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发表于 2009-3-5 10:50 |只看该作者 |倒序浏览 |打印

a Department of Cell Biology and Genetics, Erasmus University, Rotterdam, Netherlands;# V6 T- j  ^2 ?! p2 }4 x" t

b Third Department of Internal Medicine, Technical University of Munich, Germany;( L. z4 U, r( X0 O4 @
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c Max Planck Institute of Molecular Genetics, Berlin, Germany9 S" J! p3 e+ \) J. S& {

Key Words. Hematopoiesis ? Hematopoietic stem cells ? AGM ? Transwell ? Microenvironment+ ]* ?) E: z6 X
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Correspondence: Robert A.J. Oostendorp, Ph.D., III Medizinische Klinik und Poliklinik, Klinikum Rechts der Isar, Technische Universit?t München, Ismaningerstrasse 22, 81675 München, Germany. Telephone: 49-89-4140-2362; Fax: 49-89-4140-4826, e-mail: oostendorp@lrz.tum.de$ u& X0 J& H. s: }/ N; p' R
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ABSTRACT
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The first definitive hematopoietic repopulating stem cells (HSCs) are autonomously generated within the embryonic aorta, gonads, and mesonephros (AGM) region . In the midgestation mouse embryo, these HSCs are found mainly in the major blood vessels, the dorsal aorta, and the arteries . Interestingly, HSCs expand dramatically between embryonic day 10.5 (E10.5) and E11 in the dorsal aorta and subsequently can be found in the urogenital ridges , yolk sac, and liver. Thereafter, the fetal liver shows a capacity to expand but not to induce additional HSCs . Thus, the AGM microenvironment plays a unique role in the establishment of definitive hematopoiesis.
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To delineate the molecular events involved in the induction and early events of definitive hematopoiesis within the AGM microenvironment, we and others have generated stromal cell lines from this region . These lines were shown to support cultures of murine fetal liver–derived , marrow-derived , and human cord blood–derived cells . More important, these lines maintained repopulating stem cells from these sources. Because for most of the lines nonsupportive counterparts were generated in parallel, these cell lines enable the comparison of gene expression patterns required for the maintenance of murine HSCs.

In a first comparison, most investigators focused on the expression of certain growth factors, which are thought to be of importance for the maintenance of hematopoiesis in culture. It was demonstrated that hematopoiesis-supportive AGM-derived stromal cells express mRNA for many growth factors . However, factors that could distinguish hematopoiesis-supportive stromal cells from nonsupportive cells were not identified. From our own studies , it appeared that supportive cell lines expressed the chordin-like gene, the product of which is involved in the regulation of the activity of bone morphogenic protein 4 . In addition, although a specific support-promoting cytokine could not be identified, hematopoiesis-supportive stromal cells more frequently expressed high mRNA levels of thrombopoietin (TPO), stem cell factor (SCF), and interleukin (IL)–6 . Differences in cytokine expression between supportive stromal cells are also apparent. For instance, the AGM-S3 line expresses high levels of oncostatin M (OSM) but no M-CSF , whereas the endothelial DAS104-4 line expresses M-CSF . The supportive lines we have generated (i.e., EL08-1D2 and UG26-1B6) fail to express OSM, but all express G-CSF , which is not found on AGM-S3 or DAS104-4. This divergence in growth factor transcript levels or gene expression patterns suggests that soluble factors like M-CSF, G-CSF, and OSM do not suffice for the maintenance of stem cells in culture.0 c: o! F0 m: R! @( b
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For the development of clinically applicable regimens for the maintenance or expansion of stem cells, many investigators use soluble factors. Commonly used factors are TPO, Flk2-ligand (FL), SCF, IL-6, G-CSF, and IL-3. Although some regimens have met with a degree of success, most investigators reported only a moderate expansion of stem cells in culture. Indeed, many reports stress the fact that it is difficult, if not impossible, to maintain stem cells in culture without the presence of a supportive feeder layer of cells. Whether the stem cells should be in contact with these stromal cells is still a matter of debate. Both the AGM-S3 and DAS104-4 cell lines were incapable of maintaining early progenitors from fetal liver  or cord blood CD34  cells  when they were not in direct contact with the stromal cells during culture. Previously, we did not investigate the ability of the stromal cells we have generated to maintain bone marrow HSCs in noncontact cocultures. In this study, we demonstrate that the urogenital ridge–derived UG26-1B6 and the embryonic liver–derived cell line EL08-1D2 both support the maintenance of adult bone marrow stem cells in noncontact cultures. Gene expression analyses of two hematopoiesis-supportive (UG26-1B6 and EL08-1D2) and four nonsupportive cell lines (UG15-1B7, AM20-1B4, EL28-1B3, and AM30-3F4) have revealed several putative secreted molecules that might be involved in the maintenance of stem cells in noncontact culture.+ k& _. |1 z2 U* R7 D6 v" s. q: H

MATERIALS AND METHODS

We have shown that HSCs from the CD31  c-kit  Ly-6C– (CD31 -K6) marrow fraction are maintained on the urogenital ridge–derived stromal clone UG26-1B6 . In the present study, we wished to use an even more HSC-enriched population to minimize the effects of proliferating and metabolically active mature hematopoietic cells on HSC maintenance. For this purpose, we combined the CD31 -K6 stain with Hoechst 33342 staining to obtain the side population (SP) cells, which represent approximately 0.04% of adult bone marrow cells (Fig. 1B). The c-kit  Ly-6C– bone marrow cells (Fig. 1C) (95% of these cells expressed CD31) are 20- to 50-fold enriched in SP cells (K6-SP cells, Fig. 1B). For the experiments described here, we used these K6-SP cells from adult bone marrow. The human ?-globin transgene served as the donor cell marker to detect repopulation after in vivo injection into irradiated adult recipients . As determined by in vivo transplantation, the frequency of HSCs in the K6-SP population was 1 in 10 (95% confidence interval, 1 in 14 to 1 in 7; Table 2) 16 weeks after transplant.8 ^9 z/ d$ _7 x" W! c; @
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Table 2. Frequency of repopulating cells in murine bone marrow K6-SP cells4 R8 Z+ x& _* e. L; h; E" z
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Previously, it was shown that the support of hematopoiesis in cocultures with two AGM-derived cell lines, AGM-S3 and DAS 104-4, depended on direct contact of stromal cells with the hematopoietic cells . However, UG26-1B6 and the cell line that supports maintenance of E11 HSCs  and human cobblestone area-forming cells , EL08-1D2, were not previously tested in noncontact cultures for support of colony-forming cells or HSCs. Thus, we cultured Lin- or K6-SP cells in collagen-coated transwell inserts above irradiated EL08-1D2 or UG26-1B6 for 4–6 weeks.

Both Contact and Noncontact Cultures with UG26-1B6 and EL08-1D2 Support Hematopoietic Repopulating Cells

To determine the ability of six different cell lines to maintain the ability of K6-SP cells to repopulate mice, K6-SP cells were cultured on irradiated stromal cells. In the present series of experiments, we used K6-SP cells instead of CD31 -K6 cells , and each culture contained approximately 30 repopulating cells instead of 3 to 6 in our previous work. The first colonies of proliferating cells appeared after 1–2 weeks of culture. These colonies proliferated rapidly thereafter. After 4 weeks of culture, complete cultures were harvested and transplanted into at least three irradiated recipients per culture. Six and 16 weeks after transplant, PCR analyses established that cocultures from both EL08-1D2 and UG26-1B6 contained cells with repopulating ability, both at 6 weeks (not shown) and 16 weeks after transplant, whereas cocultures with either AM20-1B4 or AM30-3F4 did not (Table 3). In all cultures, the frequency of repopulating cells decreased from 1 in 10 K6-SP cells to 1 in 19 to 25 input cells on EL08-1D2 and UG26-1B6 and below 1 in 85 to 125 for the aorta/mesenchyme-derived cells AM20-1B4 and AM30-3F4. Unexpectedly, we found that both EL08-1D2 and UG26-1B6 support the maintenance of repopulating ability when K6-SP was not in direct contact with the stromal cells (Table 3). In cultures on UG26-1B6, the frequency of repopulating cells was unchanged in contact and noncontact cultures (1 in 19 and 1 in 20, respectively). Interestingly, the frequency of repopulating cells in noncontact cultures on EL08-1D2 seems to remain higher (1 in 5 input K6-SP cells) compared with the direct contact cultures (1 in 25). These results demonstrate that HSCs need not be in direct contact with EL08-1D2 or UG26-1B6 to be maintained.8 [# L+ o# z% h
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Table 3. Maintenance of murine bone marrow K6-SP cells on embryonic stromal cells
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Macroarray Analysis of Gene Expression Patterns Between HSC-Supportive and Nonsupportive Stromal Cells

The above experiments suggest that HSC maintenance can be supported by soluble factors. What soluble growth factors are produced by embryonic stromal cell clones? Our earlier studies suggested that the chordin-like gene might be expressed at a higher level in HSC-supportive cell lines. Expression levels of other growth factors (TPO, SCF, FL, G-CSF, IL-1?, IL-6, IL-11, leukemia inhibitory factor, OSM, and transforming growth factor ?  were very similar between supportive and nonsupportive stromal cells .
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To find more distinctive markers between HSC-supportive stromal cells and HSC-nonsupportive cells, we performed macroarray-based gene expression analyses. When single cell lines are compared with other cell lines, many differentially expressed cells may be found. These genes usually represent differences in cellular lineages but do not necessarily provide information about a certain function of the cell lines〞that is, whether they can support HSCs. To minimize the possibility that only cell type–specific information would be obtained, we opted to compare the combination of both EL08-1D2 and UG26-1B6, which are of different cell lineage and origin, with four different cell lines derived from different embryonic regions that did not support repopulating cells in culture (Table 3) . Our analysis (see Materials and Methods) of this combination strategy revealed 31 genes to be differentially expressed between HSC-supportive and HSC-nonsupportive cell lines. Twenty of these genes are expressed at a higher level in the two supportive cells (Table 4), and 11 genes were expressed at a lower level (Table 5). Genes with highest (more than threefold) expression differences were fibroblast growth factor-7 (FGF-7), cathepsin K, thrombospondin 2 (TSP2), pleiotrophin (PTN), and the IGFBP-3 and -4." A1 O3 _. K8 p0 o' }7 A

Table 4. Combined gene expression profile of EL08-1D2 and UG26-1B6 compared with the combined gene expression profile of UG15-1B7, EL28-1B3, AM20-1B4, and AM30-3F4: genes expressed at a higher level! W# w" N/ {) J# C- Q# q
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Table 5. Combined gene expression profile of EL08-1D2 and UG26-1B6 compared with the combined gene expression profile of UG15-1B7 and EL28-1B3, AM20-1B4, and AM30-3F4: genes expressed at a lower level4 ^0 _4 Q+ m. F' F; }

The differential expression of many of the genes found in the macroarray analyses (Tables 4 and 5) could be confirmed by RT-PCR (Fig. 2A), real-time RT-PCR (Fig. 2B), and, in some cases, ELISA (Fig. 3). Although PTN mRNA was expressed by both EL08-1D2 and UG26-1B6 as confirmed by RT-PCR and real-time PCR (Figs. 2A and 2B, respectively), we detected secretion of this 17-kDa heparin-binding protein only in the conditioned medium of EL08-1D2 (data not shown). Also, IGFBP-3 message was found in all cell lines by RT-PCR (not shown), reflecting the fact that all cell lines show a relatively high level of gene expression (Table 4). On the protein level, however, only EL08-1D2 and UG26-1B6 were found to secrete high levels of IGFBP-3, and AM20-1B4 secreted medium levels compared with the other cell lines, confirming the difference found in the macroarray analysis (Fig. 3). The array analyses additionally revealed that another IGFBP, IGFBP-4, was strongly expressed in EL08-1D2, whereas expression in UG26-1B6 was only slightly higher than in the other four stromal lines, and this was confirmed by real-time PCR (Fig. 2B). Other RT-PCR and real time-PCR experiments confirmed the relative overexpression of TSP2 and cathepsin K in EL08-1D2 and UG26-1B6. Follistatin was expressed in all cell lines, but the highest expression was found in EL08-1D2, UG26-1B6, and EL28-1B3. In addition, the soluble frizzled-related protein sFRP-2 (secreted frizzled-related protein 2) was confirmed to be overexpressed in cell lines, supporting repopulating activity by real-time PCR (Fig. 2B). Moreover, we show that GPX3 and HSP27 are relatively over-expressed in cell lines that do not support repopulating cells in culture (Figs. 2A, 2B).
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Figure 2. Confirmation of macroarray analyses at mRNA level. The expression of some candidate HSC regulatory genes (Tables 4 and 5) was determined by (A) reverse transcription-PCR (B) and real-time PCR. Total RNA was isolated, and cDNA prepared using poly-T primer from confluent irradiated stromal cell lines as described in Materials and Methods. cDNA, 0.5 μg, was then amplified using the primers described in Table 1 by PCR. In the representation of the real-time PCR, results of the cycle numbers required for a half-maximum response(Ct) was calculated as the difference (Ct) between the Cts of the housekeeping gene RPL-p0 and that of the test molecule. The bars represent the mean and SE of the mean of three independent experiments (three different passages of the cell lines indicated). The black bars depict the HSC-supportive cell lines, and the white bars the cell lines that do not support maintenance of HSC in culture. The smaller the Ct, the higher the expression level. Negative Ct represents expression levels higher than the housekeeping gene (see for instance the level of IGFBP-4 in EL08-1D2). Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPX-3, plasma glutathione peroxidase 3; HSC, hematopoietic repopulating stem cell; HSP27, heat shock protein 27; IGFBP, insulin-like growth factor-binding protein; PCR, polymerase chain reaction; PTN, pleiotrophin; RPL-p0, acid large ribosomal protein p0; sFRP-2, secreted frizzled-related protein 2; TSP2, thrombospondin 2.
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Figure 3. Confirmation of macroarray analyses at the protein level. The expression of insulin-like growth factor-binding protein (IGFBP-3) was determined by enzyme-linked immunosorbent assay(ELISA). Proteins were isolated from confluent, irradiated stromal cell lines as described in Materials and Methods. Data plot of ELISA analysis of the IGFBP-3 content in conditioned media collected from irradiated stromal cells at different time points after the first complete medium change. Shown is the analysis of conditioned media from EL08-1D2 (closed squares), UG26-1B6 (closed circles), UG15-1B7 (open circles), AM20-1B4 (open diamonds), EL28-1B3 (half-filled squares), and AM30-3F4 (open triangles).
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These studies were supported by research grants from the Koningin Wilhelmina Fonds (KWF 2001-2004 to E.A.D.), the National Institutes of Health (NIH ROI DK54077-06 to E.A.D.), the Deutsche Forschungsgemeinschaft (SFB 456, project B2, to C.P.), the European Commission (LSHB-CT-2003-503161  to U.A.N. and R.A.J.O.), and the Klinikum Rechts der Isar, Munich (KKF 24-01 to R.A.J.O.). The authors would like to thank Corn谷 Snoys (Erasmus University Rotterdam) for the excellent flow cytometric sorting performed for these studies. We also express our appreciation for the Erasmus Animal Facility and Lien Braam for animal care." @6 p  G& O8 j) a# H% p! X

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