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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

a 1st Department of Pathology,: X# C* D& w4 |+ C; I% S3 ]

b Regeneration Research Center for Intractable Diseases,( o1 R+ n8 H. `: D$ i% r' o
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c Department of Transplantation for Regeneration Therapy (Sponsored by Otsuka Pharmaceutical Co., Ltd.), Kansai Medical University, Moriguchi, Osaka, Japan;

d Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan;/ k! @' z" Q! x$ b) V9 V+ Z

e Department of Toxicology, School of Public Health, Jilin University, Changchun, Jilin, China;

f Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, California, USA
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Key Words. Neural cell adhesion molecule ? Pluripotent hemopoietic stem cells ? Bone marrow stromal cells ? PA6 ? Mouse( o/ F7 F0 u1 G7 P1 ~' `$ U/ m
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Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara@takii.kmu.ac.jp

ABSTRACT3 p( b7 w1 o3 p' g* H) X4 C
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In adult mammals, hemopoiesis is restricted to the extravascular compartment of the bone marrow (BM), where pluripotent hemopoietic stem cells (P-HSCs) and their clonogenic progeny associate intimately with distinctive stromal cell elements comprising the hemopoietic microenvironment or stem cell niches . Stromal cells produce various growth factors, cell adhesion molecules, and matrix proteins that contribute to the formation of stem cell niches, which govern the homing, growth, survival, and differentiation of HSCs . Growth factors produced by stromal cells include cytokines (e.g., monocyte colony-stimulating factor , interleukin-6, leukemia inhibiting factor , thrombopoietin, hepatocyte growth factor, and transforming growth factor ? ), ligands of receptor tyrosine kinase (e.g., stem cell factor  and Flt-3 ligand ), bone morphogeneic protein 4 , and sonic hedgehog . However, in vitro, it is difficult to enhance the self-renewal or expansion of P-HSCs and immature progenitor cells without stromal cells, even if all known exogenous growth factors and other materials are added to the culture . In contrast, long-term hemopoiesis can be maintained by only coculturing HSCs with stromal cells . Our recent reports have also demonstrated that successful BM transplantation depends on the cotransplantation of donor stromal cells ; stromal cells migrate into the recipient BM and spleen, where they support hemopoiesis. These findings have shaped the view that stromal cell–hemopoietic cell interactions in the marrow microenvironment are crucial for physiological hemopoiesis.

Current data suggest that two classes of molecules produced by stromal cells contribute to stromal cell–hemopoietic cell interactions and regulate hemopoiesis: hemopoietic growth factors and CAMs . However, stem cell niches and the mechanisms involved in controlling hemopoiesis remain largely unknown, although Zhang et al.  and Calvi et al.  have now confirmed that osteoblasts have a function in stromal cell–hemopoietic cell regulation in animals. To clarify the mechanisms underlying cell-to-cell interactions between HSCs and stromal cells, we took advantage of our previously established monoclonal antibodies (mAbs) against BM stromal cells . These mAbs were produced by inducing neonatal tolerance in rats using mouse BM stromal cell lines MC3T3-G2/PA6 (PA6) and PA6-mutant (PA6-M); the former has the capacity to support HSCs, whereas the latter has no such capacity. An anti-PA6 mAb inhibits pseudoemperipolesis and suppresses the proliferation of HSCs, suggesting that the anti-PA6 mAb reacts with molecules responsible for the interaction between HSCs and stromal cells.
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In the present study, we established a fetal BM-derived stromal cell line (FMS/PA6-P) expressing a high level of PA6 molecules, which has high hemopoiesis-supporting capacity. We report herein that neural cell adherion molecule (NCAM) is the molecule that reacts with anti-PA6 mAb and is related to the hemopoiesis-supporting capacity of stromal cells.0 k; g7 H; h: Y, w3 E5 m7 e3 `

MATERIALS AND METHODS
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Establishment and Characterization of the FMS/PA6-P Cell Line7 \/ D. U3 X$ w- f$ y

PA6-positive adherent cells sorted from the third passage of the fetal BM adherent cells were cultured in a flask containing DMEM supplemented with 10% FBS and LIF (10 ng/ml). Interestingly, although the cells were dormant for about 2 months, several cells showing the same morphology began to proliferate thereafter and formed several large colonies 2 weeks later. They were subcultured, and a cell line (FMS/PA6-P) was thus established. The FMS/PA6-P cell line showed fibroblastic morphology, although BM adherent cells showed heterogeneous morphology. The FMS/PA6-P cell line proliferated rapidly with a doubling time of 1.5 days and stopped dividing at confluency. The FMS/PA6-P cell line exhibited continuous growth and retained a stable morphology for more than 25 passages. The FMS/PA6-P cell line was negative for hematolymphoid and endothelial lineage markers (Fig. 1). However, the FMS/PA6-P cell line reacted with anti-CD44, Sca-1, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and very late antigen 4 (VLA-4) mAbs (Fig. 1), which is characteristic of hemopoiesis-supporting stromal cells . Most interestingly, a high expression of molecules recognized by the anti-PA6 mAb was observed in FMS/PA6-P cells until approximately the 20th passage.
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Figure 1. Flow cytometric analysis of FMS/PA6-P cell line. The FMS/PA6-P cells of different passages were collected by trypsin-EDTA treatment and stained with fluorescent isothiocyanate anti–ICAM-1, VCAM-1, CD11c, Mac-1, Sca-1, CD45, CD3, CD4, CD8, and PE anti-CD11a, CD31, VEGFR-2 (BD Biosciences Pharmingen). They were also stained with anti-PA6, CD44, VLA-4 mAbs, followed by PE anti-rat immunoglobulin G. The stained cells were analyzed using a FACScan. The FMS/PA6-P cell line reacts with anti-PA6, CD44, Sca-1, VCAM-1, ICAM-1, and VLA-4 mAbs but not with mAbs against surface markers of hematolymphoid cells and endothelial cells. The closed profile indicates the cells stained with isotype-matched control mAbs. Abbreviations: ICAM-1, intercellular adhesion molecule 1; mAB, monoclonal antibody; PE, phycoerythrin; Sca-1, stem cell antigen-1; VCAM-1, vascular cell adhesion molecule 1; VEGFR-2, vascular endothelial growth factor receptor 2; VLA-4, very late antigen 4.# u) \  }( D" B5 ?
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Hemopoiesis-Supporting Capacity of the FMS/PA6-P and Other Stromal Cell Lines In Vitro
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When HSCs of B6 mice were cocultured with the FMS/PA6-P cell layers without cytokines, they crawled under the stromal layer and proliferated. The proliferating cells demonstrated a cobblestone-like appearance and were referred to as CAFCs. To compare the capacity of FMS/PA6-P (eighth or 22nd), MS-5, and PA6 to sustain long-term hemopoiesis, HSCs were cultured on a monolayer of these cell lines without adding exogenous cytokines. After 7-day culture, not only were the counts of cobblestone colonies significantly higher, but the size was also larger in cultures of HSCs on FMS/PA6-P cells (eighth) than other stromal cell lines (data not shown). Hemopoiesis can be maintained for only 8 weeks on the FMS/PA6-P (22nd), MS-5, and PA6, whereas a high number of cobblestone colonies were still observed in the culture on the FMS/PA6-P (eighth) cells thereafter, and hemopoiesis could be maintained on the FMS/PA6-P (eighth) up to 26 weeks. Furthermore, as shown in Table 1, HSCs cocultured with FMS/PA6-P (eighth) showed an approximately 34.7-fold increase in total cells and 8.2-fold increase in Sca-1  CD45  cells after 4 weeks. The numbers of total cells and Sca-1  CD45  cells were significantly higher in cultures with FMS/PA6-P (eighth) than those with FMS/PA6-P (22nd), MS-5, or PA6.% v* V( a( y) I. X

Table 1. Different hematopoiesis-supporting capacities of stromal cell lines

Requirement for Cell-to-Cell Contact Between HSCs and Stromal Cells

We examined the effect of interruption of cell-to-cell contact between HSCs and stromal cells on the expansion of HSCs. As shown in Figure 2, the numbers of total hemopoietic cells, Sca-1 CD45  cells, and total CFU-C were significantly lower in non-contact cultures than those in contact cultures; HSCs cocultured with the FMS/PA6-P cell line showed approximately 2.3- and 5.3-fold increases in total hemopoietic cells and Sca-1 CD45  cells, respectively. These results demonstrated that the direct cell-to-cell contact between HSCs and FMS/PA6-P cells is essential for the maximum expansion of HSCs and progenitor cells.
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Figure 2. Effect of interruption of stromal cell contact on ex vivo expansion of HSCs. HSCs (3 x 104) were cultured on a monolayer of FMS/PA6-P (contact) or on a transwell insert placed above the stromal layer (noncontact) for 10 days and then analyzed. Data are expressed as the mean ± SD of five wells. *p & W  q+ }% \4 H" h

Expression of Cytokines and PA6 Molecule in Various Stromal Cell Lines7 r& |6 M+ r1 Q0 M4 A; v( w% ^

To further analyze the mechanism regulating the hemopoiesis-supporting ability, we first examined the cytokine expressions in various cell lines. As shown in Figure 3A, not only Flt3-L  and basic fibroblast growth factor  (which promote cell growth) but also LIF  and TGF-?1  (which negatively regulate hemopoiesis) are found in all of the stromal cell lines. In contrast, the expression level of SCF, macrophage inflammatory protein 1, insulin-like growth factor-type 1, and granulocyte-CSF (G-CSF) differed greatly among these cell lines. No difference was observed between the eighth and 22nd passage of FMS/PA6-P, although the eighth passage showed better supporting ability than the 22nd passage. The cell line FMS/NS showed no expression of G-CSF and SCF. Accordingly, it seems that both G-CSF and SCF are responsible for the hemopoiesis-supporting ability. However, G-CSF is not produced from MS-5 and PA6, indicating that G-CSF is not an essential cytokine for supporting capacity. Therefore, the differences in cytokine expression are not enough to explain the better hemopoiesis-supporting capacity in FMS/PA6-P (eighth).
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Figure 3. Expressions of PA6 molecule and cytokines in various stromal cell lines or BM adherent cells. (A): mRNA expression of cytokines in various stromal cell lines. The FMS/PA6-P (eighth and 22nd), MS-5, PA6, FMS/NS cells cultured in 25-cm2 flasks were irradiated under the same conditions as we had investigated the in vitro hemopoiesis-supporting effect of these stromal cell lines. Expression of the indicated cytokines in various stromal cell lines was analyzed using reverse transcription–polymerase chain reaction. (B): Flow cytometric analysis of PA6 expression in various cell lines and BM adherent cells. The closed profile indicates the cells stained with isotype-matched control antibodies. (C): Western blotting analysis of PA6 expression in various cell lines. PA6 molecules were expressed in a higher amount in the cell lines and BM adherent cells having the ability to support hemopoiesis than in those without such ability, and the most supportive cell line (FMS/PA6-P ) expressed the highest level of PA6 molecule. Abbreviations: bFGF, basic fibroblast growth factor; BM, bone marrow; Flt3-L, Flt-3 ligand; G-CSF, granulocyte-colony stimulating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGF-1, insulin-like growth factor type 1; LIF, leukemia inhibiting factor; MIP-1, macrophage inflammatory protein1; SCF, stem cell factor; TGF-?1, transforming growth factor ?1.

We next examined the expression of PA6 molecule in the FMS/PA6-P of different passages and other stromal cell lines by flow cytometry. As shown in Figure 3B, a high level of PA6 expression was observed in FMS/PA6-P until approximately the 20th passage, whereas a decrease in this level was observed thereafter. In contrast, no significant change was detected in the expression level of CD44, Sca-1, VCAM-1, ICAM-1, and VLA-4 among the FMS/PA6-P cells cultured for different periods (data not shown). Furthermore, although PA6 expression was observed in MS-5 and PA6 stromal cell lines, the expression level in MS-5 was lower than that in FMS/PA6-P cells (eighth) and the expression level in PA6 was even lower and was comparable with that in FMS/PA6-P cells (22nd). In the stromal cell lines showing no hemopoiesis-supporting capacity (FMS/NS), the expression level of PA6 was very low.) }( z: {4 V( ^

The PA6 molecule in various stromal cell lines was further detected by Western blotting. A molecule of approximately 140 kDa reacted with anti-PA6 mAb, and the most supportive cell line, FMS/PA6-P (eighth), expressed the highest level of PA6 (Fig. 3C), suggesting that the level of PA6 expression is positively correlated to the hemopoiesis-supporting capacity of the stromal cells.

Inhibitory Effect of Anti-PA6 Antibody for HSC Proliferation on FMS/PA6-P Cell Line
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When 1 and 0.2 μg/ml anti-PA6 mAb were added to the culture medium and 3 x 103 HSCs was inoculated on the FMS/PA6-P monolayer, the 3H-TdR uptake was only 23% and 21% of that of the isotype control (Fig. 4), respectively, reflecting that the proliferation of HSCs was suppressed to a great extent by the anti-PA6 mAb, although not completely.$ [4 G! s8 {3 H, Z4 a9 h! d
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Figure 4. Inhibitory effect of anti-PA6 mAb or anti-NCAM mAb on the proliferation of HSCs. Anti-PA6 mAb or anti-NCAM mAb at 0.2 and 1 μg/ml can significantly inhibit the proliferation of HSCs. *p

NCAM is the Molecule That Reacts with Anti-PA6 Antibody8 y+ z# d2 i2 @
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Since the PA6 gene has a deduced size of more than 3 kb, it is difficult to clone. We therefore attempted to obtain some peptide information on the PA6 protein using MALDI-TOF peptide mass fingerprinting. The molecules that react with anti-PA6 mAbs were identified by affinity chromatography on immobilized Sepharose columns coupled with anti-PA6 mAbs. The eluted materials were separated by SDS-PAGE and visualized by silver staining. A dominant protein band with an apparent molecular weight of 140 kDa was confirmed by Western blotting to be the molecules that react with anti-PA6 mAbs (Figs. 5A, 5B). Four of the 140-kDa protein bands were excised and digested with trypsin. The resulting peptides were analyzed by MALDI-TOF peptide mass fingerprinting. Sequences of the tryptic peptides exhibited 100% homology with NCAM (Table 2). Furthermore, anti-PA6 mAb does not react with the PA6 molecule after pretreatment with anti-NCAM mAb (Fig. 5C), indicating that the PA6 molecule is NCAM. Indeed, as shown in Figure 6, NCAM expression patterns of the FMS/PA6-P (eighth and 22nd), MS-5, PA6, and FMS/NS cell lines were similar to PA6 expression patterns of these cell lines in Figure 3B, confirming that the expression level of NCAM is positively correlated to the hemopoiesis-supporting capacity of stromal cell lines. This finding was further confirmed by an inhibitory effect of anti-NCAM mAb on the proliferation of HSCs cocultured with FMS/PA6-P cells, which is compatible with the inhibitory effect of anti-PA6 mAbs on the proliferation of HSCs (Fig. 4). In addition, although in very low amount, 180-kDa and 120-kDa protein bands were also detected among the proteins identified by affinity chromatography using anti-PA6 mAbs when the film was exposed for longer periods (data not shown).- i# S0 v+ U7 Y! u8 @, N
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Figure 5. Identification of the molecule that reacts with anti-PA6 mAb. (A): The FMS/PA6-P cell lysates were first precleared by immobilized bovine serum albumin columns and then loaded onto immobilized anti-PA6 mAb columns. Bound proteins were eluted with 0.5 M NH4OH, lyophilized, and subjected to SDS-PAGE using a 5%–20% polyacrylamide gel. After silver staining, a dominant 140-kDa protein band was noted to react with anti-PA6 mAb. (B): One percent of eluted proteins was subjected to SDS-PAGE at the same time, transferred onto polyvinylidene difluoride membrane, and analyzed by Western blotting. A 140-kDa protein band was revealed to react with anti-PA6 mAb with the highest intensity. (C): Inhibitory effect of anti-NCAM mAb against binding of anti-PA6 mAbs to PA6 molecule. Anti-PA6 mAb does not react with PA6 molecule on FMS/PA6-P cell line after pretreatment with anti-NCAM mAb. (1): Cells were only stained with anti-PA6 mAbs followed by PE anti-rat IgG. (2, 3): Cells were first incubated with (2) anti-NCAM mAb or (3) normal mouse IgG and then stained with anti-PA6 mAbs followed by PE anti-rat IgG. The stained cells were analyzed using a FACScan. Broken lines indicate the cells stained with isotype-matched control mAbs. Abbreviations: IgG, immunoglobulin G; mAb, monoclonal antibody; NCAM, neural cell adhesion molecule; PE, phycoerythrin; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel.6 v3 Z! q2 z, Q* x

Table 2. Identification of mouse NCAM protein by MALDI-TOF analysis9 G& h( ~! N" J6 N" M& d( j( d- c
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Figure 6. NCAM expression in various stromal cell lines. The FMS/PA6-P cells (eighth and 22nd passages), MS-5, PA6, and FMS/NS were stained with anti-NCAM monoclonal antibody, followed by phycoerythrin anti-mouse immunoglobulin G. The expression patterns of NCAM in various stromal cell lines were similar to those of PA6 molecule. The closed profile indicates the cells stained with iso-type-matched control antibodies. Abbreviation: NCAM, neural cell adhesion molecule., B5 Q) X1 ]2 u7 R# i
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NCAM Contributes to Hemopoiesis-Supporting Capacity of Stromal Cells

To investigate the role of NCAM in the hemopoiesis-supporting capacity of stromal cells, we repressed the expression of NCAM by an approach based on siRNA . Transfected NCAM siRNA specifically reduced NCAM expression in FMS/PA6-P cells expressing a high level of NCAM by 60% at the protein level 48 hours after infection, as measured by flow cytometry (Fig. 7A) and by nearly 80% at the mRNA level 24 hours after infection, as measured by RT-PCR (Fig. 7B). The maximum silencing was observed at 96 hours at the protein level (71%) (Fig. 7A) and 72 hours at the mRNA level (90%) (Fig. 7B). This finding confirmed that NCAM silencing occurred by reducing mRNA stability. Four days after transfection, NCAM expression in the NCAM siRNA-transfected FMS/PA6-P cells began to increase slightly. However, these cells maintained a low level of expression at least 13 days after transfection (data not shown).

Figure 7. Hemopoiesis-supporting activity of NCAM. (A, B): Suppression of NCAM expression by siRNA. (A): The FMS/PA6-P cells (10th passage), transfected with NCAM or control siRNA at the times indicated, were trypsinized and stained with anti-NCAM monoclonal antibody, followed by phycoerythrin anti-mouse immunoglobulin G. The tained cells were then analyzed using a FACScan. The broken lines indicate the cells stained with isotype-matched control antibodies. (B): Total RNA was extracted from the above cells at the times indicated and subjected to reverse transcription–polymerase chain reaction with NCAM and GAPDH primers. (1): FMS/PA6-P cells transfected with control siRNA; (2, 3, 4): FMS/PA6-P cells transfected with NCAM siRNA 24, 48, and 72 hours later. (C): Repression of NCAM in FMS/PA6-P cells suppresses LTC-IC frequency. The LTC-IC frequency is 1 out of 198 ± 21 HSCs in control siRNA-transfected FMS/PA6-P cells and 1 out of 614 ± 45 HSCs in NCAM siRNA-transfected FMS/PA6-P cells. (1): FMS/PA6-P cells transfected with control siRNA; (2): FMS/PA6-P cells transfected with NCAM siRNA. Error bars indicate the SD. (D): Repression of NCAM in FMS/PA6-P cells suppresses the formation of long-term CAFCs. NCAM siRNA-transfected FMS/PA6-P cells were irradiated, cultured with 1 x 103 Lin–Sca-1  HSCs, and retransfected weekly with siRNA. CAFCs were counted after 5 weeks. (1): FMS/PA6-P cells transfected with control siRNA; (2): FMS/PA6-P cells transfected with NCAM siRNA. Results are reported as a ratio of the value from control siRNA-transfected cells and are the mean ± SD from 12 wells. *p
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To assess the effect of NCAM silencing on the hemopoiesis-supporting capacity of stromal cells, the cells were irradiated 96 hours after transfection (the time of maximum silencing), and HSCs of serial cell dilutions (1 x 102 to 3 x 104 per well) were then inoculated. After 5 weeks of culture, the LTC-ICs were quantified by in vitro colony assay of all the cells collected from each well. The LTC-IC frequency was 1 of 198 ± 21 HSCs in the control siRNA-transfected FMS/PA6-P cells and 1 of 614 ± 45 HSCs in the NCAM siRNA-transfected FMS/PA6-P cells (Fig. 7C). The frequency of long-term CAFCs (Fig. 7D) was also suppressed by repression of NCAM in the FMS/PA6-P cells. Taken together, these results indicate that NCAM functions on the maintenance of HSCs.( [' U$ q1 j+ q2 B' S  A2 b5 ^
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DISCUSSION
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This work was supported by a grant from Haiteku Research Center of the Ministry of Education, a grant from the Millennium program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the Science Frontier program of the Ministry of Education, Culture, Sports, Science and Technology, a grant-in-aid for scientific research (B) 11470062, grants-in-aid for scientific research on priority areas (A)10181225 and (A)11162221, and Health and Labor Sciences research grants (Research on Human Genome, Tissue Engineering Food Biotechnology), the 21st Century Center of Excellence Program (Project Leader), the Ministry of Education, Culture, Sports, Science and Technology, a grant from the Department of Transplantation for Regeneration Therapy (Sponsored by Otsuka Pharmaceutical Company, Ltd.), a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd., and a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO). We thank S. Miura for conducting FACS sorting and K. Yasaka for helping to analyze peptide mass. We also thank Hilary Eastwick-Field and K. Ando for manuscript preparation.! {! N7 ?; e4 o+ z

DISCLOSURES
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The authors indicate no potential conflicts of interest.* e0 Q+ ~- R" J( ?2 R. R2 M

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