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First published online June 13, 2005
Stem Cells Vol. 23 No. 8 September 2005, pp. 1180 -1191
doi:10.1634/stemcells.2004-0361; www.StemCells.com
© 2005 AlphaMed Press

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Hematopoietic Progenitor Cells and Cellular Microenvironment: Behavioral and Molecular Changes upon Interaction

Wolfgang Wagnera, Rainer Saffricha, Ute Wirknerb, Volker Ecksteina, Jonathon Blakeb, Alexandra Ansorgeb, Christian Schwagerb, Frederik Weina, Katrin Miesalaa, Wilhelm Ansorgeb, Anthony D. Hoa

a Department of Medicine V, University of Heidelberg, Heidelberg, Germany;
b Biochemical Instrumentation Program, European Molecular Biology Laboratory, Heidelberg, Germany

Key Words. Hematopoietic stem cell • Microenvironment • Uropod • Adhesion • Microarray

Correspondence: Anthony D. Ho, M.D., Department of Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Telephone: 49-6221-566001; Fax: 49-6221-565813; e-mail: anthony_dick.ho{at}urz.uni-heidelberg.de


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Cell–cell contact between stem cells and cellular determinants of the microenvironment plays an essential role in controlling cell division. Using human hematopoietic progenitor cells (CD34+/CD38–) and a stroma cell line (AFT024) as a model, we have studied the initial behavioral and molecular sequel of this interaction. Time-lapse microscopy showed that CD34+/CD38– cells actively migrated toward and sought contact with stroma cells and 30% of them adhered firmly to AFT024 stroma through the uropod. CD44 and CD34 are colocalized at the site of contact. Gene expression profiles of CD34+/CD38– cells upon cultivation with or without stroma for 16, 20, 48, or 72 hours were analyzed using our human genome cDNA microarray. Chk1, egr1, and cxcl2 were among the first genes upregulated within 16 hours. Genes with the highest upregulation throughout the time course included tubulin genes, ezrin, c1qr1, fos, pcna, mcm6, ung, and dnmt1, genes that play an essential role in reorganization of the cytoskeleton system, stabilization of DNA, and methylation patterns. Our results demonstrate directed migration of CD34+/CD38– cells toward AFT024 and adhesion through the uropod and that upon interaction with supportive stroma, reorganization of the cytoskeleton system, regulation of cell division, and maintenance of genetic stability represent the most essential steps.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
The hallmark of stem cells is their dual ability to self-renew and to differentiate into multiple cell types. For adult stem cells, this duplex function is regulated by the microenvironment or the so-called stem cell niche [1–4]. Direct contact and communication between stem cells and cellular determinants in the microenvironment has been shown to play an essential role in this process [5]. This has been proven for a variety of different types of stem cells in different animal models. The cell fate of the daughter cells, that is, self-renewal versus differentiation, is governed by asymmetric cell division. The daughter cell with contact to a supportive cellular microenvironment retains the self-renewal potential, whereas the other daughter cell is destined to differentiation [6]. By analogy, cocultivation with feeder layer cells maintains the self-renewing ability of embryonic stem cell lines in vitro [7].

For hematopoietic progenitor cells (HPCs), many studies have demonstrated the vital role of a stroma feeder layer to maintain stem cell function in vitro [5, 8–10]. Many stroma cell preparations and cell lines, mostly derived from the bone marrow, have been demonstrated to serve as a feeder layer and to maintain HPCs in an undifferentiated state with varying degrees of efficiency [11, 12]. AFT024 is a cell line derived from murine fetal liver that supports the growth of long-term repopulating HPCs of murine and human origin in vitro [13, 14]. In comparison with human primary stroma cells, AFT024 cells have been shown to be more efficient and have generated consistent and reproducible results [15]. Using this cell line as a model for the HPC niche, investigations by others and our group have provided evidence that only direct cell-cell contact with AFT024 stroma was able to maintain the self-renewing ability of HPCs in vitro [5, 16, 17].

HPCs are characterized by rapid migratory activity and constantly changing morphology [18, 19]. They have been shown to migrate rapidly toward stroma cells and to extend magnupodia in the direction of the latter [20]. Several authors have reported the significance of podia formation for migration of HPCs and subsequently for the cell-cell contact between HPCs and cellular elements in the niche [20–22]. In previous reports, we have described various types of podia in CD34+ and CD34+/CD38– and in the slow-dividing fraction (SDF) of CD34+/CD38– cells [18, 23]. These different morphological types of pseudopodia might be associated with specific functions, including communication, adhesion, and homing to specific sites in the microenvironment.

To identify the essential cellular and molecular mechanisms involved in the interaction between HPCs and stroma feeder layer, we have studied the impact of cocultivation on the behavior and gene expression of HPCs using human CD34+/CD38– cells and irradiated AFT024 cells as a model system in vitro. The results will lead to a better understanding of the intrinsic and extrinsic factors regulating self-renewing divisions.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Cell Isolation
Human cord blood was collected from the umbilical cord after informed consent using guidelines approved by the Ethic Committee on the Use of Human Subjects at the University of Heidelberg. Mononuclear cells (MNCs) were isolated after centrifugation on Ficollhypaque (Biochrom KG, Berlin, Germany, http://www.biochrom.de). CD34+ cells were enriched with a monoclonal anti-CD34 antibody labeled with magnetic beads on an affinity column (Miltenyi Biotec, Bergisch-Gladbach, Germany, http://www.miltenyibiotec.com). CD34+-enriched cells were incubated with anti–CD38-phycoerythrin (PE) (Becton, Dickinson and Company [BD], San Jose, CA, http://www.bd.com) and anti–CD34-allophycocyanin (APC) (BD). The cells were washed in phosphate-buffered saline, 1% fetal calf serum (FCS), and 25 mM EDTA and stained with propidium iodide (PI) to allow exclusion of nonviable cells. CD34+/CD38– and CD34+/CD38+ populations were sorted using the automatic cell-depositing unit on a FACS-Vantage-SE flow cytometry system. Cells positive for PI were excluded. Approximately 2 x 104 to 1 x 105 CD34+/CD38– cells were isolated per cord blood sample.

Cultivation with and Without Feeder Layer
The murine fetal liver cell line AFT024 (a kind gift from I.R. Lemischka, Princeton University, Princeton, NJ) was maintained in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 20% FCS, 50 µM 2-mercaptoethanol (Bio-Rad, Hercules, CA, http://www.bio-rad.com), 1% vol/vol penicillin-streptomycin, and 1% vol/vol L-glutamin as described before [14]. Cells were grown to confluency in 24- or 96-well plates precoated with 0.1% gelatin (StemCell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com) and maintained at 33°C. AFT024 cells were then irradiated with 20 Gy 24 hours before using the feeder layer. CD34+/CD38– cells were separated in two equal fractions and cultivated either with AFT024 or without a feeder layer in 24-well plates. Both fractions were cultivated in the same media that consisted of RPMI supplemented with 20% FCS (R20), penicillin 1,000 U/ml, streptomycin 100 U/ml, and 50 µmol/l 2ME. Medium was supplemented with Flf3-ligand 10 ng/ml and thrombopoietin 10 ng/ml because these culture conditions were reported to support maximal expansion of primitive umbilical cord blood cells on AFT024 [24, 25].

Fluorescence and Time-Lapse Microscopy
To investigate morphology and interaction of the CD34+/CD38+/– cells with the AFT024 supportive layer, we stained the cells with the membrane dyes PKH26 (red) and PKH2 (green), respectively, as described previously [20, 21, 26]. An Olympus IX70 microscope (Olympus Optical, Hamburg, Germany, http://www.olympus-global.com/en/global) was used with a dual-band fluorescence filter set fluorescein isothiocyanate (FITC)/Cy3 (AHF Analysentechnik, Tübingen, Germany, http://www.ahf.de/asp/index.asp) for simultaneous acquisition of images in both fluorescence channels (x40 objective). The microscope was equipped with an incubation box to keep a constant temperature of 37°C and 5% CO2. In seven independent experiments, membrane protrusions were analyzed for each culture condition by random choice of >500 cells in >25 different fields (x40 objective). The two-sided paired t-test was used to estimate the significance of changes in cell morphology. Immunofluorescence microscopy was performed with CD44-FITC antibody (BD) and CD34-PE antibody (BD). Artificial capping of the antigen to membrane protrusions was excluded by previous fixation with 3% formaldehyde.

For time-lapse series, fluorescence and phase-contrast images were acquired with a colorview-12 camera (SIS, Münster, Germany, http://www.soft-imaging.net) every minute and stored on a PC workstation for further analysis. To reduce the influence of UV radiation, the field was changed every 2 hours over the observation period of 24 hours when using fluorescence. Image acquisition and processing was controlled by SIS AnalySIS 3.2 software. Cell migration and adhesion to the AFT024 feeder layer was estimated in 26 time-lapse recordings (212 CD34+/CD38– and 351 CD34+/CD38+ cells) in six independent experiments.

To analyze the directed migration of HPC toward AFT024 cells, a Terasaki 72-well plate was tilted at an angle of 30 degrees to allow settling of approximately 100 AFT024 cells in the lower edge. After 1 day of cultivation, the AFT024 cell layer still covered only half of the well, and the cells were then irradiated with 20 Gy to arrest proliferation. The plate was then tilted in the opposite direction at an angle of 30 degrees, and 200 to 500 CD34+/CD38– cells were allowed to settle in the part of the well without AFT024 cells. Time-lapse observation of the cells was then performed under a microscope with a 5-degree angle in a way that the HPC had to migrate uphill toward the AFT024 cells (n = 12). As controls, we have compared migration toward human fibro-blasts HS68 (CRL-1635; American Type Culture Collection, Rockville, MD, http://www.atcc.org; n = 6) or in a control without stromal cells (n = 4).

Separation of HPCs from AFT024
To determine the influence of the AFT024 microenvironment on the gene expression of CD34+/CD38– cells, the cells were cocultivated as described above and harvested after 16, 20, 48, or 72 hours by rigorous pipetting. To separate more than 5 x 104 CD34+/CD38– cells, we had to pool MNCs from up to three fresh cord blood samples for each of these time points (three samples for 16 hours; one sample for 20 hours; three samples for 48 hours; one sample for 72 hours). Separation according to forward-scatter and side-scatter was highly efficient as AFT024 cells are larger. In addition, AFT024 cells demonstrated a high autofluorescence in the PI channel, whereas residual anti–CD34-APC fluorescence in HPCs could be used to assist separation even after 3 days of coculture. Separation was controlled by microscopy. The fraction of CD34+/CD38– cells that was not cultivated on a supportive cell layer was also sorted using the same parameters to exclude differences in gene expression by a different treatment.

RNA Isolation and Probe Synthesis
Approximately 2 x 104 cells from each fraction were lysed, and total RNA was isolated using the Arcturus PicoPure RNA-Isolation Kit (Arcturus, Mountain View, CA, http://www.arctur.com). DNase treatment was performed (Qiagen, Hilden, Germany, http://www1.qiagen.com). RNA quality was controlled with the RNA 6000 Pico LabChip kit (Agilent, Waldbronn, Germany, http://www.agilent.com). Linear amplification of mRNA was performed by in vitro transcription over two rounds using the Arcturus RiboAmp Kit (Arcturus). The amplified RNA was analyzed by the RNA Nano Lab Chip kit (Agilent) and by the SpectraMAX plus photometer (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com) at 260 nm. Approximately 10µ ga RNA of each probe was mixed with spike in control RNA to assist compensation of the data. This control RNA was a mixture of two genomic sequences of arabidopsis thaliana, chloramphenicol acetyl transferase, and firefly luciferase that were previously synthesized by in vitro transcription by T7 RNA polymerase. The control RNA has been tested to show no cross-hybridization with human probes [27]. RNA samples were then incubated with 3 µg Random Primer (Invitrogen, Karlsruhe, Germany, http://www.invitrogen.com) and labeled by aminoallyl coupling using the Atlas Glass Fluorescent Labeling Kit (Clontech, Palo Alto, CA, http://www.clontech.com/clontech) and Cy3-/Cy5-monofunctional reactive dye (Amersham Biosciences, Little Chalfont, U.K., http://www1.amershambiosciences.com).

The Human Genome Microarray
For microarray analysis, the human genome microarray described before was used [23]. It represents the UnigeneSet-RZPD3 composed of 51.145 cDNA clones, a very well-characterized subset of the IMAGE cDNA clone collection (http://www.rzpd.de; http://image.llnl.gov/image). Further details about this microarray are provided under http://embl-h3r.embl.de, and the techniques for hybridization and washing of the slides have been described previously [23, 27].

Analysis of Results
The analyzed data consist of eight cohybridization datasets that represent four time points of coculture (16, 20, 48, and 72 hours) and the corresponding color-flip experiments. Furthermore, the gene expression profile of CD34+/CD38– cells versus the murine AFT024 cell line was analyzed in four cohybridizations (including color-flip experiments) to verify that the differential gene expression pattern was not a result of residual RNA of the stromal feeder layer. Slides were scanned using the GenePix 4000B Microarray-Scanner (Axon Instruments, Union City, CA, http://www.axon.com) as described before [23] and analyzed by the ChipSkipper Microarray Data Evaluation Software (Emblem Technologies, Heidelberg, Germany, http://www.emblem.de/home/index.php). Integration grids were automatically centered to the images. For each spot, total intensity and local background were calculated. Raw ratios were derived from background-reduced signals. Normalization was performed by intensity-dependent windowed median ratio centering. The resulting data were further analyzed by Excel (Microsoft, Redmond, WA, http://www.microsoft.com). Differentially regulated genes within the two corresponding color-flip hybridizations of each time point were selected by more than twofold higher signal intensity (log2 ratio > 1 or < –1) and error estimation of replica standard deviation (SD) (SD < log2 ratio). The resulting set of 2,455 sequences that were upregulated or downregulated in at least one time point was further analyzed with TIGR MeV Ver.2.2 Software (Institute of Genomic Research, Rockville, MD, http://www.tigr.org). K-means clustering was performed using Euclidean distance with eight groups (k = 8), as suggested by the figure of merit [28]. For detailed analyses, we have selected those genes with a more than twofold average differential expression in all replicates and color-flip hybridizations of the four analyzed time points (log2 ratio > 1 or < –1). The kinetics of differential expression in the different time points is presented by color code. False discovery rate (FDR) was estimated by simulations. Stochastic permutations of all experimental ratio values within each replicated hybridization were used to create sets of virtual replications. Ten thousand simulations were performed, and the average number of genes within the same filter criteria as described above was given as FDR [23]. The complete microarray data including the description of all spotted genes (according to minimum information about a micro-array experiment [MIAME] requirements [29]) is submitted to the public microarray database ArrayExpress (http://www.ebi.ac.uk/arrayexpress; accession number E-EMBL-3).

Reverse Transcription–Polymerase Chain Reaction Analysis
Differential gene expression values observed by microarray analysis were supported by results of real-time reverse transcription–polymerase chain reaction (RT-PCR) with Light Cyclertechnology (Roche Molecular Biochemicals, Mannheim, Germany, http://www.roche.com/home.html). Eleven differentially expressed genes were analyzed by RT-PCR with total RNA of five independent cord blood samples after cocultivation either with AFT024 or without stromal feeder layer for 24–72 hours. RNA was isolated as described above and reverse-transcribed by Superscript II (Gibco, Invitrogen). Semiquantitative PCR was performed with the LightCycler Master SYBR Green kit (Roche Molecular Biochemicals,) with 3 mM MgCl at 480 seconds of preincubation at 95°C followed by 50 cycles of 10 seconds at 55°C, 25 seconds at 72°C, and 10 seconds at 95°C. PCR products were subjected to melting curve analysis and to conventional agarose gel electrophoresis to exclude synthesis of unspecific products. The following primers were used: 18s rRNA primers supplied by Ambion (Austin, TX, http://www.ambion.com):18srRNA forward primer: 5'-TCAAGAACGAA AGTCGGAGG-3'; 18s rRNA reverse primer: 5'-GGACATCTAA GGGCATCACA-3'; mcm6 forward primer: 5'-GGAGCATGAT TCGTCTCTCT G-3'; mcm6 reverse primer: 5'-GTCAGCATGA CCATTGATGC-3'; alpha-tubulin forward primer: 5'-GACAGCTCTT CCACCCAGAG-3'; alpha-tubulin reverse primer: 5'-TGAAGTCCTG TGCACTGGTC-3'; dnmt1 forward primer: 5'-ATCCTCAGGG ACCACATCTG-3'; dnmt1 reverse primer: 5'-TATACCGCAG CTTCCTGGC-3'; eln forward primer: 5'-CCCCCTACTT CAGAGGCAAG-3'; eln reverse primer: 5'-GGAAGATAAG AGCACCAGCG-3'; mmp2 forward primer: 5'-ACAGCTACTT CTTCAAGGGT GC-3'; mmp2 reverse primer: 5'-CGGTGTATCG AAGGCAGTG-3'; ggh forward primer: 5'-CCAAGAAGCC CATCATCG-3'; ggh reverse primer: 5'-TGGTACAACT CTCGCACCTG-3'; chk1 forward primer: 5'-CCAGTAAACA GTGCTTCTAG TGAAGA-3'; chk1 reverse primer: 5'-TGGGGTGCCA AGTAACTGAC-3'; tuba3 forward primer: 5'-TTCAAAACCC ATCACAGAAA TG-3'; tuba3 reverse primer: 5'-TGAGGAGGTT GGTGTGGATT-3'; gamma-globin forward primer: 5'-TTGAAAGCTC TGAAT-CATGG G-3'; gamma-globin reverse primer: 5'-CTTCAAGCTC CTGGGAAATG-3'; gapdh forward primer: 5'-ATGGCACCGT CAAGGCTGAGA-3';gapdhreverseprimer:5'-GGCATGGACT GTGGTCATGA G-3'; fos forward primer: 5'-TCTGGGTCCT TCTATGCAGC-3'; fos reverse primer: 5'-GGTGAAGACG AAGGAAGACG-3'. Primers were designed within the sequences represented on the human genome microarray and synthesized by Biospring (Frankfurt, Germany, http://www.biospring.de). The amplification efficiency of PCR products was determined by calculating the slope after semi-logarithmic plotting of the values against cycle number [30]. Differential expression was calculated in relation to 18s rRNA.

Online Supplemental Material
Online supplemental material is available at http://www.poliklinik-hd.de/HPC-and-cellular-microenvironment. This section includes (a) time-lapse recordings of different fractions of HPCs cultivated either with or without AFT024; (b) demonstration of directed migration of CD34+/CD38– cells toward AFT024 (experimental setting and time-lapse recordings are presented); (c) comparison of cell morphology of CD34+/CD38– and CD34+/CD38+ cells cultivated either with or without AFT024; (d) parameters for the separation of HPCs after cocultivation on AFT024; (e) microarray data for 2455 ESTs in eight gene clusters, as presented in Figure 5Go, including Gene Ontology terms; and (f) supplemental information for the comparison of our cocultivation data with two previous published microarray studies on CD34+ cells of the bone marrow versus peripheral blood. Log2 values of differential expression ratios are compared, and the corresponding datasets are provided.



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  		Figure 5. Kinetics of differential expression. (A): The number of differentially expressed sequences increased with the time of cocultivation in the four independent experiments (log2 ratio > 1 or < –1 in the two color-flip hybridizations; log2 ratio > standard deviation). (B): A total of 2,455 sequences that passed these filter criteria at at least one time point were further analyzed by K-means clustering to assess differential expression in the time course.

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Behavior of HPC upon Cocultivation with AFT024 Stroma
Using our time-lapse monitoring system, we have analyzed podia formation, migratory activity, and adhesive behavior of HPCs. When deposited as individual CD34+/CD38– or CD34+/CD38+ cells in a 96-well plate, they were round cells with no visible podia formation. They did not show migratory activity and ultimately died within 8 hours. However, when more than 20 cells were deposited in a 96-well plate, this homotypical interaction induced podia formation and directed locomotion was observed, as previously described [18, 22]. A difference in the morphologic phenotype of CD34+/CD38– and CD34+/CD38+ cells has not been observed (see online Supplemental Material).

Directed migration of CD34+/CD38– cells toward AFT024 was demonstrated in an assay with inclination of the culture plate. AFT024 cells were grown in one corner of a Terasaki well by being slanted at an angle of 30 degrees. Approximately 200 to 500 CD34+/CD38– cells were then deposited in the opposite edge of the well and observed under a microscope that was tilted at an angle of 5 degrees. Time-lapse camera monitoring showed that after 2–5 hours, the HPCs started to migrate uphill toward the AFT024 cells and adhered to the stroma cells (Fig. 1Go). This directed migration of CD34+/CD38– cells to AFT024 cells was observed in 7 out of 12 cord blood samples. In contrast, migration to HS68 fibroblasts has not been observed in all six experiments with three different cord blood samples even after more than 10 hours (see Online Supplemental Material). However, HPCs adhered to HS68 once they were brought in direct contact with each other.



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  		Figure 1. Directed migration toward AFT024. Migration of hematopoietic progenitor cells toward stroma cells against a gravity gradient of 5-degree inclination of the culture plate was observed by time-lapse microscopy. CD34+/CD38– cells (white arrows) were initially seeded in the lower half of a Terasaki well (A). They migrated within 2 hours toward the AFT024 cells (black arrows) and established stable cell–cell contact with these supportive stroma cells (B, C) (scale bar = 100 µm). The corresponding time-lapse video is provided in the supplemental material (see Online Supplemental Material).

 
In the next step, we have analyzed podia formation and migration of HPCs upon heterotypical cell-cell contact with supportive feeder layer cells. The CD34+/CD38– population was divided into two fractions and incubated, either with or without AFT024 cells. The experiments were repeated six times using HPCs derived from independent samples (approximately 2 x 103 cells in each well). Under all culture conditions, only a very small number (<3%) of HPCs divided or became apoptotic during the observation period of 24 hours. However, those cells that divided seemed to establish intimate contact with the underlying stroma cell before cell division, extending long membrane protrusions on both poles before they rounded off and ultimately divided. Cell morphology and podia formation was assessed in the beginning of each recording. There was a trend toward more uropod-forming HPCs upon cocultivation with AFT024 (21% ± 18% without AFT024 versus 32% ± 16% with AFT024), but the difference was statistically not significant (p = .13; see Online Supplemental Material). However, upon contact with AFT024, there seemed to be higher migratory activity in HPCs as they crawled toward and over the adherent feeder cells. Especially those cells that formed prominent uropods touched and felt for contact with the surface of AFT024 cells through lamellipodia that were quickly retracted and extended at the leading edge (see Online Supplemental Material). In contrast, the uropod at the trailing edge was retracted and extended much more slowly. Approximately 30% of the CD34+/CD38– cells remained attached by the uropod to the surface of an AFT024 cell for time periods ranging from 30 minutes to more than 6 hours (Fig. 2Go). CD44 and CD34 were colocalized at this site of adhesion in both CD34+/CD38– and CD34+/CD38+ cells (Fig. 2Go). Other HPCs, especially those with elongated morphology, crawled under the AFT024 layer. These cells migrated faster compared with those that have established contact with stroma cells through uropods.



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  		Figure 2. Adhesion of CD34+/CD38+/– cells to AFT024 cells is mediated by the uropod (indicated by the arrowheads). Most of the cells with a prominent pseudopodium seemed to adhere to the feeder layer with this podium and often stayed attached for the whole observation period of >2 hours (A–H) (0–30 minutes; CD34+/CD38– [PKH26; red]; confluent AFT024 layer [PKH2; green]). CD44 (green) and CD34 (red) are localized at the site of contact at the tip of the uropod on both (I–L) CD34+/CD38– cells and (M–R) CD34+/CD38+ cells.

 
Our observation demonstrated that, upon exposure to AFT024, CD34+/CD38– cells showed higher migratory activity that was directed toward stromal cells. They rapidly sought contact with AFT024 cells and established stable contact with the latter through a uropod.

Gene Expression Profiles of HPCs and AFT024
Approximately 5 x 104 CD34+/CD38– cells were isolated from fresh human umbilical cord blood and divided into two fractions that were cocultured either with AFT024 cells or without. The cells of four individual experiments were harvested after 16, 20, 48, and 72 hours of coculture. Efficient separation of the CD34+/CD38– cells from the feeder layer cells could be achieved by a second FAC sort (Fig. 3Go; see Online Supplemental Material). Approximately 2 x 104 cells could be retrieved after coculture with or without AFT024. A total of 20–50 ng of RNA was extracted from each fraction and amplified in two rounds of in vitro transcription. We obtained approximately 80 µg of aRNA with a continuous spectrum of 200 to 700 base pairs. Cohybridization experiments of the corresponding fractions were performed using the Human Genome Microarray described above. Color-flip experiments were performed for each hybridization to compensate for any dye-specific effects, resulting in a total of eight cohybridization datasets.



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  		Figure 3. Experimental design. CD34+/CD38– cells were isolated from human umbilical cord blood. Half of this fraction was cultivated either without feeder layer or with AFT024 to assess the influence of this cellular microenvironment on the gene expression. After an incubation time of 16, 20, 48, or 72 hours, the cells were harvested by rigorous pipetting. Hematopoietic progenitor cells and feeder-layer cells were then separated by a second FACS according to forward-scatter, side-scatter, exclusion of PI-positive cells, and residual anti–CD34-APC fluorescence. Measures of the flow cytometry plots are in log10 fluorescence. Abbreviations: APC, allophycocyanin; FACS, fluorescence-activated cell sorting; PE, phycoerythrin; PI, propidium iodide.

 
As preliminary experiments, we have compared the gene expression profile of AFT024 cells against CD34+/CD38– cells. This comparison enabled us to exclude the possibility that the differential expression after cocultivation might be caused by contamination. Two samples of approximately 50 ng RNA derived from the AFT024 cells were amplified. Hybridizations of the two AFT024 samples versus CD34+CD38– samples and the corresponding color-flip experiments resulted in four cohybridization datasets. The scatter plot analysis of the murine AFT024 cells versus human HPCs showed a very heterogeneous gene expression pattern (Fig. 4Go). A group of 348 spots showed a more than fourfold higher signal in AFT024 versus CD34+/CD38– cells in the four corresponding data sets (log2 ratio > 2, FDR = 1). In contrast, the gene expression pattern of CD34+/CD38– cells that were cultivated with AFT024 versus cells without cocultivation was rather homogenous, and the 348 spots that were highly expressed in AFT024 cells were not upregulated after cocultivation. No correlation was observed between these two microarray experiments. The Pearson’s correlation coefficient of the mean log2 ratios in all 51,147 spots of the two different experiments was r = –0.008. Thus, the gene expression profile after coculture and separation by cell sorting was not caused by contamination.



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  		Figure 4. Scatter plot analysis. These scatter plots show the relative signal intensities of the red and green channels of representative experiments with the human genome microarray (only one of two slides demonstrated). This allows global survey of differential gene expression. Lines indicate twofold, fourfold, and eightfold ratio of upregulation or downregulation. Dark spots represent spike in controls. (A): CD34+/CD38– cells (hematopoietic progenitor cells [HPCs]) that were cultivated without feeder layer in comparison with cultivation with AFT024 for 20 hours revealed a low number of differentially expressed genes. (B): In contrast, cohybridization of human HPCs and murine AFT024 cells demonstrated a quite heterogeneous pattern. Data analysis did not reveal any correlation between these two experiments. Thus, differential gene expression of HPCs after cocultivation is not based on contamination by RNA of AFT024 cells. Measure of scatter plots is log10 fluorescence in the Cy3 and Cy5 channel.

 
Gene Expression Profiles of HPCs upon Coculture
The influence of exposure to irradiated stroma cells on the gene expression profiles of CD34+/CD38– cells was analyzed. For each defined time interval of cocultivation (16, 20, 48, and 72 hours), genes that were at least more than twofold upregulated in both corresponding color-flip experiments (mean log2 ratio > 1 or < –1; SD < mean log2 ratio) were noted. A total of 2,455 spots fulfilled these criteria in at least one time point. Figure 5Go demonstrates the increase in number of differentially upregulated genes with each of the time intervals of cocultivation. Differential expression within the time course is demonstrated by K-means cluster analysis according to Euclidean distance. A few genes showed the highest differential expression within the first hours of cocultivation. This group included chk1, egr1, and cxcl2 (cluster 1). DNA (cytosine-5-)-methyltransferase 1 (dnmt1) was highly upregulated at 48 hours (cluster 7). Other sequences were increasingly upregulated or downregulated during the observation period of 72 hours. Some sequences were upregulated only at 72 hours, and these included osteopontin (spp1) and major histocompatibility complex, class II, DR, which are associated with primitive HPCs [23]. CD44 was not upregulated (mean log2 ratio, –0.02 ± 0.22); however, localization at the side of contact as a result of rearrangement does not necessarily correlate with differential gene expression. The corresponding datasets for each of the eight clusters (including Gene Ontology terms) are available in the online supplemental material (see Online Supplemental Material).

We have selected those spots that showed an average of more than twofold higher differential expression over the whole four time points of the observation period. Ninety-five spots fulfilled this criteria (mean log2 ratio > 1, FDR = 6). Among these were 42 genes that have been well characterized and are listed in Figure 6Go. These genes included tubulin, ezrin, proto-oncogene proteins c-fos and v-fos, proliferating cell nuclear antigen (pcna), uracil-DNA glycolase (ung), gamma-glutamyl hydrolase (ggh), mini-chromosome maintenance deficient 6 (mcm6), and complement component 1 q subcomponent receptor 1 (c1qr1). In contrast, an at least twofold lower expression after coculture with AFT024 was found in 154 spots (mean log2 ratio < –1, FDR = 19). Among these were 13 genes that are well characterized, which are listed in Figure 6Go. They included genes coding for various hemoglobin sequences, collagenase type IV (mmp2), and elastin (eln).



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  		Figure 6. Differential gene expression in hematopoietic progenitor cells after cocultivation with AFT024. This table summarizes all characterized genes with a more than twofold mean differential expression in the four time points (mean log2 ratio > 1 or < –1 in eight cohybridization data sets).

 
To validate the microarray data, differential gene expression of 11 genes was examined by semiquantitative RT-PCR in at least three independent experiments. In accordance with observations in microarray analysis, gapdh, ggh, tuba1, tuba 3, dnmt, mcm6, fos, and chk1 showed higher expression in HPCs after coculture, whereas eln, mmp2, and gamma-globin (hbg) were downregulated (Fig. 7Go).



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  		Figure 7. Comparison of the microarray results and reverse transcription–polymerase chain reaction (RT-PCR) results of selected genes. Semiquantitative RT-PCR by LightCycler analysis was used to analyze differential gene expression of 11 genes in relation to 18s rRNA. Right-pointing bars indicate a higher expression in CD34+/CD38– cells upon coculture. Left-pointing bars indicate a higher expression upon cultivation without supportive cellular layer. Mean values and standard deviations of at least three experiments are provided.

 
Comparison with the Gene Expression Profile of Marrow-Derived CD34+ Cells
The gene expression profiles of CD34+ cells derived from bone marrow versus G-CSF–mobilized peripheral blood have been reported by other authors. The differential gene expressions observed in these studies might represent a consequence of their derivation from different microenvironments (bone marrow vs. peripheral blood). We have compared the differential expression of those genes that were highly expressed in the bone marrow versus peripheral blood with the results found in our present study. Corresponding genes were identified by identical Unigene names and mapping to Unigene clusters. If more than one cDNA on the Human Genome Microarray corresponded to the same gene, those spots with the lowest SDs were selected for further analysis. Comparing the datasets, we discovered corresponding spots for 50 of the 53 genes identified by Steidl et al. [31] and 48 of the 66 genes identified by Graf et al. [32]. Log2 ratios of the genes as presented in the corresponding manuscripts were then plotted against the mean log2 ratios, as observed in our experiments. Despite the differences in experimental designs, starting materials, and platforms, there were several genes that were upregulated in CD34+/CD38– cells upon coculture with stroma cells, as well as in CD34+ cells from the bone marrow. These included pcna, pold1, top2A, lig1, cks1b, mcm2, mcm6, and mlh1. Datasets of these comparisons are available as online supplemental material (see Online Supplemental Material).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
Although numerous studies have demonstrated the vital role of stroma feeder layers for maintenance of multipotency of HPCs in vitro [5, 8–10], little is known about the precise cellular and molecular mechanisms of this interaction. For human HPCs, many stroma cell preparations and cell lines, mostly derived from the bone marrow, have been shown to serve as a feeder layer and to maintain HPCs in an undifferentiated state with varying degrees of efficiency [11, 12]. Irradiated AFT024 feeder layer consistently support the growth and maintenance of human HPCs in vitro [13, 14, 33]. As a model system, we have used this cell line as a feeder layer and the CD34+/CD38– cell fraction that is highly enriched in HPCs [34–36]. In a series of reports, other investigators and our group have provided evidence that direct contact with irradiated AFT024 maintained the stem cell function of CD34+/CD38– cells, increased the number of asymmetric divisions, and recruited more CD34+/CD38– cells into cell cycle versus those exposed to cytokines alone [5, 14, 16, 17]. Here, we have used this well-defined model system to analyze behavioral and molecular changes upon interaction with a cellular microenvironment. Further studies are concurrently under way to characterize the sequelae of interactions between HPCs and other feeder layers, such as human mesenchymal stem cells and fibroblasts. These experiments will determine the specific influence of a supportive versus a nonsupportive stroma to exclude the possibility that the described changes were due to adhesion alone.

A few studies have shown that HPCs are characterized by rapid migratory activity and constantly changing morphology [18–22]. We have previously demonstrated that the SDF of CD34+/CD38– cells was further enriched in HPCs with self-renewing capacity and they displayed significantly more podia formation and migratory activity compared with the more committed progenitor cells found among the fast-dividing fraction [23, 37]. In this study, we have demonstrated that HPCs migrated toward AFT024 cells and subsequently established stable contact to stroma cells through the uropod. Similar experiments with HS68 fibroblasts, which do not support hematopoiesis, showed that no directed migration of HPCs toward HS68 cells could be observed. HPCs seeded directly on a cellular layer of nonsupportive HS68 fibroblasts also established stable contact. These results indicated that CD34+/CD38– cells migrate toward specific signals released by the supportive AFT024 cells. We have not been able to characterize these signals yet. These might be chemokines secreted by AFT024. However, direct contact through extremely elongated podia of up to 300 µm from HPCs might also play a role [22]. Brief interactions of podia with cells of stroma feeder layer and consistent extension of predominant proteopodia in the direction of a stroma cell have previously been reported by Frimberger et al. [20]. In contrast to our observations, they claimed that podia formation was observed only among mobile cells but not in cells that adhered to a stromal cell. Other authors have reported that the uropod is rich in various intercellular adhesion molecules and CD44 [19, 38, 39]. CD44 is known to bind fibronectin and hyaluronic acid and is essential for homing and proliferation of hematopoietic stem cells [21, 39–41]. We have found that CD44 and CD34 were colocalized at the site of contact with stromal cells in both CD34+/CD38– cells and CD34+/CD38+ cells. These results provide further evidence that the uropod plays a significant role in adhesion of HPCs and that this mechanism is not restricted to the very immature fraction of CD34+/CD38– cells. Thus, adhesion of HPCs is mediated by the trailing edge, whereas rapidly extended proteopodia at the leading edge get in touch with surrounding stromal cells. These observations indicate that directed movement is initiated along this axis and a series of adhesive interactions may guide HPCs to their niche in the bone marrow [42–45].

Several attempts have been made to identify the gene expression profiles of stem cells using microarray technology. In most of these studies, the target population was separated from their native stem cell niche before analysis [23, 46–49]. In a published meta-analysis of our previous work and three other studies on hematopoietic stem cells, we have shown that, despite the use of different starting materials, with derivation from different species, applying very different platforms and methods of analysis, an interesting overlap of genes predominantly expressed in primitive subset of HPCs could be identified [23]. This included fzd6, mdr1, rom (rbpms), jak3, and hoxa9. Other studies have focused on the specific molecular makeup of the hematopoietic stem cell niche. Hackney et al. [50] have analyzed the gene expression profiles of AFT024 cells in comparison with other fetal liver–derived lines of varying stem cell support. Several genes that potentially influenced stem cell function were highly expressed in AFT024 cells, underscoring the hypothesis that many pathways might be involved in supporting stem cell function [4, 50, 51].

In this study, we have analyzed changes in the global gene expression profile upon interaction with AFT024 stroma. Several upregulated genes play a role in cell adhesion, reorganization of cytoskeleton system, maintenance of methylation patterns, stabilization of DNA during proliferation, and repair. Adhesion molecules that were upregulated included CD69 (early T-cell activation antigen p60) that functions as a signal-transmitting receptor and plays a role in lymphocyte development [52]. Overexpression of genes coding for tubulin {alpha}, tubulin ß, and ezrin might indicate a reorganization of the cytoskeleton system upon interaction with the cellular microenvironment. Proliferating cell nuclear antigen (pcna) is involved in the control of DNA replication and interacts with DNA (cytosin-5)-methyltransferase (dnmt1) that was also upregulated after cocultivation with AFT024 [53]. Dnmt1 is responsible for maintaining methylation patterns during embryonic development [54–56]. Thus, upregulation of dnmt1 would prevent epigenetic changes in HPCs that could result in a loss of stem cell function. Fos protooncogene has a critical role in regulation of proliferation and differentiation and can regulate gene expression indirectly through alterations in DNA methylation through activation of dnmt1 [57]. Uracil-DNA glycosylase (ung) prevents mutagenesis by eliminating uracil from DNA molecules [58]. A few genes characteristic for primitive HPCs were again overexpressed, consistent with our previous report [23]. These included the receptor for the complement component molecule C1q (c1qr1) and HLA-DR. Osteopontin (spp1) was also upregulated 4.4-fold in HPCs (log2 ratio = 2.09) upon cocultivation with AFT024 for 72 hours. We have previously demonstrated that spp1 was expressed higher in CD34+/CD38– and SDF, characteristic of the primitive self-renewing fraction [23]. Osteopontin has previously been demonstrated to be highly upregulated in CD14+ cells upon 3 days of coculture with the stromal cell line HS-27a [59]. Among the gene sequences that were downregulated after cocultivation on AFT024 were various hemoglobin genes. Our previous experiments also demonstrated that various hemoglobin genes were expressed higher in the more committed progenitors, and these results indicate that HPCs cultivated without stroma showed an intrinsic propensity to differentiate along the erythrocyte lineage.

As a function of time after coincubation over 72 hours, an increasing number of genes were overexpressed versus CD34+/CD38– cells cultured in media alone. A few genes were upregulated already within the first 24 hours of cocultivation (16 and 20 hours), and these included the chk1 checkpoint homologue (or chek1), early growth-response protein 1 (egr1), and macrophage inflammatory protein-2-alpha precursor (cxcl2). Egr1 plays an essential role as a nuclear signal transducer and in myeloid differentiation [60], whereas cxcl2 (or mip2) acts as a cytokine [61]. Chk1 is an evolutionarily conserved serine/threonine kinase, which is essential for mammalian development and viability [62]. Although further activated in response to DNA damage or stalled replication, chk1 is active even in unperturbed cell cycles [63]. As such, chk1 is critically involved in restraining cyclin-B-cdk1 kinase activity and thereby in preventing premature mitotic entry of cells that have not yet completed S phase or corrected errors that have occurred during DNA replication [64, 65]. A centrosome-associated pool of chk1 is required to limit precocious centrosomal cdk1 activation [65]. Hence, centrosome-associated chk1 might control the proper timing of cell division upon interaction with stroma. Our observations provide the first insight into the regulating mechanisms upon interaction with a cellular microenvironment within the first hours. More comprehensive analysis with examination of the specific function of each of these genes will be the subsequent steps. However, microarray provides the essential initial information on which genes or family of genes are vital, and these results are compatible with observations of other authors.

Other authors have reported on the differential gene expression profiles of CD34+ cells derived from the bone marrow compared with those from G-CSF–mobilized blood [31, 32, 66–68]. As CD34+ cells derived from the bone marrow were probably still under the influence of the natural niche, we have compared our findings with the differential gene expression profiles described in these reports. Several genes showed overlapping upregulation. These included again genes involved in regulation of DNA stability during cell division and DNA repair: pcna, pold1, top2a, lig1, cks1b, mcm2, mcm6, and mlh1. Pcna has previously been reported to be expressed higher in CD34+ cells of the bone marrow versus peripheral blood [69]. Its binding partners DNA polymerase delta (pold1) and dnmt1 were among the highest upregulated genes in these comparisons. CDC28 protein kinase regulatory subunit 1B (cks1b), topoisomerase II (top2a), DNA ligase 1 (lig1), and DNA mismatch repair protein mlh1 have been shown to play an important role for maintaining the integrity of the DNA during replication and DNA repair [70]. DNA replication licensing factors mcm2 and mcm6 possess controlling function for DNA replication during the S phase. All of these observations support the notion that the most essential influence of a cellular microenvironment is to maintain DNA stability and that gene expression in CD34+ cells derived from the marrow was similar to those cocultured with AFT024 in vitro.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Our observations demonstrate directed migration of HPCs toward supportive stromal cells and their adhesion through the uropod. Gene expression analysis supports the hypothesis that upon exposure to a cellular environment, regulation of cell division, reorganization of the cytoskeleton system, maintenance of genetic stability, and methylation patterns represent the most essential steps. This study serves as a basis to further characterize regulative mechanisms of the stem cell niche.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
We thank Dr. Ihor Lemischka for donating the AFT024 cell line for this study, Dr. Bernhard Korn and the Resource Center and Primary Database (RZPD) for the supply of the IMAGE clones and their sequence verification, and Kerstin Horsch for excellent technical assistance. Supported by Deutsche Forschungsgemeinschaft (DFG) HO 914/3-2, Bundesministerium für Bildung und Forschung (BMBF) 01GN0107, NGFN2 EP-S19T01, and Siebeneicher Stiftung, Germany.


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 Results
 Discussion
 Conclusion
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