Gene-Expression Profiling of CD34 Hematopoietic Cells Expanded in a Collagen I - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:10 |只看该作者 |倒序浏览 |打印

作者：Joachim Oswald, Christine Steudel, Katrin Salchert, Brigitte Joergensen, Christian Thiede, Gerhard Ehninger, Carsten Werner, Martin Bornhuser作者单位：Medizinische Klinik und Poliklinik I, University Hospital Carl Gustav Carus, Leibniz Institute of Polymer Research Dresden  Max Bergmann Center of Biomaterials, Dresden, Germany

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      【摘要】/ ?3 b7 m, \2 N/ W6 h5 f) u, S
   CD34  hematopoietic stem/progenitor cells (HSCs) reside in the bone marrow in close proximity to the endosteal bone surface, surrounded by osteoblasts, stromal cells, and various extracellular matrix molecules. We used a bioartificial matrix of fibrillar collagen I, the major matrix component of bone, as a scaffold for ex vivo expansion of HSCs. CD34  HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in the presence of fms-like tyrosine kinase-3 ligand, stem cell factor, and interleukin (IL)-3. After 7 days of culture, the cell number, number of colony-forming units (CFU-C), and gene-expression profile of the cultured cells were assessed. Although the total expansion factor of CD34  cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of CFU-C was greater than in control suspension cultures. Gene-expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in the presence of collagen I. Among these, genes for several growth factors, cytokines, and chemokines (e.g., IL-8 and macrophage inhibitory protein 1) could be confirmed using quantitative polymerase chain reaction. Furthermore, greater expression levels of the negative cell-cycle regulator BTG2/TIS21 and an inhibitor of the mitogen-activated protein kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34  cord blood cells in a culture system containing a three-dimensional collagen I matrix induces a qualitative change in the gene-expression profile of cultivated HSCs. ; j6 n" H( I) s& @
      【关键词】 Cord blood Hematopoietic stem/progenitor cells Collagen I Matrix gene expression6 V7 q; K$ ?, |* i) d
   INTRODUCTION
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Hematopoietic stem/progenitor cells (HSCs) receive critical signals for proliferation and differentiation from the bone marrow microenvironment, where cytokines, stromal cells, and extracellular matrix (ECM) molecules act in close proximity to HSCs .; @) N( G) Z- f3 B# h

Despite the obvious limits of ex vivo suspension culture to preserve or amplify early stem cells, ex vivo expansion currently appears to be the only viable option to increase the number of transplantable HSCs from cord blood. Three-dimensional culture carriers based on fibrillar ECM assemblies of collagen and other biopolymers may overcome the limits of ex vivo culture by presenting both soluble factors and adhesive contacts. In this context, we analyzed the effect of fibrillar collagen I on the gene-expression profile of CD34  HSCs after 7 days of culture. We found significant differences in the molecular signature between suspension cultures and HSC cultures in collagen gels induced by semisolid culture conditions that may mimic some of the anatomical constraints of the hematopoietic niche.

MATERIALS AND METHODS

Isolation of HSCs from Cord Blood; L, C; x4 |0 q# O9 K* y

CD34  cells were isolated from umbilical cord blood after informed consent of the child-bearing mother. The study was approved by the institutional review board of the university hospital in Dresden. Progenitor cell enrichment was performed using the magnetic affinity cell sorting (MACS) CD34 progenitor cell isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany, http://www.miltenyibiotec.com), following the manufacturer¡¯s recommendations. Briefly, cells were washed and resuspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA, Baxter, M¨¹nchen, Germany, http://www.baxter.de) and 5 mmol/l EDTA (Sigma, Steinheim, Germany, http://www.sigma-aldrich.com). Cells were incubated first with QBEND-10 antibody (mouse anti-human CD34) (Miltenyi Biotec) in the presence of human IgG as a blocking reagent and then with an anti-mouse antibody coupled with MACS microbeads. Labeled cells were filtered through a 30-µm nylon mesh (Miltenyi Biotec) before loading onto a column installed in the magnetic field. Trapped cells were eluted after the column was removed from the magnet. The positive fraction was loaded on a second column and again eluted with cold buffer. The purity of the CD34  population was in the range of 95 to 98%, as evaluated by flow cytometry using a fluorescence-activated cells sorter, FACScalibur (Becton-Dickinson, San Jose, CA, http://www.bd.com). After ex vivo expansion, cells were stained with a phycoerythrin (PE)-labeled anti-CD34 antibody (Becton-Dickinson) and with the respective isotype control and analyzed subsequently using flow cytometry.
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Reconstitution of Fibrillar Collagen+ K8 e0 S9 z' R2 X) ^
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Collagen fibrils were reconstituted from a sterile solution of purified, pepsin-solubilized bovine dermal collagen in 0.012 N HCl (Vitrogen, Cohesion Technologies, Palo Alto, CA, http://www.cohesiontech.com), as previously described . Briefly, eight parts of the acid collagen solution (3.0 mg/ml) were mixed with one part tenfold-concentrated PBS (Sigma) and one part 0.1 M NaOH on ice. The pH of the mixture was tested to be 7.4 and the concentration of the collagen solution was adjusted by the addition of chilled culture medium (CellGro SCGM, Cell-Genix, Freiburg, Germany, http://www.cellgenix.com). Formation of fibrils was initiated by a temperature shift to 37¡ãC. Collagen I gels were equilibrated with medium several hours before adding cells.5 `& R4 O6 y. `) y% p

Scanning Electron Microscopy
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Adhesion and characteristic shapes of CD34  selected cells on the collagen I sample surfaces were investigated using scanning electron microscopy (SEM). Therefore, collagen I was immobilized on adhesive polymer films, as described elsewhere , and CD34 -selected cells were cultivated for several days. The samples were washed with PBS (pH 7.4) to remove nonadherent cells. Subsequently, the adherent cells were fixed with 2% glutaraldehyde (Serva Electrophoresis GmbH, Heidelberg, Germany, http://www.serva.de) in PBS for 1 hour, rinsed with PBS, and dehydrated with a graded ethanol series. Samples were critical point-dried (CPD 030; BALTEC, Schalksmuehle, Germany, http://www.baltec.ch), gold-coated with a sputter coater (SCD 050; BALTEC), and examined using an SEM microscope (XL 30 ESEM FEG; FEI-Philips, Eindhoven, Netherlands, http://www.feicompany.com).

CD34  Culture in Collagen I Gels
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CD34 -selected cord blood cells were split to allow for comparisons between HSCs grown in suspension and those grown in collagen I fibrils from the same donor considering intersample variability. CD34 -enriched cells were cultured in six-well cell culture plates containing 3 ml serum-free medium (CellGro SCGM, CellGenix) at 37¡ãC and 5% CO2 in the presence of 300 ng/ml fms-like tyrosine kinase-3 ligand (FLT3-L), 50 ng/ml interleukin 3 (IL-3) (both from R&D Systems, Inc., Mannheim, Germany, http://www.rd-systems.com), and 300 ng/ml stem cell factor (SCF) (Cell Systems, St. Katharinen, Germany, http://www.cellsystems.de), as described previously . In a second set of experiments, cytokine concentrations were reduced to 100 ng/ml for SCF, 100 ng/ml for FLT3-L, and 50 ng/ml for IL-3. After 7 days of culture, cells were dissolved from the gels using collagenase (Biochrom, Berlin, Germany, http://www.biochrom.de) at a concentration of 50 mg/ml. Control cells grown in suspension were similarly treated with collagenase. Two independent experiments were performed including three repetitions. An expansion factor was calculated on the basis of the total number of CD34  cells determined by flow cytometry after cultivation divided by the starting number of CD34  cells counted with a Casy Cell Counter (Schärfe Systems, Reutlingen, Germany, http://www.casy-technology.com). The significance of differences was calculated based on the Mann-Whitney test. Differences were considered significant at the 95% confidence level (p
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For colony-forming unit-culture (CFU-C) assays, 1,000 cells were plated in 1-ml petri dishes containing complete methylcellulose medium consisting of Iscove¡¯s modified Dulbecco¡¯s medium (IMDM) with 30% fetal bovine serum, 3 U/ml erythropoietin, 50 ng/ml SCF, 20 ng/ml GM-CSF, 20 ng/ml IL-3, 20 ng/ml IL-6, and 20 ng/ml G-CSF (Methocult GF H4435, Stem Cell Technologies, Vancouver, Canada, http://www.stemcell.com). Cultures were incubated at 37¡ãC and 5% CO2. The total number of colonies was scored as CFU-C after 14 days. All assays were done in triplicate.; U; F* u. i3 D9 Q0 M9 S5 n
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Microarrays; j' _7 K) t4 C0 ]% Z1 w

Cells were cultivated as described above from three independent cord blood donations in a 12-well microtiter plate (TPP Biochrom, Berlin, http://www.biochrom.de). Before nucleic acid preparation, HSCs from three independent cord blood units cultured in suspension or in collagen I fibrils were pooled, and high-quality RNA was isolated from each pool with the RNeasy Mini Kit (Qiagen, Hilden, Germany, http://www.qiagen.com) according to the manufacturers instructions. In total, three pools of cells cultivated in the presence of collagen I and three controls, that is, cells cultivated in liquid suspension culture, were analyzed. Labeling for gene-expression profiling was performed according to the Affymetrix GeneChip Eukaryotic Small Sample Target Labeling Assay Version II (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). The detailed protocol for fragmentation, hybridization, washing, staining, and further processing of the arrays is available from Affymetrix. Briefly, in the first amplification cycle, 100 ng total RNA was transcribed into first-strand cDNA using Superscript II RT (Invitrogen Life Technologies, Karlsruhe, Germany, http://www.invitrogen.com) in the presence of T7-(dT)24 primer (5'-GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG-(dT)24-3', Affymetrix). After second-strand cDNA synthesis, the doubled-stranded cDNA was purified by ethanol precipitation, followed by in vitro transcription for cRNA amplification using Ambion MEGAscript T7 Kit (Ambion, Austin, TX, http://www.ambion.com) and cRNA cleanup with RNeasy columns (Qiagen). In the second amplification round, 250 ng cRNA was transcribed into first-strand cDNA using Superscript II RT in the presence of random primers (Invitrogen). Synthesis of the second-strand cDNA was done with T7-(dT)24 primer and DNA polymerase I from E. coli (Invitrogen) followed by ethanol precipitation to purify the double-stranded cDNA. Finally the cRNA was amplified and labeled by in vitro transcription with Enzo BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY, http://www.enzolifesciences.com) and purified with RNeasy columns. The generated biotin-labeled cRNA was fragmented by metal-induced hydrolysis, and 10 µg was hybridized to the GeneChip array (HG-U133A, Affymetrix). After washing and staining, the fluorescence intensity was measured for each array and normalized to the average fluorescence of the entire microarray. Image analysis was performed using the Microarray Analysis Suite 5.0 (Affymetrix).$ M. p1 B1 R9 v1 g9 [
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Data Analysis
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Data analysis was done with the Gene Spring Software version 6.0 (Silicon Genetics, Redwood City, CA, http://www.silicongenetics.com). To identify the most differentially regulated genes, a comparison analysis was performed. Restrictions required that the raw data had to be greater than 16 to eliminate change within the background noise, the genes had to be classified with "P" for present by Affymetrix data analysis not less than twice in three replicates, and the difference in expression had to be greater than 1.5-fold. The data discussed in this publication have been deposited in the National Center for Biotechnology Information¡¯s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE3003  .

Real-Time Polymerase Chain Reaction. ]8 Q5 u% @5 b: C* H% p7 L+ t) P
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To confirm differentially expressed genes identified by the microarray analysis, real-time polymerase chain reaction (PCR) was performed for five selected genes with Assays-on-Demand based on Taqman technology (Hs00174103_m1, Hs00358879_ m1, Hs00198887_m1, Hs00174097, and Hs00234142; Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany, http://www.applera.com). Briefly, 1 µg total RNA was converted to cDNA in the presence of random primers and RNAse inhibitor (RNAsin, Promega, Mannheim, Germany, http://www.promega.com) by reverse transcription (RT) with M-MLV Reverse Transcriptase. The Assays-on-Demand were performed according to the instructions of the supplier with 1 µl of cDNA and in replicates. GAPDH (Hs99999905_m1; Applied Biosystems) was used as the reference to adjust for different levels of inhibition during RT and PCR and for differences in the amount of total RNA initially added to the reaction.
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RESULTS AND DISCUSSION* u) L1 A1 k5 \. d% q
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With our study, we aimed to analyze the impact of fibrillar collagen I on the in vitro expansion of cord blood-derived CD34  cells. Using SEM, CD34 -selected HSCs were found to intermingle with the fibrillar collagen I matrix (Fig. 1B¨CD). Two HSCs seeded on a planar surface coated with nonfibrillar collagen (tropocollagen) are shown as a control (Fig. 1A). The strong interaction between HSCs and this variant of biopolymer matrix was demonstrated in previous studies in which we used time-lapsed microscopy to quantify the migration of adult HSCs on different types of collagen-glycosaminoglycan cofibrils. In those experiments, we showed that HSCs established intimate contact with the fibrillar substrates, which resulted in reduced mobility of the cells ." C" a* ^6 l( u
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Figure 1. Scanning electron microscopy (SEM) analysis of CD34 -selected hematopoietic stem/progenitor cells (HSCs). Shown are CD34 -selected HSCs on a planar surface coated with nonfibrillar tropocollagen (A), as control, or in intimate contact with fibrillar collagen I (B¨CD). The SEM micrograph in (C) shows that the collagen I fibrils directly attach to the cell surface, which eventually can lead to total coverage of the HSCs by the matrix (D).
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We subsequently examined the effects of various densities of fibrillar collagen I on proliferation and clonogenicity of CD34  HSCs. Purified CD34  cord blood-derived HSCs were embedded in gels reconstituted from various concentrations of collagen I and cultivated for 1 the week in presence of SCF, FLT3-L, and IL-3 to reach optimal expansion. After 7 days, the collagen I gels were solubilized using collagenase, and cells were counted and stained for CD34 antigen for subsequent flow cytometry analysis. In total, three independent experiments were performed. The percentage of cells expressing CD34  decreased with time, from >95% to a median of 45% (range, 32%¨C52%) after 7 days of expansion, with no relevant differences between cultures in collagen I and controls. Although the expansion factor for CD34  cells showed a high degree of biological variation among the three different samples, no major difference between cultivation in the presence or absence of fibrillar collagen I was detectable (Fig. 2A). Probably as a result of technical reasons, the number of HSCs recovered from the collagen I matrix was 0 to 5% (median, 3.5%) lower than in controls, which could be one explanation for the lower expansion factors calculated. In parallel, the clonogenicity after expansion was subsequently tested using a semisolid medium containing growth factors. The total number of colonies was again comparable in all tested collagen I preparations (Fig. 2B).3 [. A  {' F5 ]* E7 a' w" j# b
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Figure 2. Expansion of hematopoietic stem/progenitor cells (HSCs) in a collagen matrix. Shown are CD34  expansion (A) and the number of myeloid colony-forming units (CFU-C) per 1,000 cells (B) after ex vivo expansion in the presence of various concentrations of collagen I. CD34  expansion (C) and the number of CFU-C per 1,000 cells (D) after HSC cultivation in the presence of lower amounts of cytokines are also shown. Asterisks indicate statistical significance (p
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In a second set of experiments, the expansion and the number of CFU-C were assessed after cultivation of CD34  cells in the presence of lower levels of cytokines (100 ng/ml SCF and FLT3-L and 50 ng/ml IL-3). The total expansion factor for CD34  cells was lower in the collagen I-containing samples than in the controls (Fig. 2C). In contrast, the number of clonogenic progenitors was clearly greater after CD34  cells had been cultivated on collagen I gels (Fig. 2D). These data indicate that direct contact with the matrix molecule collagen I preserves or even increases the number of clonogenic progenitors detectable after secondary plating of cells in CFU assays. Direct contact of HSCs with fibronectin has been shown to cause a block in the S-phase of the cell cycle . The exact mechanism(s) of cell-cycle regulation in HSCs by surrounding cells and matrix molecules is still unclear. With our experimental system, we showed that collagen I is one important factor in the natural hematopoietic environment influencing both proliferation and colony-forming capacity in vitro.
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To gain a deeper insight into the effects of fibrillar collagen I on the fate of cultivated HSCs, we performed a gene-expression analysis using the Affymetrix microarray system. Samples from three independent donors were cultivated and pooled as described in Materials and Methods. Data from three chips for HSCs cultivated in the presence or absence of fibrillar collagen I were analyzed with the GeneSpring software. Table 1 summarizes the 50 genes that were upregulated the most in the presence of collagen I. Lists of all genes that were more than 1.5-fold up- or downregulated after culture in the collagen I matrix are provided as supplemental online data. Interestingly, a high number of cytokines or chemokines was overexpressed. Among these, IL-8, IL-1, and IL-1ß, as well as tumor necrosis factor- (TNF-) and microphage inhibitory protein 1 (MIP-1), have been shown previously to orchestrate the proliferation and mobilization of HSCs  pointed to osteoblast-like cells in the bone marrow as the source of MIP-1 regulated by the secretion of IL-1ß and TNF-. One might speculate that a matrix of collagen I imitates the hematopoietic stem cell niche microenvironment to a certain extent, thereby inducing the secretion of some of these mediators by HSCs and/or progenitor cells. On the other hand, HSCs that are physically attached to the matrix might start to upregulate genes and secrete the respective cytokines and chemokines associated with stem cell mobilization and proliferation in an autocrine/paracrine fashion to compensate for the constraints of the collagen gel./ L3 C' o* r( w

Table 1. First 50 most upregulated genes in CD34  hematopoietic cells expanded in a collagen matrix& r2 Y& J1 M0 A% \9 ]1 N2 ~
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Gene expression data from five selected genes were confirmed with real-time PCR (Fig. 3). We detected two important regulators of the cell cycle in the collagen I-cultivated cell population. DUSP2 is a dual specific phosphatase that inhibits kinases of the mitogen-activated protein kinase (MAPK) pathway, which is a crucial signaling pathway for various cytokines and growth factors . DUSP2 comprises an important negative regulator of the MAPK pathway, and overexpression in the presence of collagen I could be one reason for the somewhat delayed proliferation observed in our experimental system.

Figure 3. Gene-expression analysis using quantitative polymerase chain reaction (PCR). Shown are the results of a real-time reverse transcription-PCR analysis of five selected genes that were overexpressed in the presence of fibrillar collagen I.( D+ \8 k( Q) P. m" O3 _, Q# S; P+ P
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BTG2/TIS21 has been characterized as an antiproliferative gene, which can be induced by tetradecanoyl phorbol acetate .
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Although special care was taken to prevent artifacts, we cannot exclude the influence of the chemical and physical factors involved in cell harvest and analysis on gene expression in our experimental setting. In addition, the large biological variation associated with the use of primary HSCs isolated from cord blood needs to be considered when further interpreting the presented results.

In summary, our data show that in addition to stroma cells and osteoblasts, ECM components supplied in a three-dimensional culture system have a significant impact on gene expression of ex vivo cultivated CD34  HSCs. Paracrine/autocrine secretion of cytokines and chemokines by HSCs and progenitor cells regulates migratory potential and proliferation within the ECM matrix. Culture systems incorporating fibrillar collagen I in combination with other relevant ECM components, like the glycophosphoprotein osteopontin, which has recently been shown to be a major regulator of the HSC niche , may allow for a more physiological ex vivo expansion of cord blood-derived HSCs without losing pluripotency. Furthermore, our culture system might help to study the expression of auto-crine/paracrine factors in a serum-free and defined culture system.9 Y* T+ p4 A6 f) d

DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS8 A& S6 M8 e, U2 u0 S

We thank Uwe Streller for scanning electron microscopy. This work was supported in part by the German Ministry of Education and Science (BMBF grant 03N4028 to C.W. and M.B.) and by the Deutsche Forschungsgemeinschaft (SFB 655 to M.B. and C.W.). J.O. is currently affiliated with the Institute for Bioanorganic and Radiopharmaceutical Chemistry, Research Center Rossendorf, Dresden, Germany.6 }5 s# T+ x! R9 L
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