemokine Receptors CXCR1, CXCR2, CXCR4, and CCR5 on Primitive Hematopoietic Cells throughout Human Development

Recent studies evaluating the response of human progenitors to chemokines in transwell migration assays or direct addition of chemokines to in vitro colony-forming unit assays, have suggested that CXCR1, CXCR2, CXCR4, and CCR5 signaling may be important in primitive human hematopoietic cell regulation [2, 24, 25]. In order to quantitatively evaluate the capability of primitive blood cells to signal through CXCR1, CXCR2, CXCR4, or CCR5 during fetal, natal and adult stages of human development, hematopoietic populations depleted of Lin cells were isolated from circulating FB [26], full-term CB, and adult sources of hematopoietic tissue derived from BM and MPB. Human Lin cells were stained with fluorochrome-conjugated mAbs against specific epitopes of human chemokine receptors and analyzed by flow cytometry. Chemokine receptors CXCR1 and CCR5 were detected on 0.5%-3% of Lin cells from FB, BM, and MPB, implying their ability to signal on primitive cells throughout human development (Figs. 1A, B). CXCR2 was detected on 2% of adult BM and MPB Lin cells but was not identified on FB Lin cells, suggesting that this receptor may not play an important role in primitive human hematopoiesis in utero (Fig. 1C). An almost 10-fold greater proportion of Lin cells expressed CXCR4 compared to other chemokine receptors examined, illustrating that CXCR4 signaling is more prevalent in both early and late hematopoietic sources of FB, BM, and MPB cells (Fig. 1D).

Figure Figure 1..

CXCR1, CCR5, CXCR2, and CXCR4 are expressed on primitive human blood cells throughout human ontogeny.Primitive Lincells isolated from human FB, BM, G-CSF-MPB and umbilical CB were stained with antihuman CD34 and CD38 fluorochrome-conjugated antibodies in combination with antibodies recognizing A) CXCR1, B) CCR5, C) CXCR2, or D) CXCR4. Each bar graph gives the percentage of Lin(hatched), CD34+CD38Lin(filled), and CD34CD38Lin(clear) cells expressing chemokine receptors. Percentages are given as mean ± SE values. Fluorescence values < isotype controls are labeled undetectable (UD).

Based on these results, we further evaluated rare CD34+CD38Lin and CD34CD38Lin subpopulations previously shown to be enriched for cells capable of repopulating ability [21, 22, 27-29], to determine the possible expression of CXCR1, CXCR2, CXCR4, or CCR5 on candidate human stem cells. While CXCR1, CCR5, and CXCR2 expression was demonstrated on FB, BM, and MPB Lin cells, the low frequency of positive Lin cells precluded the detection of significant levels of these chemokine receptors on stringently gated CD34+CD38Lin and CD34CD38Lin subsets (Figs. 1A, B, C). In contrast, the percentage of CXCR4+ cells within subsets derived from fetal or adult sources ranged from 6% to 17%, with the exception of MPB CD34CD38Lin subfractions which contained 45 + 4.3% CXCR4-expressing cells (Fig. 1D). These data suggest that while chemokine receptors CXCR1, CXCR2, and CCR5 may play a role in primitive human blood cells, the proportion of cells utilizing signaling pathways mediated by these chemokine receptors is relatively low compared to cells signaling through the CXCR4 receptor.

Chemokine Receptor Expression Indicates that Full-Term Human CB Represents a Unique Source of Primitive Hematopoietic Cells

Human CB is highly enriched for both in vitro-detected progenitors and pluripotent repopulating cells [21, 22, 30-32], suggesting that CB may represent an alternative clinical source of stem cells for hematopoietic reconstitution [27, 33-35]. Accordingly, we examined the chemokine-signaling potential of human CB Lin cells. The proportion of CB Lin cells expressing CXCR1, CCR5, CXCR2, or CXCR4 was three- to sevenfold higher than that detected among prenatal and adult sources (Fig. 1 A-D). In contrast to FB, BM, and MPB, chemokine receptors CXCR1, CCR5, and CXCR2 were expressed on 5.4% to 18.7 % of CD34+CD38Lin and CD34CD38Lin CB cells (Fig. 1 A-C). In addition, CXCR4+ cells were more abundant within highly purified CB populations comprising 81.6% + 5.1 of the CD34+CD38Lin population and 62.9% + 8.7 of CD34CD38Lin cells (Fig. 1D). This distinct proportion of chemokine receptors detected among progenitor and stem cell-enriched subfractions of human CB suggests that chemokine signaling may have an expanded role on primitive human cells within this neonatal source compared to other periods of human ontogeny.

CXCR4-Expressing Cells are Enriched in Novel CD7Lin Hematopoietic Subpopulations

Both human blood progenitors and cells committed to the lymphoid and myeloid lineages express the cell-surface antigen CD7 [36]. Recently, our group has discovered that cells devoid of CD7 expression within the human CB CD34+CD38Lin and CD34CD38Lin subfractions are enriched for pluripotent repopulating function [37]. Since CXCR4 was shown to be abundantly expressed among CD34CD38Lin and CD34+CD38Lin subsets, novel CD7 subfractions from both CD34+ and CD34 stem cell-enriched populations were examined for CXCR4 expression. Primitive human cells expressing CXCR4 were detected among CD34+CD38LinCD7 and CD34CD38LinCD7 subfractions derived from adult MPB (13.0% + 5.8; 54.7% + 17.2) and BM (5.5% + 0.3; 15.4% + 3.7) sources as well as neonatal (80.2% + 4.8; 81.8% + 3.8) and in utero circulating FB (11.1% + 7.0; 26.3% + 13.9) (Fig. 2). These results further substantiate an essential role for CXCR4 signaling in human hematopoiesis and indicate that high levels of CXCR4 expression are limited to full-term human CB hematopoiesis alone.

Figure Figure 2..

CXCR4 is expressed on cells within newly identified populations enriched with human blood repopulating cells.Purified Lincells stained with a combination of CD34, CD38, CD7, and CXCR4 fluorochrome-conjugated mAbs were analyzed by FACS. The percentage of CD34+CD38CD7-Lin(filled) and CD34CD38CD7Lin(clear) cells expressing CXCR4 are given as mean ± SE values from three or more experiments using MPB, BM, CB, and FB.

Chemokine Receptors CCR7, CCR8, and Bonzo (STRL133) Are Expressed within Rare Subsets of CD34CD38CD7Lin and CD34+CD38CD7Lin Human CB Cells

Ligands for the chemokine receptors CCR7 (macrophage inflammatory protein-3β, SLC) and CCR8 (I-309) have been suggested to modulate the migration and proliferation of human hematopoietic progenitors in vitro [2, 38]. In order to further our understanding of chemokine signaling of primitive human CB cells, RT-PCR was used to examine CCR7 and CCR8 expression on highly purified CD34+CD38CD7Lin and CD34CD38CD7Lin subsets derived from human CB. In addition, stem cell-enriched CB subfractions were further examined for the expression of Bonzo (STRL133) and BOB (GPR15), chemokine receptors recently identified on mature human leukocytes that have yet to be examined among primitive blood cells [10]. Expression of CCR7 was detected in both highly purified CD34CD38CD7Lin and CD34+CD38CD7Lin populations, while CCR8 and Bonzo could be detected only in the primitive CD34Lin subfractions (Fig. 3A). In contrast, the chemokine receptor BOB was not detected in either stem cell-enriched subfraction (Fig. 3A). Chemokine receptor expression of both CCR8 and Bonzo suggests that chemokines I-309 and the unknown ligand for Bonzo may mediate signaling specific to CD34 human stem cells, while CCR7-mediated signaling represents a pathway involved in both CD34+ and CD34 hematopoietic stem cells.

Figure Figure 3..

Chemokine and retroviral receptor expression of CB populations enriched for repopulating capacity.Highly enriched CD34+CD38CD7Linand CD34CD38CD7Linpopulations were purified from CB Lincells. RT-PCR for chemokine and retroviral receptors A) CCR7, CCR8, Bonzo, BOB (n = 3), and B) GALVR1, and GALVR2 (n = 3) was performed on cDNA derived from these populations. RT-PCR for β-glucuronidase (bottom panel) was used as a lower limit of detection and a positive control for the integrity of cDNA in each population. Human fetal cDNA was used in PCR reactions as a positive control.

Since chemokine receptors are used for retroviral adhesion and internalization [4, 9-11], the identification of CCR7, CCR8, and Bonzo among stem cell-enriched populations in this study suggests their potential use in specialized retroviral pseudotyping for the successful transfer of genes into human repopulating cells. Currently, the gibbon ape leukemia virus receptors 1 and 2 (GALVR1 Pit1) and GALVR2 (Pit2, AmphoR) are the most widely accepted pseudotypes for retroviral vehicles used in clinical transduction of primitive human repopulating cells [13, 17]. Studies have shown that GALV receptors are detected at low levels on CD34+ cells in the human and that higher efficiencies of retroviral transduction can be correlated to enhanced expression of the GALVR transcript [39, 40]. Based on these correlations, recently identified CD34+CD38CD7Lin and CD34CD38CD7Lin populations were examined for GALVR1 and GALVR2 expression and compared to expression of chemokine receptors CCR7, CCR8, and Bonzo. GALVR1 was detected among CD34+CD38CD7Lin and CD34CD38CD7Lin subpopulations while GALVR2 was undetectable (Fig.3B). The identification of GALVR1 expression within the CD34CD38CD7Lin subfraction represents the first association of this retroviral receptor with primitive human CD34 cells, and suggests that GALVR1-pseudotyped retroviruses may allow the transduction of human stem cells within this novel subpopulation. Therefore, our data illustrate that specific retroviral gene transfer into human CD34 stem cells may be augmented using Bonzo or CCR8 as retroviral pseudotypes. Based on the results of this comparative analysis between GALVR retroviral receptors to chemokine receptor expression on hematopoietic populations enriched with primitive blood cells, we suggest that CCR7, CCR8, and Bonzo may provide alternative envelope pseudotypes for retroviral gene transfer into candidate human hematopoietic repopulating cells.

Discussion

Our present study demonstrates that CXCR4 is a dominantly expressed chemokine receptor on primitive human blood cells within CD34+CD38Lin repopulating cell-enriched populations from MPB, BM, CB, and FB representing all stages of human development. This was further substantiated by the expression of CXCR4 among CD7 subsets shown to provide additional enrichment of human repopulating stem cells [37]. Identification of primitive CXCR4+ cells in human BM, MPB, and CB provides further support for the role of this chemokine receptor and its downstream signals in human hematopoietic stem cell physiology. Although the role of CXCR4 in the human fetus has not yet been demonstrated, its function is well established in embryonic development of the mouse [7, 8, 41, 42]. The presence of rare CD34+CD38LinCXCR4+ cells in circulating human FB suggests that the function of CXCR4 signaling on human stem cells is conserved during initial stages of blood formation in the human. Overall, this study adds to the body of evidence supporting a critical and conserved role for CXCR4 signaling in the regulation of primitive CD34+ stem cells throughout human development, and suggests that CXCR4 may provide a useful retrovirus pseudotype for stem cell gene transfer in the clinic.

A number of groups have identified hematopoietic repopulating cells within the CD34 subfraction of both mouse and human tissues, suggesting that BM repopulating activity arises from a compartment that is comprised of both CD34+ and CD34 stem cells [22, 37, 43, 44]. Recent studies in the mouse have indicated that repopulating cells within the CD34 subfraction represent a “resting population” of stem cells that play an important role in long-term reconstitution [45]. In addition, mesenchymal stem cells with the ability to adopt several different cell fates have been identified within CD34 hematopoietic subfractions [46, 47]. These studies have determined a potentially crucial role for primitive CD34 cells during hematopoietic transplantation in the clinic as well as in the treatment of multiple tissue disorders. Our data demonstrate the expression of CXCR4, CCR7, CCR8, and Bonzo (STRL133) on novel CD34 subsets, illustrating a potential role for these chemokine-signaling pathways in human CD34 stem cells. As CXCR4 and CCR7 expression was also identified among CD34+CD38CD7Lin cells, this study indicates that CXCR4 and CCR7 signaling may play an important role in the human stem cell compartment. Alternatively, CCR8 and Bonzo expression was exclusive to the CD34CD38CD7Lin hematopoietic subset, suggesting a role for induced signaling from I-309 and the unknown Bonzo ligands in CD34 stem cells. Therefore, the current study is the first to associate chemokine signaling with primitive CD34 blood cells in the human, thereby providing insights into the mechanisms that regulate this uncharacterized population of primitive blood cells. In addition, the potential use of these receptors for retroviral gene transfer into CD34 stem cells may provide an important means for both the study of this population and its relationship to CD34+ repopulating cells in the human.

Human CB represents an easily assessable source of primitive hematopoietic cells expressing chemokine receptors. As the frequency of progenitor cells within primitive hematopoietic subfractions decreases with the age of the donor [31, 48], the differences in chemokine receptor expression shown here do not correlate with the clonal capacity of each ontogenic source. Therefore, the proportion of chemokine receptors among full-term human CB subsets of CD34+CD38Lin and CD34CD38Lin cells indicates a fundamental difference in the function of chemokine signaling from this hematopoietic tissue. Our demonstration of high levels of chemokine receptor expression among stem cell-enriched subsets of CB is unique to human hematopoietic ontogeny and suggests that primitive CB populations may provide an important source for studying the effects of chemokine signals on human stem and progenitor cells, but may not represent adult stem cell function.

Our data provide insights into potential chemokine signaling required for physiological regulation of primitive blood cells throughout human ontogeny, and suggests alternative candidate pseudotypes for retroviral-mediated gene transfer into both CD34+ and CD34 candidate human repopulating stem cell fractions. In addition, our analysis identifies novel candidate chemokine signaling pathways to be examined in future studies using in vivo stem cell detection assays to provide definitive evidence for the presence of chemokine receptors on the surface of human blood repopulating cells. Accordingly, we are developing mAbs that will allow the isolation and analysis of primitive CCR7- and CCR8-expressing populations for functional analysis of these rare human subsets in both CD34 and CD34+ stem cell fractions.

Acknowledgements

Supported by grants from the Medical Research Council of Canada, Ontario, Canada and Bayer Inc., and a scholarship award (#MSH-35681) to M.B. from the Medical Research Council of Canada.

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