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发表于 2009-3-5 10:49 |只看该作者 |倒序浏览 |打印

a Department of Medicine V, University of Heidelberg, Heidelberg, Germany;

b Biochemical Instrumentation Program, European Molecular Biology Laboratory, Heidelberg, Germany, r8 K% |' i( R
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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@urz.uni-heidelberg.de; b: H  E+ }1 x7 j
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ABSTRACT& O* p6 D8 u7 U3 r

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 . Direct contact and communication between stem cells and cellular determinants in the microenvironment has been shown to play an essential role in this process . 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 . By analogy, cocultivation with feeder layer cells maintains the self-renewing ability of embryonic stem cell lines in vitro .

For hematopoietic progenitor cells (HPCs), many studies have demonstrated the vital role of a stroma feeder layer to maintain stem cell function in vitro . 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 . 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 . In comparison with human primary stroma cells, AFT024 cells have been shown to be more efficient and have generated consistent and reproducible results . 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 .
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HPCs are characterized by rapid migratory activity and constantly changing morphology . They have been shown to migrate rapidly toward stroma cells and to extend magnupodia in the direction of the latter . 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 . 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 . These different morphological types of pseudopodia might be associated with specific functions, including communication, adhesion, and homing to specific sites in the microenvironment.. ]- @) l! [  L
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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.# N4 @5 T. x1 U& }% x
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MATERIALS AND METHODS
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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 . A difference in the morphologic phenotype of CD34 /CD38– and CD34 /CD38  cells has not been observed (see online Supplemental Material).( }* ~( j! F* @1 }9 |! t3 B
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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. 1). 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.

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).
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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 (" ?/ W5 P' D+ X
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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– ; confluent AFT024 layer ). 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.
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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
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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. 3; 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.

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.: V% f; d; R% P7 j! u1 r7 w

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. 4). 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.0 _7 J( r0 {$ X5 h3 V

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 ) 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.
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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 4 \8 V+ w- b" o2 v  ?% I

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 6. 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 * a: r. Z+ Z. C3 k/ M
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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 9 [, O5 i9 R, }

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. 7).; i$ Z  c- U( m: B6 i4 h

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.( k/ N' f) K) B4 q' z

Comparison with the Gene Expression Profile of Marrow-Derived CD34  Cells; {8 \3 `; t1 D" g8 `

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.  and 48 of the 66 genes identified by Graf et al. . 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).
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DISCUSSION9 f! ?, K. G3 J9 \& X% ~' Q

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.2 y  D8 O; G3 v6 m5 \2 Q% n
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