Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
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Originally published online as doi:10.1189/jlb.0504273 on January 26, 2005

Published online before print January 26, 2005
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(Journal of Leukocyte Biology. 2005;77:634-643.)
© 2005 by Society for Leukocyte Biology

Phenotypic and functional characteristics of CD34+ cells are related to their anatomical environment: is their versatility a prerequisite for their bio-availability?

Jean-Jacques Lataillade*, Denis Clay{dagger}, Catherine David*, Laetitia Boutin*, Bernadette Guerton{dagger}, Michel Drouet{ddagger}, Françis Hérodin{ddagger} and Marie-Caroline Le Bousse-Kerdilès{dagger},1

* Département Recherches, Centre de Transfusion Sanguine des Armées Jean Julliard, Clamart, Cedex, France;
{dagger} Institut National de la Santé et de la Recherche Médicale, Unité 602, Institut André Lwoff, Hôpital Paul Brousse, Villejuif, France; and
{ddagger} Centre de Recherche du Service de Santé des Armées, La Tronche, Cedex, France

1 Correspondence: INSERM U602, Hôpital Paul Brousse, 14, Avenue Paul Vaillant Couturier, 94800 Villejuif, France. E-mail: lebousse{at}vjf.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human CD34+ hematopoietic progenitors (HP) are mainly resident in adult bone marrow (BM). However, their recent revelation in nonhematopoietic tissues implies their circulation through peripheral blood (PB). The intimate mechanisms of this physiological process are not yet understood. Our results showed that steady-state CD34+ HP exhibit a differential phenotypic profile according to their BM versus PB localization. We demonstrated that this phenotype could be modulated by incubation in the presence of their counterpart mononuclear cells (MNC) through cell interactions and cytokine production. Such a modulation mainly concerns migration-mediated cytokine and chemokine receptors as well as some adhesion molecules and partly results from MNC specificity. These phenotypic profiles are associated with distinct cell-cycle position, cloning efficiency, and migration capacity of CD34+ cells from the different anatomical sources. We therefore propose a definition for a circulating versus resident CD34+ cell profile, which mostly depends on their cellular environment. We suggest that blood would represent a supply of cells for which phenotypic and functional characteristics would be a prerequisite for their bio-availability.

Key Words: mononuclear cells • CD34+ progenitors • unmobilized peripheral blood • bone marrow • adhesion molecules • chemokines • cytokines • receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD34+ hematopoietic progenitors (HP) from various sources such as bone marrow (BM), cord blood, and mobilized peripheral blood (mPB) are currently used for hematopoietic transplantation. Their presence in low number in the PB from normal, unmobilized subjects demonstrates their continuous circulation under physiological conditions [1 ]. Unlike the numerous papers reporting data on HP from mPB, few groups have searched for the existence of a potential relationship between the phenotypic and functional characteristics of unmobilized circulating HP and those of their medullar sedentary counterparts [2 3 4 5 ]. Recent papers have underlined the crucial role of membrane receptors for cytokines and chemokines as well as of adhesion molecules in the circulation of hematopoietic cells [5 6 7 ]. Among these receptors, a key participation of the CXC chemokine receptor 4 (CXCR4) in the migration/homing of HP has been reported [8 ]. CXCR4 is differentially expressed on resident BM and circulating CD34+ cells, and its expression is clearly modulated by environmental cells [9 10 11 ]. The versatility of its expression prompted us to analyze the modulation of receptors involved in cell migration/proliferation, to compare the cycle and activation status, the cloning efficiency, and the migration capacity of CD34+ cells according to their BM versus PB localization, and to look for the contribution of environmental mononuclear cell (MNC) interactions in this process. Our results show for the first time a relationship between phenotypic and functional profiles and the resident/circulating status of steady-state (SS) CD34+ progenitors. We suggest that the phenotypic and functional versatility of progenitors could be a prerequisite for their bio-availability, allowing them to respond rapidly to environmental signals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell sample collection
Unmobilized PB samples were obtained from 400 healthy adult donors. MNC-enriched buffy coats (60 ml/sample) were prepared by centrifugation at 2300 g for 20 min at 20°C. BM cells were collected from 20 normal donors undergoing hip prothesis surgery and were recovered using a Potter grinder and several washes with RPMI 1640 [9 ].

All samples were obtained with informed consent from donors at the "Centre de Transfusion Sanguine des Armées Jean-Julliard" (Clamart, Cedex, France), the "Services d’Hématologie Clinique et de Chirurgie Orthopédique de l’Hôpital d’Instruction des Armées Percy" (Clamart, Cedex, France), and the "Service de Chirurgie Orthopédique du CHR d’Aulnay-Sous-Bois" (France).

Immunomagnetic purification of CD34+ cells
PB MNC (PBMC) and BM MNC were isolated individually by centrifugation on a Ficoll density gradient (d=1.077 g/ml; Seromed, ATGC Biotechnologies, Marne la Vallée, France) at 700 g for 30 min at 20°C. MNC expressing the CD34 antigen were immunomagnetically selected directly after density gradient separation (SS) or after an overnight incubation (18–20 h; Inc.+; Fig. 1 ). For that purpose, the BM MNC or PBMC fraction (5x106 cells/ml) containing accessory cells was incubated in Iscove’s modified Dulbecco’s medium (IMDM) containing 2% human serum albumin (HSA) in 75 cm2 plastic culture flasks (Falcon, Becton Dickinson, Le Pont de Claix, France) at 37°C in a 5% CO2/95% air atmosphere. In some experiments, we used untreated culture adhesion-free flasks (Iwaki flask, ATGC Biotechnologies) to analyze the role of cell adhesion on the plastic surface. Nonadherent cells, collected by washing the flask three times with phosphate-buffered saline (PBS)/HSA or MNC, recovered directly after Ficoll centrifugation, were incubated for 15 min at 4°C with 50 µl blocking reagent [human immunoglobulin G (IgG)] and 50 µl CD34 antibody (QBEND/10, mouse IGg1) per 108 cells. Cells were washed once in PBS/5 mM EDTA/0.5% HSA and incubated for 15 min at 4°C with colloidal magnetic cell sorter (MACS) microbeads (50 µl/108 cells) directed against the haptenized QBEND/10. The labeled cells were separated individually by using an AutoMACS® system (Miltenyi Biotec, Auburn, CA). Cell number and viability (>97%) were assessed with a hemacytometer and Trypan blue. For the SS and Inc.+ procedures, MACS purification produced a 95–98% pure CD34+ cell preparation. The SS and Inc.+ CD34+ cells accounted for 0.2 ± 0.05% and 0.18 ± 0.07%, respectively, of the low-density PB cells and for 1.3 ± 0.8% and 1.1 ± 0.12% of the low-density BM cells. A pool of CD34+ cell samples from eight buffy coats was used for PB experiments, whereas BM CD34+ cells were individually processed.



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  		Figure 1. Typical procedure for PB CD34+ cell purification. A typical technical procedure used to purify CD34+ cells from unmobilized PB is shown. A similar procedure was carried out for BM samples. A total of 420 samples from PB and BM was processed, respectively. Each PB experiment was performed on a pool of eight CD34+ cell samples, whereas BM experiments were performed on individual CD34+ cell samples.

 
CD34+ cell cultures
In some experiments, to analyze the specific role of MNC from PB or BM source on CD34+ cell phenotype, SS CD34+ cells from PB or BM were cross-cultured for 12 h with the supernatant of BM MNC or PBMC (5x106 cells/ml), obtained after an overnight incubation.

To study the involvement of diffusible cytokines released from MNC, we also performed Transwell assays in which CD34+ cells and MNC were separated by a 0.2-µ pore membrane and overnight-cultured in RPMI medium.

Immunophenotyping and flow cytometry analysis
Two- or three-color flow cytometry assays were performed using an ELITE flow cytometer (Beckman Coulter/Immunotech, Roissy CDG, France) and Expo 32 data acquisition software (Beckman Coulter/Immunotech). The instrument was calibrated using beads (Beckman Coulter/Immunotech) according to the manufacturer’s instructions.

To avoid nonspecific antibody binding, we have saturated the Fc receptors by incubating cells in a flow cytometry PBS buffer containing 0.5% polyvalent human Ig and 2% HSA. Cells (5x104 cells/40 µl PBS/0.5% HSA) were then incubated in 96-well culture plates containing 10 µg/ml (saturating concentration) allophycocyanin (APC)-conjugated anti-CD34 (8G12) monoclonal antibody (mAb) and fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated mAb against receptor antigens (Table 1 ). For Tpo receptor (TpoR) detection, we used a biotin-conjugated Tpo revealed by a streptavidin-PE. The receptor profiles were also characterized on CD34+CD38– cells by using a FITC- or PE-conjugated mouse anti-human CD38 (T16). Incubations were performed on ice for 20 min in the dark and were followed by two washes with ice-cold PBS/0.5% HSA. Immunostained cells were immediately analyzed by flow cytometry. For each sample, images from 1 x 104 cells were acquired in listmode. Forward- and side-scattering (FSC and SSC, respectively) and two to three fluorescence signals were stored in listmode data files and analyzed on a computer with WinMDI software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA). The cell phenotype was studied by constructing a gate for living CD34+ cells (R1) excluding CD19+ B lymphoid precursors (R0), debris, and autofluorescent cells on a FSC/SSC plot (Fig. 2 ). Total CD34+ cells (R2) and CD34+CD38– cell subset (R3) were selected for receptor profile analyze. The percentage of CD34+ cells that reacted with the different antibodies was calculated by comparison with each isotype control. Positive cells were defined by a marker set on the negative isotype control. When receptors were expressed weakly, the specificity was attested by a PE amplification procedure using a polyclonal rabbit antibody anti-PE (Biogenesis, Valbiotech, Paris, France) revealed by a PE-conjugated donkey anti-rabbit antibody (Jackson Immunological Research, Tebu-bio). In multiple staining, compensation was adjusted using the single-stained cell samples. Mean fluorescence intensity (MFI) was expressed in arbitrary units (AU). With the Beckman Coulter/Immunotech flow cytometer, MFI that ranged from 1 to 10 and from 10 to 100 corresponds to the second and the third log of the dot plot scale, respectively.


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  		Table 1. Antibodies Used to Phenotype PB and BM CD34+ Cells

 


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  		Figure 2. Flow cytometry procedure for analyzing CD34+ (R2) and CD34+CD38– (R3) receptor profiles. BM and PB CD34+ cells were purified immediately after density gradient separation (SS) or after incubation in the presence of MNC (Inc.+). Purified cells were stained with CD34-APC and CD38-FITC antibodies and analyzed by two-color cytometry. An R1 region was first drawn for excluding dead cells and BM CD34+CD19+ B lymphoid precursors (R0) on a FSC/SSC dot plot. A second region (R2), corresponding to total CD34+ cells, was drawn on a second CD34-APC/CD38-FITC dot plot, gated on R1. A third region (R3), corresponding to the CD34+CD38– cell subset, was further drawn. The arbitrary quadrants were drawn based on isotype-matched, negative control profiles.

 
Erythroid [burst-forming units-erythroid (BFU-E)], granulocytic [colony-forming units-granulocytic (CFU-G)], CFU-monocytic (CFU-M), CFU-granulo-monocytic (CFU-GM), and CFU-multilineage (CFU-Mix) colony assays
Progenitors were assessed in methylcellulose medium as described previously [9 ]. Briefly, Inc.+ or Inc.– CD34+ cells freshly purified from PB or BM (500 cells/ml) were plated on methylcellulose culture medium containing a cocktail of cytokines (Stem {alpha} ID, Stem Alpha, St. Clément les Places, France). Duplicate 35 mm2 tissue-culture dishes containing 1 ml cell suspension were incubated at 37°C in a 4.5% CO2/95.5% air atmosphere. BFU-E, CFU-G, CFU-M, CFU-GM, and CFU-Mix were scored on day 14 using an inverted microscope.

In vitro CD34+ migration assays in Boyden chambers
Analysis of cell migration was performed by seeding 1 x 105 SS or Inc.+ CD34+ from PB or BM in the top compartment of a Boyden chamber. The bottom compartment contained 125 ng/ml stromal cell-derived factor-1 (SDF-1) in RPMI 10% fetal calf serum and was separated from the top compartment by a membrane with 5 µm pores. Cells were allowed to migrate for 4 h at 37°C in humidified atmosphere, and migrated cells present in the bottom compartment were numbered.

Cell-cycle fractionation with propidium iodide (PI) and Ki67
CD34+ cell-cycle phases were discriminated by analyzing simultaneous Ki67 antigen expression and DNA content as described previously [10 , 12 ]. Cells (1x105) were fixed in 70% ice-cold ethanol, permeabilized using PermaCyte-FPTM (BioErgonomics, CliniSciences, Montrouge, France), and then immunostained using FITC-labeled anti-Ki67 (Beckman-Coulter/Immunotech) or its isotype IgG control. After two washes in PermaCyte-FPTM, cells were stained with PI. The cell histogram FL-2 was divided into three regions according to cell-cycle phase: G0, G1, [S+G2/M]. Doublets were eliminated by gating on a peak/area plot of PI fluorescence. The data were analyzed with WinCycle software (Phoenix Flow Systems, San Diego, CA). Specificity of the Ki67 antibody binding was evaluated by using a FluoroTrolTM-DNAplus cell-cycle control kit (BioErgonomics, CliniSciences).

Cytokine bead array (CBA®) assay
The cytokine production occurring during the overnight incubation of the PBMC or BM MNC fraction prior to CD34+ separation was quantified in MNC supernatants by using the Inflammation Human BDTM CBA kit, according to the manufacturer’s instructions (Becton Dickinson). The human inflammation CBA assay used a series of particles with discrete fluorescence intensities to simultaneously detect and quantify by flow cytometry multiple cytokines such as IL-8, IL-1ß, IL-6, IL-10, TNF-{alpha}, and IL-12. The data files were analyzed using BD® CBA calibration and analysis software. Cytokine concentrations were determined based on known concentration values in a set of calibration files.

Statistical analysis
Data were expressed as means ± SD. The significance of differences between groups was determined by Student’s t-test for paired samples. A P value <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differential membrane receptor expression of CD34+ cells according to their circulating or BM resident status
We first explored the membrane expression of migration-mediated cytokine and chemokine receptors, adhesion molecules, and hematopoietic-related cytokine receptors on SS CD34+ cells according to their circulating (SSPB) versus resident BM (SSBM) status.

Whereas the expression levels of cytokine receptors were globally low, the percentages of expressing cells were distinct between SSPB and SSBM CD34+ progenitors. The majority of migration-mediated cytokine receptors, such as c-Kit, GM-CSFR{alpha}, and G-CSFR, was less expressed in PB than in BM (Fig. 3I ). This differential expression concerns the percentage of expressing cells and the MFI. Whatever the source of HP, most of the chemokine receptors were expressed at a lower level than those of cytokines (Fig. 3 I-IV) . Especially, the percentage of CXCR4-expressing cells was lower in SSPB than in SSBM CD34+ cells (8.8%±3.6 vs. 56%±12.3), as described previously [9 ]. In contrast, we detected a higher expression on SSPB than SSBM CD34+ cells of receptors to inflammatory cytokines such as IFN-R{alpha}/IFN-Rß (39.9%±7.6/17.6%±1.7 vs. 11%±6.2/11.9%±3.7), TNF-RI/TNF-RII (35.8%±10.8/31.9%±12.3 vs. 10.4%±8.7/12.4%±7.6), and CCR1 (40.1%±11.5 vs. 3.3%±1.3; Fig. 3 III-IV ). Adhesion molecules of the integrin family were also heterogeneously expressed between SSBM and SSPB CD34+ cells (Fig. 3V) . In contrast to CD49f({alpha}6) and CD18(ß2), which were less expressed in SSBM (2.6%±0.9; 6.8%±1.4, respectively) than in SSPB CD34+ cells (21.6%±9.2; 52%±20, respectively), CD29(ß1), CD49d({alpha}4), CD49e({alpha}5), CD11a({alpha}L), as well as CD44 were more expressed in SSBM (74.8%±12.6; 84.1%±16.1; 78.8%±19.1; 99.2%±15.5; 79.5%±12.1, respectively) than in SSPB CD34+ cells (40.1%±5.4; 56.5%±9.5; 59.5%±8.8; 59.7%±11.2; 58.6%±5.4, respectively).



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  		Figure 3. Cytokine, chemokine, and adhesion receptor profiles on CD34+ cells according to their resident/circulating SS status. A graphic representation of flow cytometry analysis of cytokine, chemokine, and adhesion receptor expression by CD34+ cells is shown. CD34+ cells were purified from BM or unmobilized PB at SS. After purification, CD34+ cells were labeled with antibodies against cytokine and chemokine receptors or adhesion membrane molecules, as described in Table 1 . Results are expressed as percentage of expressing cells and as MFI expressed in AU (mean±SD). The results shown are based on 20–50 independent experiments. *Significant difference in the receptor expression between BM and PB CD34+ cells; P < 0.05.

 
Incubation in the presence of MNC specifically modulates the membrane receptor expression of CD34+ cells
The differential expression of migration-mediated cytokine and chemokine receptors as well as of adhesion molecules between SSPB and SSBM CD34+ cells led us to investigate whether interactions occurring within the cellular microenvironment could influence the surface receptor expression. Therefore, we analyzed the phenotypic profile of BM and PB CD34+ cells that were purified after an overnight incubation with their counterpart MNC (Inc.+; Fig. 4A and 4B ). We showed that such a procedure greatly increased the expression of migration-mediated cytokine receptors (c-kit, GM-CSFR{alpha}, and G-CSFR) on Inc.+ PB CD34+ cells (66.9%±2.4, 18.3%±2.5, and 17.2%±2.7, respectively) as compared with SSPB CD34+ cells (14.2%±4, 13.4%±2.4, and 8.4%±2.4, respectively; Figs. 3 and 4 , group I). As described previously for CXCR4 [9 , 11 ] most of the chemokine receptors were also up-regulated in Inc.+ PB CD34+ cells as compared with SSPB CD34+ cells (Figs. 3 and 4 , group IV). Similarly, the expression of CD49d, CD49e, CD11a, CD29, and CD44 adhesion molecules was increased strikingly in Inc.+ PB CD34+ cells (80.5%±20.4; 88.2%±16.3; 95.5%±10.1; 71.2%±6.3; 90.1%±10.5, respectively; Fig. 4 , group V) as compared with SSPB CD34+ cells (56.5%±9.5; 59.5%±8.8; 59.7%±11.2; 40.1%±5.4; 58.6%±5.4, respectively; Fig. 3 , group V).



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  		Figure 4. Modulation of cytokine, chemokine, and adhesion receptor phenotypic profile on BM or PB CD34+ cells after an overnight incubation in the presence of MNC. A graphic representation of flow cytometry analysis of cytokine, chemokine, and adhesion receptor expression by CD34+ cells is shown. CD34+ cells were purified from BM or unmobilized PB after an overnight incubation (Inc.+) in the presence of accessory MNC from BM (A and D) or PB (B and C). After purification, CD34+ cells were labeled with antibodies against cytokine and chemokine receptors or adhesion membrane molecules as described in Table 1 . Results are expressed as percentage of expressing cells and as MFI expressed in AU (mean±SD). The results shown are based on 10–50 independent experiments. *, Significant difference in the receptor expression between SS and Inc.+ CD34+ cells; P < 0.05.

 
We also studied the influence of an overnight incubation in the presence of accessory MNC on the expression profile of CD34+CD38– cells from PB and BM. We found that except for CXCR4, this incubation process induced a similar up-regulation of migration-mediated cytokine and chemokine receptors than the one observed on total CD34+ cells (data not shown; ref. [9 ]).

We further explored the potential causative role of MNC from different origins (PB or BM) in the change of HP expression phenotype by crossed cultures in which PB CD34+ cells were incubated with BM MNC and vice versa. We showed that whatever the anatomical source of CD34+ cells, their surface receptor expression was influenced mostly by exposure to PBMC. Actually, in contrast to results showed in Figure 4B , incubation of PB CD34+ cells with BM MNC did not increase the expression of c-Kit, Flt3, CXCR4, CXCR3, and CD11a (Fig. 4B and 4D) . In the same way, incubation of BM CD34+ cells with PBMC inversed the modulation described for c-Kit, CXCR3, and CD11a (Fig. 4A and 4C) . It is interesting that the expression of CXCR4 by CD34+ BM was more up-regulated when incubated in the presence of PBMC than in the presence of BM MNC.

Involvement of adhesion and of cytokines produced by MNC in the membrane receptor modulation on PB CD34+ cells
The role of diffusible factors produced by accessory MNC was analyzed by Transwell assays. We showed that up-regulation of c-Kit (4.7%±0.6–55.9%±3.5; P≤0.05) and CXCR4 (12.1%±3.6–55.6%±3.7; P≤0.05) was observed when purified CD34+ cells were in contact with diffusible growth factors such as IL-6 and stem cell factor. Our results, demonstrating that PBMC or BM MNC differentially produced cytokines during the overnight incubation, strengthened this hypothesis. Actually, supernatants obtained from the PBMC overnight incubation contained higher levels of IL-8, IL-1ß, IL-6, IL-10, and to a lesser extent, TNF-{alpha} than BM MNC supernatants (Fig. 5 ). The significant c-kit, GM-CSFR, and CXCR4 overexpression, observed when SSPB CD34+ cells were incubated overnight in the presence of IL-6, IL-8, and TNF-{alpha} after their immunomagnetic purification, further confirmed the involvement of at least these growth factors in such a modulation process (Table 2 ).



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  		Figure 5. Cytokine production by MNC during overnight incubation. A representative CBA dot plot shows the cytokine production (IL-8, IL-1ß, IL-6, IL-10, TNF-{alpha}, and IL-12) by MNC isolated from BM or PB. MNC were overnight-incubated at 37°C prior to supernatant harvesting. Results are expressed as MFI values assigned to the FL2 histogram and transformed by the software into concentration values in pg/ml (n=3). *Significant difference between BM and PB values; P < 0.05.

 

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  		Table 2. Cytokine Treatment Increases Receptor Expression on SSPB CD34+ Cells

 
We also analyzed the role of cell adhesion in the modulation of PB CD34+ cell receptor expression by performing an overnight incubation on adhesion-free plastic support prior to the CD34+ cell purification. In these experimental conditions, c-kit and CXCR4 were not up-regulated, demonstrating that the adhesion process participated in the receptor modulation as well (data not shown).

Relationship between cell-cycle position, receptor expression, and the resident (BM)/circulating (PB) status of CD34+ cells
We wondered whether cellular environment could influence cell cycling and activation and whether receptor expression modulation was related to cell-cycle position. We showed that a 4°C overnight storage maintained SSPB CD34+ cells in G0. In contrast, a 37°C incubation of MNC prior to CD34+ cell purification (Inc.+) raised the percentage of SSPB CD34+ cycling cells (Fig. 6A ) and significantly increased their global tyrosine phosphorylation level (48.5±3.5 AU vs. 105.5±13.5 AU; P≤0.005). These results are in favor of a relationship among cellular environment, activation, and cell cycling. The higher proportion of cycling cells [10 ] (Fig. 6A) and tyrosine phosphorylation level in resident BM (180±20 AU) as compared with circulating CD34+ cells (40±10 AU) reinforced this assumption. By using Ki67 staining to discriminate G0 from G1 + S/G2M phases, we demonstrated that G1 + S/G2M Inc.+ PB CD34+ cells expressed the c-kit, GM-CSFR, G-CSFR, and CXCR4 (Fig. 6B) . This suggest that the receptor overexpression observed in Inc.+ PB CD34+ cells could be related to their characteristic cell-cycling induction after an overnight incubation (Fig. 6A) .



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  		Figure 6. Relationship among cell-cycle position, phenotype, and resident/circulating status of CD34+ cells. (A) PB and BM CD34+ cells were processed for cell-cycle fractionation by using simultaneous staining for DNA content (PI) and for Ki67 expression, as indicated in Materials and Methods. Results are based on four independent experiments and expressed as mean ± SD percentage of cells in cell-cycle phases. *P < 0.05 versus Inc.+ PB. (B) Cell-cycle status of purified Inc.+ PB CD34+ cells, coexpressing cKit, GM-CSFR, G-CSFR, or CXCR4, was determined by using a dual staining for Ki67 expression and for each receptor expression. A graphical representation of three independent experiments shows the MFI (mean±SD) of positive cells for cKit, GM-CSFR, G-CSFR, and CXCR4 according to their cell-cycle position. *, P < 0.05, versus G0 Inc.+ PBCD34+.

 
CD34+ HP exhibit functional differences in terms of clonogenicity and migration capacity according to their circulating versus resident status
We further analyzed the clonogenicity and migration capacity of CD34+ cells according to their PB or BM origin. We showed that the plating efficiency of SS CD34+ cells was higher in BM than in PB (Table 3 ). It is interesting that we observed a different proportion of erythroid (54±7 vs. 27±5, respectively) and granulo-macrophagic (23±3 vs. 91±4, respectively) colonies between SSPB and SSBM. After incubation in the presence of MNC, the global plating efficiency increased in similar proportion in BM or PB CD34+ cells (Table 3) .


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  		Table 3. Plating Efficiency of CD34+ Cells According to Their Resident (BM)/Circulating (PB) Status

 
At SS, the migration capacity of CD34+ cells was higher when purified from BM than from PB (Fig. 7 ). On the contrary, after incubation in the presence of accessory MNC, the percentage of CD34+ migrating cells was significantly increased in Inc.+ PB as compared with Inc.+BM (Fig. 7) .



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  		Figure 7. Migration capacity of CD34+ cells according to their resident (BM)/circulating (PB) status. SS and Inc.+ CD34+ cells from PB or BM were isolated as described previously in Materials and Methods. In vitro migration assays were performed by seeding 1 x 105 cells in the top compartment of the Boyden chamber, in which the bottom compartment contained 125 ng/ml SDF-1. The number of migrating cells after 4 h incubation at 37°C was evaluated by hemacytometer count. Results are expressed as mean ± SD percentage of migrating cells. *, Significant difference between SSBM and SSPB cells; P < .05; **, significant difference between Inc.+ PB and SSPB cells; P <.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous results [9 ] showing the differential expression of CXCR4 between medullar and peripheral CD34+ cells incited us to analyze the implication of anatomical distribution on the phenotypic and functional status of CD34+ cells. With the aim to define an identity card of circulating versus nested medullar CD34+ cells, we first compared the expression of cytokine and chemokine receptors as well as of adhesion molecules between SSPB CD34+ cells and their medullar counterparts. In the present study, we showed that the main receptors involved in HP trafficking, such as CXCR4, c-kit, GM-CSFR, and G-CSFR, were weakly expressed on SSPB CD34+ cells as compared with SSBM CD34+ cells. We demonstrated that this low expression was associated with a reduced responsiveness to SDF-1, which is in agreement with their circulating status [13 ]. Incubation in the presence of MNC induced an overexpression of migration-mediated cytokine receptors on PB Inc.+ CD34+ cells correlated with a higher proportion of migrating CD34+ cells. In contrast, in BM CD34+ cells, neither the expression of migration-related receptors nor the migration capacity was modified by incubation with MNC. All together, these results confirm the relationship between migration capacity and related receptor expression and suggest that versatility of their expression could affect the CD34+ circulating status.

The differential expression of adhesion molecules that we demonstrated between circulating and medullar CD34+ cells is also in agreement with their role in HP homing and nesting processes [14 , 15 ]. Actually, apart from CD49f({alpha}6) and CD18(ß2), the majority of adhesion molecules such as CD29(ß1), CD49d({alpha}4), CD49e({alpha}5), CD11a({alpha}L), as well as CD44 was expressed higher in SSBM than in SSPB CD34+ cells. It is interesting that under G-CSF-stimulation, the reversible down-regulation of the CD49d integrin participated in mobilization and homing of CD34+ cells [16 ]. In a recent paper, Lapidot and co-workers [17 ] reported that CD44 and its hyaluronic acid ligand cooperated with SDF-1 in the trafficking of CD34+ cell to the BM, demonstrating a cross-talk between CD44 and CXCR4. Our results showing that like CXCR4, CD44 is expressed lower in percentage and expression level in SSPB CD34+ cells than in SSBM counterparts and that both receptors are similarly up-regulated in Inc.+ PB CD34+ cells reinforce the cooperative role of these molecules in the HP homing/circulating process.

Concerning the expression of hematopoietic-related cytokine receptors, the marked difference of IL-3R, EpoR, and G-CSFR{alpha} expressions observed between SSPB and SSBM CD34+ cells may explain, at least partially, their differential BFU-E and CFU-GM clonogenicity [18 ]. The higher expression of receptors to inflammatory cytokines such as IFN-R{alpha}/IFN-Rß, TNF-RI/TNF-RII, and CCR1 on SSPB, as compared with SSBM CD34+ cells, tallies with the need for a rapid mobilization to the inflammation sites where their ligands are produced.

It is interesting to note that whatever the source of cells, the chemokine receptor expression was mostly lower than those of cytokines. In contrast to the exponential curve of cytokine biological activity, the specific bell-shaped curve of the chemokine response that is associated with the desensitization regulation mechanism of their receptors could account for such a low expression [9 ].

Altogether, these results led us to suggest that humoral and cellular interactions related to the anatomical environment could participate in the distinct phenotypic and functional pattern between circulating PB and resident BM progenitors. Actually, several and nonexclusive hypotheses could explain such distinct patterns; a difference in the maturation level between PB and BM cells can be evoked [19 ]. Our results, demonstrating that whatever the anatomical source of progenitors the phenotype of primitive CD34+CD38– cells behaved like more mature CD34+ cells do not support this hypothesis. A second hypothesis lies on a difference in cell cycling and activation level between PB and BM CD34+ cells. Our previous results demonstrating that the percentage of cycling cells was higher in BM than in PB CD34+ cells egged us on to analyze the influence of cell environment on HP cycling [10 ]. We showed that in contrast to a 4°C overnight storage that maintained SSPB CD34+ cells in G0, a 37°C incubation of MNC prior to CD34+ purification (Inc.+) significantly raised the percentage of SSPB CD34+ cells into cycle. Such an increase was not observed on CD34+ cells from SSBM, which were already activated and in cycle. The higher proportion of cycling cells [10 ] and tyrosine phosphorylation level, which we observed in SSBM as compared with circulating CD34+ cells, is in agreement with these results. It has been reported that quiescent CD34+ cells adhered less efficiently to stroma than cycling cells, likely contributing to their higher circulation [3 ]. Such a superior migratory ability of G0/G1 CD34+ cells may have important implication for their homing and engraftment [20 ]. A disparity within PB versus BM accessory MNC, which could account for the different expression profile between BM and PB CD34+ cells, can be proposed as a third hypothesis. Actually, upon incubation in the presence of autologous MNC, CD34+ cells and especially PB CD34+ cells exhibited marked receptor expression modifications as compared with SS CD34+ cells. These modulations mainly occurred when PB CD34+ cells were incubated in the presence of autologous MNC, demonstrating a potent influence of PBMC in this process. The higher growth factor levels detected by CBA in PBMC as compared with BM MNC supernatants could account for the superior stimulatory effect of PBMC. Beside the effect of adhesion process and of cell contact, these data suggest that growth factors produced by MNC within a specific environment play a major role in the versatility of the CD34+ cell profile.

In conclusion, our results suggest a relationship between the phenotypic profile and functional properties of CD34+ cells and their circulating (PB)/resident (BM) status. Our study, which is the first one to be performed on CD34+ cells from unmobilized SSPB, reinforces recent results from Steidl et al. [21 ], obtained with mPB, and extends the understanding of the physiology of stem cell circulation and highlights the significance of the versatility of membrane receptor expression in this process. Circulation by itself would not be a mode of transport exclusively but might represent a cellular compartment in which quiescent progenitors are preserved from stromal-derived proliferation and differentiation signals. We therefore propose a definition for a circulating cell profile and suggest that blood may represent a supply of cells for which phenotypic and functional versatility would be a prerequisite for their bio-availability. Such a proposal could be of relevance in view of the debate surrounding the stem-cell plasticity concept.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the DSP (Direction des Systèmes de Forces et de la Prospective, Direction Générale à l’Armement, Ministère de la Défense, No. 99CO032) and the ANRB (Association Nouvelles Recherches Biomédicales). We are indebted to Dr. Loïc Niel, Pr. Sylvain Rigal, and Dr. Yann Masse for continuously supplying BM and blood samples from normal subjects.

Received May 5, 2004; revised December 22, 2004; accepted December 29, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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