NCAM Contributes to Hemopoiesis-Supporting Capacity of Stromal Cell Lines

¹ First Department of Pathology, Kansai Medical University, Moriguchi, Osaka, Japan; Department of Toxicology, School of Public Health, Jilin University, Changchun, Jilin, China
² First Department of Pathology, Regeneration Research Center for Intractable Diseases, Department of Transplantation for Regeneration Therapy, Kansai Medical University, Moriguchi, Osaka, Japan
³ Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan
⁴ First Department of Pathology, Kansai Medical University, Moriguchi, Osaka, Japan, Regeneration Research Center for Intractable Diseases, Kansai Medical Univ., Moriguchi, Osaka, Japan
⁵ First Department of Pathology, Kansai Medical University, Moriguchi, Osaka, Japan
⁶ Department of Toxicology, School of Public Health, Jilin University, Changchun, Jilin, China
⁷ Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, California, USA

^* To whom correspondence should be addressed. E-mail: ikehara{at}takii.kmu.ac.jp.

	Abstract

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 cell (CAFC) and long-term culture-initiating cell (LTC-IC) were significantly suppressed by repression of NCAM in the FMS/PA6-P cells using NCAM siRNA. Our findings clearly indicate that NCAM functions on the maintenance of hematopoietic stem cells.

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