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First published online July 28, 2005
Stem Cells Vol. 23 No. 9 October 2005, pp. 1389 -1399
doi:10.1634/stemcells.2004-0343; www.StemCells.com
© 2005 AlphaMed Press

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Neural Cell Adhesion Molecule Contributes to Hemopoiesis-Supporting Capacity of Stromal Cell Lines

Xiaoli Wanga,e, Hiroko Hishaa,b,c, Shigeru Taketanid, Muneo Inabaa,b, Qiang Lia, Wenhao Cuia, Changye Songa, Tianxue Fana, Yunze Cuia, Kequan Guoa, Guoxiang Yanga, Hongxue Fane, Zhexiong Lianf, M. Eric Gershwinf, Susumu Ikeharaa,b,c

a 1st Department of Pathology,
b Regeneration Research Center for Intractable Diseases,
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;
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

Key Words. Neural cell adhesion molecule • Pluripotent hemopoietic stem cells • Bone marrow stromal cells • PA6 • Mouse

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{at}takii.kmu.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To clarify mechanisms underlying cell-to-cell interactions between hemopoietic stem cells (HSCs) and stromal cells, we established a stromal cell line (FMS/PA6-P) from day-16 fetal bone marrow (BM) adherent cells using an anti-PA6 monoclonal antibody (mAb) specific for BM stromal cells. Importantly, this FMS/PA6-P cell line, showing homogenous fibroblastic morphology, is absent from hematolymphoid and endothelial lineage markers and maintains a high level of expression of PA6 molecule, recognized by the anti-PA6 mAb, for approximately 20 passages. Further, the cell line expressing a high level of PA6 molecule has a better hemopoiesis-supporting capacity in vitro than other stromal cell lines such as PA6 and MS-5. In fact, the PA6 molecule is closely related to the hemopoiesis-supporting capacity of the stromal cells because the proliferation of HSCs was suppressed to a great extent by the anti-PA6 mAb. Affinity chromatography and mass peptide fingerprinting revealed that the protein reacting with the anti-PA6 mAb is neural cell adhesion molecule (NCAM). The frequencies of long-term cobblestone area–forming cells and long-term culture-initiating cells were significantly suppressed by repression of NCAM in the FMS/PA6-P cells using NCAM small interfering RNA. Our findings clearly indicate that NCAM functions on the maintenance of HSCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 [1]. 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 [2–6]. Growth factors produced by stromal cells include cytokines (e.g., monocyte colony-stimulating factor [M-CSF], interleukin-6, leukemia inhibiting factor [LIF], thrombopoietin, hepatocyte growth factor, and transforming growth factor ß [TGF-ß]), ligands of receptor tyrosine kinase (e.g., stem cell factor [SCF] and Flt-3 ligand [Flt3-L]), bone morphogeneic protein 4 [7], and sonic hedgehog [8]. 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 [9–12]. In contrast, long-term hemopoiesis can be maintained by only coculturing HSCs with stromal cells [13–18]. Our recent reports have also demonstrated that successful BM transplantation depends on the cotransplantation of donor stromal cells [19–22]; 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 [23–26]. However, stem cell niches and the mechanisms involved in controlling hemopoiesis remain largely unknown, although Zhang et al. [27] and Calvi et al. [28] 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 [29]. 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.

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.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Seven- to 8-week-old C3H and C57BL/6 (B6) mice and pregnant B6 mice with 14-day fetuses were purchased from Shizuoka Experimental Animal Laboratory (Hamamatsu, Japan). All mice were maintained in a pathogen-free environment.

Establishment of FMS/PA6-P Cell Line
The femurs, tibias, and humeri of B6 fetuses on day 16 of gestation were removed and diced. The resultant bone pieces were inoculated in a 25-cm2 flask containing Dulbecco’s modified Eagle’s medium (DMEM) (low glucose [LG]) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2 in air. Half of the medium in the culture flask was replaced with fresh medium every 3 days. When the adherent cells reached 70%–80% confluence, they were collected by trypsin-EDTA treatment and subcultured with 2 x 105 cells per flask (25 cm2) until the third passage.

Fetal BM-adherent cells at the third passage were incubated with anti-PA6 mAb [29] and thence with phycoerythrin (PE) anti-rat immunoglobulin G (IgG) (Serotec Ltd., Oxford, U.K., http://www.serotec.com). PA6-positive BM-adherent cells were then sorted using EPICS ALTRA (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). PA6-positive BM adherent cells (1 x 105 cells per 25-cm2 flask) were cultured with DMEM (LG) containing LIF (10 ng/ml; Chemicon, Temecula, CA, http://www.chemicon.com), which is thought to be required for the successful expansion of rodent mesenchymal stem cells (also termed multipotent adult progenitor cells) [30]. When the PA6-positive BM-adherent cells reached 70%–80% confluence after weekly medium change, they were collected by trypsin-EDTA treatment and subcultured. Finally, two types of stromal cell lines were established: One demonstrated fibroblastic morphology and the other endothelial morphology. The former has the capacity to support hemopoiesis and was named FMS/PA6-P, whereas the latter has no supportive activity and was named FMS/NS. The phenotypes of FMS/PA6-P cell line were analyzed as described in the figure legends.

Purification of HSCs
Lin– BM cells were obtained from B6 mice as described previously [31], among which stem cell antigen 1 (Sca-1)–positive cells were contained at a concentration of 26%. Sca-1+ cells were separated from Lin– BM cells using a magnetic cell separation system (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany; http://www.miltenyibiotec.com). Lin– Sca-1+ cells were considered to be HSCs.

Culture of HSCs on the FMS/PA6-P Cell Line or Other Stromal Cell Lines
Stromal cell lines, such as MS-5, which is established by irradiating adherent cells in long-term BM cultures of C3H mouse [32], and PA6, a clonal preadipose cell line that is established from newborn mouse calvaria, have the capacity to support HSCs in vitro [33]. Therefore, we compared the hemopoiesis-supporting capacity of the FMS/PA6-P cells with these stromal cell lines. Monolayers of the confluent FMS/PA6-P cells (eighth or 22nd) or other stromal cell lines were prepared in flasks and irradiated (20 Gy), and the HSCs (1.3 x 105 in 8 ml Iscove’s modified Dulbecco’s medium [IMDM] containing 10% FBS) were then inoculated. Each week, the medium in the flask containing nonadherent cells was completely removed and replaced with fresh medium. The nonadherent cells were stained with fluorescent isothiocyanate anti–Sca-1 and PE anti-CD45 mAbs (BD Biosciences Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). The stained cells were analyzed by a FACScan (BD, Mountain View, CA). Sca-1+CD45+ cells were considered to be HSCs.

Transwell Culture
HSCs (3 x 104) were cultured on confluent FMS/PA6-P monolayer (eighth passage, 20-Gy irradiated, contact) or on a transwell insert (Intercell, KURABO, Osaka, Japan, http://www.kurabo.co.jp/english) placed above the stromal layer (noncontact) for 10 days. Nonadherent cells were counted and assessed in methylcellulose assay (Methocult GF M3434; Stem Cell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com).

Analyses of the Effect of Anti-PA6 or Anti-NCAM Antibody on the Proliferation of HSCs
Four hours after confluent FMS/PA6-P cells were irradiated (20 Gy), the culture medium was replaced with IMDM supplemented with 10% FBS and various concentrations (0.2 and 1 µg/ml) of anti-PA6 or anti-NCAM mAb (clone: N-CAM 13; BD Biosciences Pharmingen) or corresponding isotype mAbs. After reaction for 2 hours, 3 x 103 or 1 x 103 HSCs were added to the wells. The culture was then incubated for 6 days and pulsed with 0.5 µCi of 3H-TdR for the last 20 hours of culturing period. Results are presented as the mean counts per minute ± SD of 12 wells.

Cytokine Production
The production of cytokines was estimated using reverse transcription–polymerase chain reaction (RT-PCR) assays with previously defined primers [34–38]. Amplifications (30 to 35 times) were performed according to established protocols and using known controls [34–38].

Western Blotting Analyses of PA6 Expression in Various Stromal Cell Lines
Lysates of stromal cells were sonicated and denatured with sodium dodecyl sulfate (SDS) in the presence of ß-mercaptoethanol and proteins separated by SDS-polyacrylamide gel (SDS-PAGE); 20 µg of protein was loaded per lane. The separated proteins were transferred onto nitrocellulose membrane and stained with anti-PA6 mAb, and the membrane was stained with a secondary horse-radish peroxidase–labeled conjugate (chicken anti-rat; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com). Chemiluminescence detection was performed with the Amersham ECL plus Detection Kit (Amersham Biosciences UK Ltd., Bucking-hamshire, U.K., http://www.amershambiosciences.com).

Affinity Chromatography
Bovine serum albumin (BSA) (5 mg/ml; Sigma, St. Louis, http://www.sigmaaldrich.com) and rat anti-mouse PA6 mAb (5 mg/ml) were coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). Cell lysates of FMS/PA6-P cells were first loaded onto a BSA-coupled column, and then the precleared cell lysates were loaded onto a rat anti-mouse PA6 mAb-coupled column. Bound proteins were eluted with 0.5 M NH4OH and lyophilized. One percent proteins were subjected to SDS-PAGE using a 5%–20% polyacrylamide gel, transferred onto polyvinylidene difluoride membrane, and analyzed by Western blotting. The remaining proteins were divided into five fractions and simultaneously separated by SDS-PAGE.

Protein Identification by Peptide Mass Fingerprinting
Four 140-kDa protein bands, which were isolated by affinity chromatography and recognized by Western blotting using anti-PA6 mAb, were excised from silver-stained gels, digested with trypsin, desalted, and concentrated using Ziptipc18 (Millipore, Billerica, MA, http://www.millipore.com). The samples were eluted with 0.1 mg/ml {alpha}-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 1% trifluoroacetic acid, and the peptide masses were determined with a Voyager-DE PRO matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com). A peak list was compiled with Data Explorer and used for peak selection; the resulting peptide mass fingerprint was used to search the nonredundant NCBI protein database using the MS-Fit search engine (University of California at San Francisco).

Inhibitory Effect of Anti-NCAM mAb Against Binding of Anti-PA6 mAbs to PA6 Molecule
The FMS/PA6-P cell line was divided into four aliquots. The first aliquot was stained with PE anti-rat IgG. The second was stained with anti-PA6 mAbs, followed by PE anti-rat IgG. The third was stained with anti-NCAM mAbs and then anti-PA6 mAbs, followed by PE anti-rat IgG. The fourth was stained with normal mouse IgG and then anti-PA6 mAbs, followed by PE anti-rat IgG. The stained cells were analyzed using a FACScan.

siRNA Transfection
NCAM small interfering RNA (siRNA), annealed, desalted, and pooled and in the 2'-hydroxyl form, was provided by Dharmacon (siGENOME SMARTpool reagent M-044153-00-0005, mouse NCAM; Chicago, http://www.dharmacon.com). The FMS/PA6-P cells (8 to 10 passages) were trypsinized and plated in a 96-well plate at 3 x 103 cells per well for 18–24 hours before transfection. NCAM siRNA or control siRNA (Ambion, Austin, TX, http://www.ambion.com) was transfected into cells using the oligofectamine reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Cationic lipid complexes, prepared by incubating 2.5 pmol of indicated siRNA with 0.6 µl oligofectamine in DMEM (LG) with a total volume of 20 µl for 20 minutes, were added to the wells in a final volume of 100 µl in DMEM (LG). After 24 hours of incubation, 50 µl DMEM (LG) containing 30% FBS was added to the wells. NCAM gene and protein expression was analyzed 24 hours later. The following primers were used to detect NCAM: sense, CAGTCT-GTCACCCTGGTGTGTGATG; and antisense, TCTGGGGT-CACCTCCAGATAGC, with an expected product size of 734 bp.

Hemopoiesis-Supporting Assay of the FMS/PA6-P Cells Transfected with NCAM siRNA
For the long-term culture-initiating cell (LTC-IC) assay, a monolayer of FMS/PA6-P cells (in a 96-well plate) transfected with NCAM siRNA or control siRNA was irradiated (20 Gy) and various numbers of HSCs (3 x 104, 1 x 104, 3 x 103, 1 x 103, 3 x 102, 1 x 102 per well) were inoculated. Each week, the medium (IMDM containing 10% FBS) in the wells was completely removed and the cells were retransfected with the indicated siRNA. However, during retransfection, the culture was incubated with cationic lipid complexes for only 4 hours. After 5 weeks, all cells were trypsinized and subjected to an in vitro colony assay using Methocult GF M3434. The colony-forming unit in culture (CFU-C), including CFU-G, CFU-GM, and CFU-M, was determined after 12 days. The LTC-IC frequency was determined by Poisson statistics at 37% negative wells.

For the long-term cobblestone area–forming cell (CAFC) assay, 1 x 103 HSCs were inoculated on the FMS/PA6-P cells transfected with siRNA. The stromal cell line was transfected with siRNA every week, as described above. After 5 weeks, long-term CAFCs were counted under phase-contrast microscopy.

All experiments were carried out three or more times, and reproducible results were obtained. Representative data are shown in the tables and figures.

Statistics
Statistical differences in all experiments were analyzed by Student’s two-tailed t-test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment and Characterization of the FMS/PA6-P Cell Line
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. 1Go). 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. 1Go), which is characteristic of hemopoiesis-supporting stromal cells [27, 39–42]. 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.

 
Hemopoiesis-Supporting Capacity of the FMS/PA6-P and Other Stromal Cell Lines In Vitro
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 1Go, 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.


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  		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 2Go, 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 < .001; ***p < .05. Abbreviations: CFU-C, colony forming unit in culture; HSC, hemopoietic stem cell; Sca-1, stem cell antigen 1.

 
Expression of Cytokines and PA6 Molecule in Various Stromal Cell Lines
To further analyze the mechanism regulating the hemopoiesis-supporting ability, we first examined the cytokine expressions in various cell lines. As shown in Figure 3AGo, not only Flt3-L [43, 44] and basic fibroblast growth factor [45, 46] (which promote cell growth) but also LIF [47] and TGF-ß1 [48, 49] (which negatively regulate hemopoiesis) are found in all of the stromal cell lines. In contrast, the expression level of SCF, macrophage inflammatory protein 1{alpha}, 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 [eighth]) 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{alpha}, macrophage inflammatory protein1{alpha}; SCF, stem cell factor; TGF-ß1, transforming growth factor ß1.

 
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 3BGo, 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.

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. 3CGo), 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
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. 4Go), respectively, reflecting that the proliferation of HSCs was suppressed to a great extent by the anti-PA6 mAb, although not completely.



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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 < .001; **p < .01; ***p < .05. Abbreviations: 3H-TdR, 3H-thymidine; cpm, count per minute; HSC, hemopoietic stem cell; IgG, immunoglobulin G; mAb, monoclonal antibody; NCAM, neural cell adhesion molecule.

 
NCAM is the Molecule That Reacts with Anti-PA6 Antibody
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, 5BGo). 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 2Go). Furthermore, anti-PA6 mAb does not react with the PA6 molecule after pretreatment with anti-NCAM mAb (Fig. 5CGo), indicating that the PA6 molecule is NCAM. Indeed, as shown in Figure 6Go, 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 3BGo, 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. 4Go). 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).



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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.

 

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  		Table 2. Identification of mouse NCAM protein by MALDI-TOF analysis
 


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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.

 
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 [50]. 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. 7AGo) and by nearly 80% at the mRNA level 24 hours after infection, as measured by RT-PCR (Fig. 7BGo). The maximum silencing was observed at 96 hours at the protein level (71%) (Fig. 7AGo) and 72 hours at the mRNA level (90%) (Fig. 7BGo). 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).



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  		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 < .001. Abbreviations: CAFC, cobblestone area-forming cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSC, hemopoietic stem cell; LTC-IC, long-term culture-initiating cell; NCAM, neural cell adhesion molecule; siRNA, small interfering RNA.

 
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. 7CGo). The frequency of long-term CAFCs (Fig. 7DGo) 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.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The interaction between stromal cells and hemopoietic cells plays an important role in the proliferation and differentiation of HSCs and progenitors. However, because of the complexity of cell-to-cell interaction, the precise molecular mechanisms underlying the crosstalk between stromal cells and HSCs are relatively poorly defined. MAbs provide a useful means for isolating a defined population of cells from heterogeneous components. The anti-PA6 mAb is reactive with stromal cells but not hemopoietic cells [29]. Using this antibody, we established a fetal BM–derived and PA6-positive stromal cell line (FMS/PA6-P) expressing a high level of molecules that react with the anti-PA6 mAb and demonstrate that NCAM is the molecule that reacts with the anti-PA6 mAb and contributes to the hemopoiesis-supporting capacity of stromal cells.

The FMS/PA6-P cell line has a significant level of expression of the PA6 molecule and better capacity to support long-term hemopoiesis in vitro. This long-term hemopoiesis-supporting capacity cannot be attributed to the cytokines produced by the FMS/PA6-P cells because the conditioned medium shows much lower hemopoiesis-supporting capacity (Fig. 2Go), demonstrating that the FMS/PA6-P cell line is a suitable stromal cell line for studying cell-to-cell interactions between HSCs and stromal cells.

We therefore focused on the relationship between the expression level of PA6 and the hemopoiesis-supporting capacity of the stromal cells. The FMS/PA6-P cell line maintained a high level of PA6 expression until approximately the 20th passage and gradually decreased thereafter; the cells from the 22nd passage showed a decreased level of PA6 (Fig. 3BGo). In contrast, the expression of other surface molecules, including Sca-1, VCAM-1, ICAM-1, VLA-4, and CD44, did not change with an increase in the number of passages (data not shown). We have also found that the FMS/PA6-P cells gradually lose their ability to support hemopoiesis from the 19th passage and reduce to only one eighth of their original capacity at the 22nd passage (Table 1Go). The expression patterns of cytokines that are known to be related to the hemopoiesis were not significantly altered in different passages (Fig. 3AGo).

Some stromal cell lines, such as MS-5 [32] and PA6 [33], have been reported to have the capacity to support HSCs in vitro. If PA6 is really required for the support of hemopoiesis, all supportive cell lines should express this protein. We have found that the PA6 molecule is expressed positively in the third-passage fetal BM–adherent cells, MS-5 and PA6, but very weakly in a nonsupportive cell line, FMS/NS (Fig. 3BGo). We then compared the hemopoiesis-supporting capacity of FMS/PA6-P cells with that of MS-5 and PA6. Both the numbers of total cells and HSCs/progenitor cells in cultures on MS-5 were significantly lower than those in cultures on FMS/PA6-P showing high PA6 expression, although the numbers were higher than those in cultures on PA6 (Table 1Go). Moreover, the addition of anti-PA6 antibody to the culture medium significantly inhibited the proliferation of HSCs and progenitor cells (Fig. 4Go), further underscoring the importance of the PA6 molecule to hemopoiesis.

NCAM, a neural cell adhesion molecule of the Ig superfamily, is a transmembrane glycoprotein in mice [51]. It exists in three isoforms of different sizes, NCAM-180, -140, and -120, with identical extracellular domains. NCAM is expressed during development in a variety of tissues, including nonneuronal tissues, and in adults, it is expressed in neural cells, Schwann cells, skeletal myoblasts, cardiomyocytes, natural killer (NK) cells, spermcells, and thymocytes. Most research concerning NCAM has focused on the neural system because of its expression on the surface of almost all neural cell types throughout the central and peripheral nervous system, subserving neuron–neuron and neuron–glia adhesion [52, 53]. NCAM plays a pivotal role in early brain development, synaptic plasticity, and memory consolidation [54, 55] and promotes neurite outgrowth and fasciculation [56, 57]. Several earlier reports indicated that NCAM is a unique molecule with a homophilic binding like cadherin. In the hemopoietic system, however, its expression and function are not well elucidated except that NCAM is regarded as an NK cell marker. Here in, NCAM was uncovered as the molecule that reacts with anti-PA6 mAbs using MALDI-TOF mass peptide fingerprinting (Fig. 5Go, Table 2Go) and plays an important role in supporting HSCs (Figs. 7C, 7DGo). We can consider some possible ways by which the PA6 molecule (NCAM) would support the HSCs. First, it may support hemopoiesis through heterophilic interaction because we have found that NCAM is not expressed in any HSC/progenitor cells in the BM, including Lin–Sca-1+, Lin–CD34–/+, Lin–CD38–/+, or Lin–c-kitlow/+ (data not shown). It has also been reported that, in the neural system, NCAM can promote neurite outgrowth not only upon homophilic but also upon heterophilic interaction with other molecules on adjacent cell surfaces and in the extracellular matrix [52, 53]. For example, heterophilic interaction between NCAM and N-syndecan, a membrane-bound heparan sulphate (HS) proteoglycan, participates in the neuronal migration from the rat olfactory placode [58]. Because HS present on the surface of hemopoietic stromal cells has an important role in the control of adhesion and growth of hemopoietic stem and progenitor cells [59], it is possible that NCAM exerts its hemopoiesis-supporting effect through binding to HS. Second, NCAM might act directly on HSCs to maintain the HSCs in an undifferentiated state for a long time, like notch ligand [6]. Indeed, several reports show that the deficit in stromal cell–stem cell interaction results in the initiation of stem cell differentiation and a significant reduction in early stem cell numbers. Third, the interactions among stromal cells via NCAM may enhance the hemopoietic supportive capacity of the stromal cells by, for example, upregulating production and secretion of hemopoietic factors. However, the mechanism by which NCAM stimulates hemopoiesis in vitro and in vivo has not been elucidated. Our further investigation will focus on the ligand or receptor of NCAM on HSCs and the mechanisms underlying the interaction between HSCs and stromal cells via NCAM, which will improve our understanding of its function and the mechanisms by which the hemopoietic environment regulates hemopoiesis.

The FMS/PA6-P cells also expressed other surface molecules, such as Sca-1 [27], which functions as a regulator of hemopoietic progenitor cell proliferation, and VCAM-1 [39], ICAM-1 [40], and CD44 [41], which have been shown to mediate the interaction between hemopoietic cells and stromal cells. They play a critical role in normal hemopoiesis by providing signals to elicit the proliferation and differentiation of HSCs. These data suggested that these molecules combined with NCAM might facilitate the interaction between HSCs and FMS/PA6-P cells. We have also found that ICAM-1 is not expressed on MS-5 or PA6, but a high level of ICAM-1 is expressed in FMS/NS (data not shown), suggesting that ICAM-1 is not essential for the functional interaction between HSCs/progenitors and stromal cells. This is consistent with the reports of others [60].

In conclusion, we have shown that the FMS/PA6-P cell line has high hemopoiesis-supporting capacity in vitro, that NCAM is the molecule involved in the cell-to-cell interaction between HSCs and stromal cells, and that NCAM is required for the maintenance of an undifferentiated state of HSCs. This finding will be of great importance in further understanding the mechanisms underlying the interaction between HSCs and stromal cells and the regulation of hemopoiesis.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.

DISCLOSURES
The authors indicate no potential conflicts of interest.


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Received December 3, 2004; accepted for publication May 9, 2005.



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