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Originally published online as doi:10.1189/jlb.0603287 on November 21, 2003

Published online before print November 21, 2003
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(Journal of Leukocyte Biology. 2004;75:314-323.)
© 2004 by Society for Leukocyte Biology

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

Yuk Yin Ng*,{dagger}, Berris van Kessel*, Henk M. Lokhorst*, Miranda R. M. Baert{dagger}, Caroline M. M. van den Burg{dagger}, Andries C. Bloem* and Frank J. T. Staal{dagger},1

* Departments of Hematology and Immunology, University Medical Center Utrecht, The Netherlands; and
{dagger} Department of Immunology, Erasmus University Medical Center Rotterdam, The Netherlands

1 Correspondence: Department of Immunology, Erasmus University Medical Center Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: f.staal{at}erasmusmc.nl


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ABSTRACT
 
The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34+ cells and PBSC significantly differ from BM CD34+ cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34+ cells, and CD34+ PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34+ cells contained the highest frequency of CAFCwk6 (3.6- to tenfold higher than BM CD34+ cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34+, with a higher proportion of CD45+ cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34+ SC and 64 genes between BM CD34+ cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34+ HSC and leads to a better understanding of the discrepancy among the SC sources.

Key Words: peripheral blood stem cell • G-CSF • umbilical cord blood • bone marrow • microarray


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INTRODUCTION
 
Hematopoiesis is sustained by a small compartment of hematopoietic stem cells (HSC) and committed progenitors cells. It is a tightly regulated process, which involves self-renewal, proliferation, and differentiation of CD34+ HSC, which have the unique capacity to restore hematopoiesis after bone marrow (BM) ablation [1 ]. CD34+ cells are often used for SC transplantation to support high-dose chemotherapy for various malignancies and are considered as ideal targets for gene therapy. Additionally, HSC may be capable of differentiating into nonhematopoietic cells such as vascular endothelial cells, hepatocytes, and cardiomyocytes, although this is a controversial issue [2 3 4 5 6 ]. Therefore, CD34+ cells may serve as a therapeutic tool for the treatment of hematological and perhaps of nonhematological disorders. To date, three different SC sources are used clinically: CD34-enriched cells from BM, umbilical cord blood (UCB), and granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood SC (PBSC).

Conventionally, BM has been used as the main source of CD34+ SC, but currently, UCB and PBSC are frequently being used as alternative sources of SC. Clinically, it has been demonstrated that in addition to providing more rapid recovery of neutrophils and platelets, an increase of incidence of graft-versus-host disease (GVHD) was observed with PBSC grafts [7 , 8 ]. In contrast, UCB transplantations are associated with low severity of GVHD, but compared with PBSC or BM grafts, the hematological recoveries are often delayed [9 ]. The delayed engraftment after UCB transplantations is probably a result of the limited number of total CD34+ stem/progenitor cells present in the (partially purified) grafts, which are necessary for rapid hematological recovery, not as a result of the maturation time of SC. However, it is well established that UCB contains the largest percentage of the most immature, pluripotent CD34+CD38- SC, a subset with highly enriched, true SC activity; reacts more strongly to in vitro cytokine stimulation; and produces more progeny cells than its adult counterparts [10 , 11 ]. Furthermore, in vivo studies have demonstrated that the engraftment capacity of UCB CD34+ was significantly greater than CD34+ SC from BM and PBSC [12 ]. Indeed, the frequency of severe combined immunodeficiency (SCID)-repopulating cells is highest in UCB and lowest in PBSC [13 , 14 ].

The molecular basis for the different biological properties of CD34+ cells from various SC sources is poorly understood. Characterization of gene-expression profiles of CD34+ cells might lead to a better understanding the discrepancy among these SC sources and more importantly, to elucidate the regulation of normal and pathological hematopoiesis [15 16 17 ]. Several studies on the gene-expression profile of human CD34+ cells have been performed and mostly focus on a limited number of genes [18 19 20 ]. Recently, Steidl et al. [21 ] and Graf et al. [22 ] have mapped the gene-expression profile of CD34+ from PBSC and BM using cDNA microarray technology. Both groups showed that PBSC CD34+ cell SC sources significantly differed in expression of genes involved in cell adhesion, cell cycle, and apoptosis. A large-scale comparison of the gene-expression profile among all clinically relevant SC sources (BM, UCB, PBSC) has not yet been described.

In the present study, we investigated functional differences among the BM, UCB CD34+ SC, and PBSC in two established assays for primitive progenitor cells: the in vitro cobblestone area-forming cells (CAFC) assay and the in vivo nonobese diabetic (NOD)/SCID repopulation assay. In attempt to explain the functional differences observed, a comparative analysis of a gene-expression profile using Affymetrix microarray technology was performed to translate the differences into the molecular phenotype among BM, UCB CD34+ SC, and PBSC.


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MATERIALS AND METHODS
 
Isolation of CD34+ cells from different BM, PBSC, and cord blood
UCB (n=10) and BM (n=5) samples were obtained from informed and consenting healthy donors. G-CSF-mobilized PBSC (n=5) samples were obtained from harvests for allogeneic SC transplantations. Mononuclear cells from all samples were isolated by density centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). CD34+ cells were enriched using the magnetic cell sorter CD34+ progenitor cells (Miltenyi Biotech, Bergisch-Gladbach, Germany), according to the manufacturer’s instructions. The purity of the enrichment was determined by staining with monoclonal antibodies (mAb) against CD34-fluorescein isothiocyanate (FITC; HPCA-2, Becton Dickinson, Mountain View, CA), CD38-phycoerythrin (PE; HB-7, Becton Dickinson), and CD45-PE-Cy5 (IMMU19-2, Coulter, Miami, FL). Flow cytometry was performed on the FACSCalibur and was analyzed with the CellQuest software (Becton Dickinson). For microarray experiments, we have performed two experiments with isolated CD34+ cells from different donors. Thus, duplicate biological samples were used and separately hybridized to the arrays.

CAFC assay
Freshly isolated CD34+ SC were cultured at limiting dilution in the CAFC assay as described previously [10 , 24 ]. Briefly, confluent monolayers of the FBMD-1 cell line were established in 96-well plates (precoated with 0.2% gelatin) in CAFC medium consisting of Iscove’s modified Dulbecco’s medium supplemented with 5% fetal calf serum (FCS), 20% horse serum, 10-5 M hydrocortisone 21-hemisuccinate (Sigma Chemical Co., St. Louis, MO), and Pen/Strep. Twelve dilutions (15 replicate wells per dilution) were seeded on FBMD-1. The CD34+ cells were cultured in CAFC medium in the presence of 10 ng/ml interleukin (IL)-3 (PeproTech, Rocky Hill, NJ) and 20 ng/ml G-CSF (Amgen, Thousands Oaks, CA). The number of CAFC was determined at 14 days (CAFCwk2) and 42 days (CAFCwk6) after culture.

In vivo repopulation assay with NOD/SCID mice
Male NOD/SCID mice were a kind gift from Dr. Robbert Benner from the Department of Immunology, Erasmus University Medical Center, Rotterdam, The Netherlands. Mice were maintained at the shared animal facility at the University of Utrecht (The Netherlands) under sterile and air-filtered conditions. Ten- to 12-week-old mice were sublethally irradiated (350 cGy) and transplanted with CD34+ cells. CD34+ cells (105–1x106 per mouse) were injected intravenously via lateral tail vein.

Thirty-five days after transplantation, mice were killed by cervical dislocation. Before, PB was obtained by heart puncture and collected in heparin-containing microtubes. Erythrocytes were depleted by treatment with lysis buffer containing of NH4Cl (8.3 g/L) and KHCO3 (1 g/L) on ice for 5 min and were immediately washed twice with RPMI medium supplemented with 10% FCS. Single-cell suspension was made from spleen using open chamber filter (NPBI, The Netherlands). BM cells were collected by flushing femora with RPMI medium and were washed twice with 1% FCS/phosphate-buffered saline (PBS). The phenotype of the cells was determined by staining with mAb against the following antigens: CD4, CD8, CD10, CD15, CD19, CD20, CD33, CD34, CD38, CD42a, CD45, and human leukocyte antigen (HLA)-DR (Becton Dickinson); CD3 and CD14 (Immunotech, Marseille, France); and Ery-1 (Central Laboratory of the Netherlands, Amsterdam). As controls, FITC-, PE-, peridinin chlorophyll protein-, and antigen-presenting cell-conjugated mouse immunoglobulin G (IgG)1 and IgG2b were used. Flow cytometric analysis was performed on the FACSCalibur and analyzed with CellQuest software (Becton Dickinson).

Total RNA isolation
Total RNA of SC was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA), according to the manufacturer’s instruction. In brief, 10 x 106 CD34+ SC were pelleted, lysed in RLT buffer containing 1% ß-mercaptoethanol, and passed through a QiaShredder (Qiagen) to prevent cell clumps. RNA was quantified by spectrophotometric analysis at 260 nm and 280 nm, and the quality of all RNA samples was assessed by electrophoresis on a 1.2% agarose gel containing formaldehyde/formamide.

cDNA synthesis, in vitro transcription, hybridization, washing, staining, and scanning of Affymetrix microarrays
All procedures were performed according to the standard protocol for the Affymetrix HG-U95av2 microarray with slight modifications. Briefly, cDNA was prepared from 5 µg total RNA using the Superscript II Choice System (Invitrogen, Breda, The Netherlands), incorporating the T7-(dT24) primer [5'-GCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGA GGC GG-(dT)24-3' (Genset, La Jolla, CA)]. In vitro transcription was performed to generate biotinylated cRNA for probing on the microarrays with the BioArray High Yield transcript labeling kit (Enzo Diagnostics, Farmingdale, NY). After purification, 20 µg newly synthesized cRNA was fragmented in buffer containing potassium and magnesium for 35 min at 94°C and was hybridized for 16 h at 45°C. After hybridization, the chips were washed using the Affymetrix fluidics station in buffer containing biotinylated antistreptavidin antibody and stained with streptavidin PE. The intensity of the fluorescence was measured in an Affymetrix scanner at 570 nm.

Cell-cycle and apoptosis analysis of CD34+ cells from various SC sources
The cell-cycle activity of CD34+ derived from BM, PBSC, and UCB was measured with propidium iodide (PI) staining. Briefly, 0.75 x 106 CD34+ cells were washed and resuspended in 500 µl cold PBS (pH 7.4). Ice cold 70% ethanol was added carefully to a final volume of 5 ml and fixed for 2 h at 4°C. Afterward, cells were washed twice with PBS, resuspended in 400 µl PI/RNase/Triton X-100 solution, and incubated for 30 min at room temperature. Stained cells were analyzed by flow cytometry. The level of apoptotic activity of CD34+ cells was assessed using the Annexin V-PE apoptosis detection kit (BD PharMingen, San Diego, CA), according to the manufacturer’s specifications. In brief, CD34+ cells were washed twice with cold PBS and then resuspended in 1x binding buffer at a concentration of 1 x 106 cells/ml. Cells (1x105) were aliquoted and incubated with Annexin and 7-amino-actinomycin (7-AAD) for 15 min at room temperature. Afterward, 1x binding buffer was added to the cell suspension. Stained cells were analyzed by flow cytometry.

Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
cDNA was reverse-transcribed from 1 µg total RNA using avian myloblastosis virus–RT (Promega Benelux, Leiden, The Netherlands) and random hexamer primers in a 20-µl reaction volume. After transcription, cDNA sample was added to 100 µl. All real-time PCR was performed on the ABI PRISM 7700 sequence detector (Applied Biosystems, Foster City, CA), and specific primers and probe were designed with Primer Express software (PerkinElmer, Wellesley, MA). Two reaction mixtures were made for the amplification. For amplification of porphobilinogen deaminase (PBGD), glucocorticoid-induced leuzine zipper (GILZ), and c-Fos, the PCR reaction mixture consisted of 1x buffer A (Applied Biosystems), 5 mM MgCl2, 0.4 mM dNTPs (Pharmacia), 300 nM forward and reverse primers, 100 mM probe, 0.75 U AmpliTaq Gold DNA polymerase (PerkinElmer), and 5 µl template. For elastase, IL-8, and Jun-B, PCR reactions were performed using the SYBR Green PCR core reagent kit (Applied Biosystems). The amplification of the target genes was as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min (Table 1 ).


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  		Table 1. Oligonucleotide Primer and Probes for Real-Time Quantitative RT-PCR (RQ-PCR)


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RESULTS
 
In vitro and in vivo SC assay
To investigate the functional differences in vitro among BM, UCB CD34+ SC, and PBSC, we assessed the frequency of primitive SC/progenitors with long-term engraftment capacity using the CAFC assay (CAFCwk6) and in SCID/NOD mice. These assays are widely used to monitor SC activity, although other assays also exit [colony-forming units and long-term culture (LTC)-initiating cells], which are useful to measure short-term repopulation in vitro. Freshly isolated CD34+ cells from BM, UCB, and PBSC were seeded in limiting dilution onto FBMD-1 stromal cells for LTC. We observed that UCB CD34+ cells contain significantly (3.6-fold) more CAFCwk6 cells than BM CD34+ cells, and the frequency of mature progenitors, CAFCwk2, only differed slightly. It is interesting that the frequency of CAFCwk6 in PBSC CD34+ cells was markedly lower, suggesting that PBSC CD34+ cells have diminished long-term engraftment capacity compared with BM and UCB CD34+ cells but behave similarly in short-term, repopulation assays (Fig. 1 ).



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  		Figure 1. CAFC assays. Freshly isolated CD34+ cells from UCB (n=2) and BM (n=4) were seeded in limiting dilution on a monolayer of FBMD-1 cell line and culture in the presence of IL-3 and G-CSF LTC. The presence of cobblestone colonies was determined at week 2 (CAFCwk2) and week 6 (CAFCwk6).

In vivo-repopulating SC assays were performed to determine differences in engraftment capacity between UCB and BM. To this end, CD34+ cells from both the SC sources were injected into immunodeficient NOD/SCID mice. Five-weeks post-transplantation, engraftment of human cells was determined by the presence of human CD45+ cells in the mouse BM. As expected, CD45+ cells (33–65%) were detected in the BM of three out of three mice injected with 100,000 UCB CD34+ cells. In contrast, a higher cell dose (1x106) of BM CD34+ cells was needed to obtain engraftment, and the proportion of CD45+ cells (0.6–14%) was significantly lower compared with UCB CD34+ cells (Fig. 2 ). As a result of the low frequency of CAFCwk6 detected in CD34+ PBSC and a recent study by Civin and co-workers [23 ], reporting that CD34+ PBSC have very low, if at all detectable, NOD/SCID repopulation capacity, we decided not to perform PBSC NOD/SCID transplantation experiments. In conclusion, the functional SC capacity of CD34+ cells from the three different sources can be ranked as UCB > BM > PBSC.



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  		Figure 2. In vivo engraftment of NOD/SCID mice transplanted with BM (n=4) and UCB CD34+ cells (n=3). The figure represents the proportion of CD45+ cells recovered from the femora of NOD/SCID mice transplanted with 1 x 105 UCB CD34+ cells and 1 x 106 BM CD34+ cells, respectively.

Comparative analysis of gene expression among various SC sources
In an attempt to provide a molecular basis for the functional differences, we analyzed the gene expression of CD34+ cells using high-density DNA microarrays covering over 12,000 human transcripts [25 , 26 ]. CD34+ cells were isolated from BM (n=5), G-CSF-mobilized PBSC (n=5), and UCB (n=10) using magnetic beads and were cryopreserved until use. The purity of the CD34+ cells was 90–95%. The data reported here are the averages of two sets of three microarrays, comprised of pooled CD34+ cells from each of the SC sources from different individuals. We compared the gene-expression profiles of CD34+ HSC by declaring BM as a baseline for comparison, and the relative differences in gene-expression levels with UCB and PBSC were determined (Fig. 3 ). Further, a numerical threshold of 2.5-fold was set to identify target genes, which were differentially expressed among the SC sources. This threshold was chosen, as a 2.5-fold difference can be validated by other assays such as quantitative real-time RT-PCR. Based on these criteria, a comparative analysis showed that in UCB CD34+ cells, 51 genes were differentially expressed compared with BM CD34+ cells. Twenty-one genes showed significantly decreased expression in UCB CD34+ cells, whereas the expression of 30 genes was increased. On the contrary, 64 genes were found differentially expressed between BM CD34+ cells and PBSC. Twenty-five genes showed decreased expression, and 39 genes were increased (Fig. 4 ).



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  		Figure 3. Comparative analysis of gene-expression profiles between UCB, PBSC, and BM CD34+ cells. For comparison, the gene-expression profile of BM CD34+ cells was used as baseline (x-axis). Also, the samples were scaled to an arbitrary value of 1000 to normalize for differences in overall intensity. Dashed lines, Two-, three-, ten-, and 30-fold difference in fluorescence intensity. CB = UCB, LF = PBSC



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  		Figure 4. Differential gene-expression profiles between BM and UCB CD34+ cells. An arbitrary 2.0-fold threshold in difference was set to identify the gene to be considered differentially expressed. In UCB versus BM cells, 51 genes were expressed differently than BM CD34+ cells.

Hierarchical cluster analysis demonstrated that BM and in particular, PBSC from different donors are highly similar, and the UCB samples varied much more data not shown. Nevertheless, clusters of SC source-specific genes can readily be discerned. These signature profiles largely consist of the genes described below, as they are differentially expressed among different SC sources. It is striking that the two UCB and BM experiments (composed of different pooled samples) cluster together in this analysis, whereas the two PBSC experiments form a separate cluster. This division can also be seen in the gene dot plots (Fig. 3) , where differences between PBSC and BM CD34+ cells are greater than between UCB and BM CD34+ cells. The reasons why the two different preparations of UCB CD34+ cells vary between experiments are unclear but may reflect an inherently greater heterogeneity of various subpopulations of CD34+ cells.

A considerable number of targets of CCAAT1 enhancer-binding protein (C/EBP) transcription factor family [27 , 28 ] were expressed markedly lower in UCB CD34+ cells and PBSC than BM CD34+ cells, and the expression of C/EBP itself was also 2.4-fold and 2.5-fold decreased in UCB CD34+ cells and PBSC, respectively (data not shown). Among these target genes, we found the expression level of neutrophil elastase, myeloperoxidase, and cathepsin G was four- to 14-fold lower in UCB CD34+ cells and PBSC than BM CD34+ cells (Tables 2 and 3 ). It is interesting that megakaryocytic-related genes such as GPIIb and PF4 also showed decreased expression in UCB CD34+ cells and PBSC. These data suggest that BM CD34+ cells contain significantly more progenitors of myeloid lineages than other SC sources. Furthermore, higher expression of genes encoding growth factors (SC growth factor, epidermal growth factor-like AR), chemokines (connective tissue-activation peptide, PF4), and their receptors (CXCR-4) was detected in BM CD34+ cells, except for IL-8, underscoring the importance of interaction between SC and the microenvironment.


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  		Table 2. Differential Gene Expression between UCB and BM CD34+ Cells


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  		Table 3. Differential Gene Expression between PBSC and BM CD34+ Cells

It is striking that the number of cell cycle-related genes encoding phosphatases, endonucleases, and mitotic checkpoint proteins with differential expression is slightly higher in BM CD34+ cells than in PBSC CD34+ and to a lesser extent, with UCB CD34+ cells. These genes are expressed during the cell-cycle progression and DNA synthesis. This indicates that the difference in cell-cycle activity between BM and PBSC CD34+ cells is more pronounced than between PBSC and UCB CD34+ cells. To functionally check these differences in gene expression of cell-cycle-related genes, we investigated the cell-cycle profile of the various CD34+ cell types using the DNA dye PI. Cell-cycle analysis revealed that BM CD34+ cells proliferate more (5.3% in G2/M phase) than PBSC (1.1%) or UCB (0.3%) CD34+ cells (Fig. 5A ), confirming published data about cell-cycle analysis [29 , 30 ] and entirely consistent with the gene-expression profiles described here.



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  		Figure 5. Differences in cell-cycle and apoptotic activities of CD34+ cells derived from various SC sources. For cell-cycle analysis (A), cryopreserved CD34+ cells from BM, PBSC, and UCB (CBMC) were fixed with 70% ethanol and incubated with PI. Annexin V- and 7-AAD-staining assessed the level of apoptosis of CD34+ cells (B).

A higher expression level of antiapoptotic genes was also detected in BM CD34+ cells. Among them, GILZ and clusterin expression were significantly (3.6- to 7.8-fold) increased in BM CD34+ cells. Accordingly, expression of the proapoptotic genes TRAIL in PBSC cells and Annexin II ligand in UCB CD34+ cells was increased. Together, this indicates that BM CD34+ cells have a lower apoptotic activity compared with UCB and PBSC CD34+ cells. We investigated whether these changes in gene expression were reflected in the percentage of apoptotic cells in the various SC sources. To this end, purified CD34+ cells were stained with Annexin V and anti-CD34 and analyzed by flow cytometry. These analyses showed (Fig. 5B) that PBSC have the highest percentage of apoptotic cells [14.8%, followed by UCB (9.5%) and BM (5.7%)], fully in line with the microarray data showing up-regulated expression of proapoptotic genes in UCB and PBSC compared with BM CD34+ cells.

Furthermore, a significant number of genes with a higher expression in UCB CD34+ cells were encoding for transcription factors. These transcription factors have been demonstrated to inhibit the differentiation by preventing activation of lineage-specific genes and are preferentially found in immature SC and progenitors. GATA-2, Id1, and jun-B were expressed higher in UCB CD34+ than BM CD34+ cells. Moreover, nonhematopoietic lineage-associated genes, such as Tie (endothelial cells) and NAP-22 (neuronal cells), were also detected in UCB CD34+ cells.

Validation of DNA microarray data
To validate the results obtained by DNA microarray, we selected four target genes, which are differentially expressed, and assessed their expression level in CD34+ cells by quantitative real-time RT-PCR. Elastase showed lower expression on microarray in the UCB CD34+ cells than BM CD34+ cells, and three genes, IL-8, c-fos, and jun-B, were significantly increased in BM CD34+ cells. Quantitative real-time RT-PCR confirmed the microarray data, although the quantitative fold difference was usually higher by RT-PCR than microarray-based detection (Fig. 6 ). This probably reflects the technical difficulties in detecting relatively small differences in gene expression quantitatively by both assays.



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  		Figure 6. Validation of microarray data by quantitative real-time RT-PCR. To corroborate the data obtained by the microarray, we assessed the expression of elastase, c-fos, jun-B, and IL-8 by quantitative real-time RT-PCR. The bar graph represents the comparison of the fold-change differences detected by both measurements.


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DISCUSSION
 
BM has traditionally been the main source for CD34+ SC, but in recent years, UCB and G-CSF-mobilized PBSC have been shown to be very useful as alternative SC sources for clinical and research applications. However, several groups including ours have demonstrated that CD34+ cells from these SC sources differ in terms of reactivity to in vitro cytokine stimulation, their potential to generate mature blood cells, and their capacity for in vivo engraftment. The molecular phenotype of CD34+ SC, the regulatory mechanisms to remain immature, cell cycle, mobilization, and differentiation into blood-cell lineages are only partially understood. Thus, comparative analyses among SC sources might explain the molecular causes underlying their differences in functional characteristics. To address this issue, we quantified the number of SC in BM and UCB CD34+ cells and PBSC using a CAFC assay and assessed the in vivo engraftment capacity of BM and UCB CD34+ cells by a NOD/SCID repopulation assay. As expected, CAFC confirmed that UCB CD34+ cells contained the highest frequency of SC with long-term engraftment capability (CAFCwk6), and moreover, the engraftment capacity was also significantly greater than BM CD34+ with a higher proportion of CD45+ cells detected in the recipients at a lower cell dose.

To determine how functional differences were reflected by changes of molecular phenotype of SC, we have used Affymetrix microarrays to assess differences in the gene-expression profile of CD34+ cells among the SC sources. Comparative analysis with UCB CD34+ cells was done by setting the gene-expression profile of BM CD34+ cells as baseline and was demonstrated that UCB CD34+ cells expressed 51 genes differentially. Twenty-one genes were markedly decreased, and 30 genes showed increased expression. Comparison of PBSC and BM CD34+ cells resulted in slightly more differences; 64 genes were detected with differential expression. Most elevated genes have been described to play important roles in diverse biological processes such as cell adhesion/migration, proliferation, differentiation, and apoptosis.

For instance, UCB CD34+ cells and PBSC showed reduced expression of neutrophil elastase, myeloperoxidase, and cathepsin G and lower CXCR-4 than BM CD34+ cells. Elastase and cathepsin G are proteases, which are mainly found in the granule of neutrophils, and their biological activity involves proteolytic degradation of proteins. Recently, it has been demonstrated that inhibition of elastase activity prevents mobilization of BM CD34+ cells into PB by reducing the degradation of stroma-derived factor-1 (SDF-1), and stimulation with G-CSF resulted in increased degradation of SDF-1. CXCR-4, the ligand for SDF-1, plays an important role in homing and the engraftment of CD34+ SC to the BM, and any disruption of the interaction between SDF-1 and CXCR-4 will likely have a negative impact on the engraftment capability of CD34+ cells [31 , 32 ]. UCB CD34+ cells may need to home to the BM, not egress from it, and therefore, elastase activity needs to be low. The low expression on PBSC may reflect a negative regulation of these genes after successful mobilization. Petit et al. [32 ] show increased CRCX-4 expression on BM cells after mobilization (5 days); this is not necessarily reflected by expression of this receptor on those cells that have reached the periphery (i.e., the PBSC). In fact, the actual PBSC should have lost/lowered CRCX-4 expression to further differentiate and home back to the BM after transplantation, consistent with the differential effects reported on BM and PB SDF-1 expression after G-CSF stimulation [32 ]. Together, these data indicate that high expression of elastase and CXCR-4 in BM CD34+ cells is important for the SC to maintain the interaction with the BM stroma and when needed, the migration into PB.

We also found that BM CD34+ cells expressed higher level of megakaryocytic-related genes, such as CTPIII, PF4, and GPIIb (CD41b). PF4 is a member of CXC-type chemokine and regulates the production of megakaryocytic progenitors [33 ]. Han et al. [34 ] demonstrated that PF4 together with other CXC-type chemokines, IL-8 and NAP-2, were able to support survival of myeloid progenitor cells and reduced their sensitivity to cytotoxic agents. CTPIII is a precursor protein, which will result in NAP-2 after cleavage with cathepsin G and elastase. It is suggestive that BM CD34+ cells contain more myeloid progenitor cells than PBSC cells reflected by the expression of these myeloid-related genes. We hypothesized that PBSC CD34+ at least would also contain a significant number of myeloid progenitors cells as a result of the mobilization regime with G-CSF. So far, no strong indications from the gene-expression profile support this hypothesis. Only a slightly increased expression of CD33 (1.6-fold) and CD15 (2.9 fold) was detected in PBSC CD34+ cells compared with BM CD34+ cells. Thus, PBSC likely represent a SC source enriched for progenitor cells, not so much mature myeloid cells. This is in agreement with reports describing PBSC as efficacious HSC grafts.

Many groups have demonstrated that UCB CD34+ cells and PBSC have markedly lower cell-cycling activity than BM CD34+ cells [29 , 30 ]. Virtually all the cells with a primitive phenotype, CD34+CD38- and CD34+Thy-1+, detected in UCB and PBSC are in G0/G1 phase of the cell cycle [35 36 37 ]. This is probably caused by the loss of contact of the CD34+ cells with the BM microenvironment. Here, in line with previous studies, we found the BM CD34+ cells showed slightly higher expression levels of S/G2/M-related genes, including HMG-2, FEN1, MCM7, ZWINT, and BTG1, than UCB CD34+ cells and PBSC.

In our study, the (anti) apoptotic-related genes such as death receptors, caspases, and bcl proteins were not differentially expressed between BM CD34+ cells and UCB CD34+ cells, but a higher expression level of GILZ and clusterin (apolipoprotein J) was found in BM CD34+ cells than UCB CD34+ cells. GILZ has been demonstrated to protect T cells from apoptosis induced by treatment with anti-CD3 antibody [38 ], and clusterin has been shown antiapoptotic against a variety of stimuli such as Fas-mediated apoptosis [39 ]. Consistent with that increased expression of proapoptotic genes, Annexin II ligand and TRAIL were detected in UCB CD34+ cells and PBSC, respectively. The results of these studies suggest that BM CD34+ cells also have lower apoptotic activity than UCB CD34+ cells.

It is interesting that several transcriptional regulatory genes were expressed differentially among BM, UCB CD34+ cells, and PBSC. Increased expression of transcription factors including GATA-2, Id1, Fos proteins (c-Fos and FosB), LIM-related proteins, and jun-B was detected in UCB CD34+ cells. It is known that GATA-2 and Id1 act as negative regulators of early hematopoiesis and inhibit differentiation by preventing activation of lineage-specific genes [40 41 42 ]. The expression level of GATA-2 and Id1 has been detected highest in the progenitor cells and declines during differentiation. Oh et al. [43 ] assessed the gene expression of several transcription factors in CD34+ subpopulation cells using RT-PCR. These investigators showed that some of these transcription factors, such as c-Fos and c-jun, were expressed differentially between CD34+CD38- and CD34+CD38+ SC from various SC sources, surprisingly with the highest level in BM. Further, prolonged expression of c-Fos resulted in preservation of HSC in G0/G1 cell-cycle status [44 ]. We hypothesize that the differential expression of these transcription factors as seen in our study was a result of the difference in number of immature SC present in BM and UCB CD34+ cells. As expected, CAFC and NOD/SCID repopulation assay confirmed that UCB CD34+ cells contained the highest frequency of SC with long-term engraftment capability. The high expression of Id and GATA genes probably plays a role in maintaining an immature progenitor phenotype, and the Fos and jun transcription factors lead to higher proliferation of UCB compared with BM and PBSC. Expression of these types of genes may help maintenance and proliferation of more immature HSC and consequently, in self-renewal.

In summary, our attempt to explain the functional differences among UCB, BM CD34+ SC, and PBSC by comparing the gene-expression profiles showed significant differences among the SC sources, mainly in terms of key transcription factors, cell-cycle-related genes, apoptotic genes, and genes involved in homing and adhesion of SC. Comparative analysis of the gene-expression profile among the SC sources showed that the BM CD34+ cells contained more myeloid progenitors than UCB CD34+ cells and are in line with intensive cross-talk and interactions with the stroma microenvironment. It is also clear that BM CD34+ cells have slightly greater cell-cycle activity and low apoptotic activity, potentially as a result of the interaction with the stroma. On the contrary, UCB CD34+ cells consist of more quiescent SC with long-term engraftment capacity, as confirmed here by in vitro and in vivo SC assays, and might have the potential to differentiate into nonhematopoietic cells types.


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ACKNOWLEDGEMENTS
 
This study is supported in part by research funding from Biomed EEC, Biotech Demonstration Project (Y. Y. N. and B. v. K.), the Dutch Heart Foundation (C. M. M. v. d. B.), the Royal Dutch Academy for Arts and Sciences (F. J. T. S.), and the Bekales Foundation (F. J. T. S.). The authors thank E. Borst for his technical assistance with quantitative real-time RT-PCR.

Received June 23, 2003; revised September 30, 2003; accepted October 8, 2003.


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