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Identification of side population cells of salivary glands
International Congress Series 1284 (2005) 83 – 84
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Identification of side population cells of salivary glands Kenji Mishima, Hiroko Inoue, Ichiro Saito * Tsurumi University School of Dental Medicine, Yokohama, Japan
Abstract. Side population cells (SP cells) have a highly enriched stem cell activity and can proliferate to regenerate damaged tissues. In this study, we identified SP cells in mouse salivary glands to apply them for the cell therapy of hypofunction in damaged salivary glands. SP cells were isolated from mouse salivary glands by flow cytometry, based on the efflux of fluorescent dye, Hoechst 33342. The frequency of the SP cells was approximately 0.5% and showed epithelium-like sheet formation on culture dishes. Isolated SP cells expressed Bcrp1, which is a marker molecule for SP cells from diverse sources. Here we identified and characterized SP cells in salivary glands for clinical uses. D 2005 Elsevier B.V. All rights reserved. Keywords: Tissue stem cell; SP cells; Salivary glands
Destruction of salivary glands was caused by Sjogren syndrome (SS) and radiation therapy for head and neck cancer. This leads to loss of secretory function in salivary glands, with which patients suffer from considerable morbidity. As a novel strategy to treat this condition, we have chosen to apply cell transfer therapy with stem cells. A technique based on the exclusion of the DNA binding dye Hoechst 33342 has been used to identify and purify, using a fluorescence-activated cell sorter (FACS), a subpopulation of cells, termed SP cells. Hoechst dye exclusion of SP cells is due to Bcrp1 [1]. Here, we identified SP cells in mouse salivary glands and examined the expression of Bcrp1. Salivary tissues obtained from C57BL/6J (6-week-old male) were digested for 1 h at 37 8C with collagenase and hyaluronidase. Following enzyme digestion, the salivary organoids were isolated and disaggregated in trypsin–EDTA. Isolated epithelial cells were incubated in 5 Ag/ml of Hoechst 33342, and subsequently, reserpine was added to * Corresponding author. E-mail address: [email protected] (I. Saito). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.014
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K. Mishima et al. / International Congress Series 1284 (2005) 83–84
Fig. 1. Flow-cytometric analysis (A) and RT-PCR analyses for Bcrp1 expression in SP and MP cells (B). (A) SP cells present in salivary glands and addition of reserpine prevented Hoechst exclusion from SP cells. (B) Bcrp1 expression of SP cells was higher than that of MP cells. Bone marrow was used for positive control.
samples. Cells were incubated for 90 min at 37 8C and SP cells and non-SP cells (main population, MP cells) were sorted on a BD FACS Vantage SE cell sorter. RT-PCR analysis of Bcrp1 in SP and MP cells were carried out. Density dot-plot analysis (Hoechst red vs. Hoechst blue) confirmed the presence of SP cells in salivary glands, (Fig. 1A). The formation of the SP population was blocked by reserpine, a potent functional inhibitor of several ABC transporters. SP cells comprised about 0.5% (range 0.2–1.0%) in salivary glands (Fig. 1A). The higher expression of Bcrp1 mRNA in SP cells was detected by RT-PCR analysis as compared with MP cells (see Fig. 1B). SP cells have been identified from various types of adult tissues [2–4]. In this study, we identified SP cells in salivary glands of adult mice and showed that these cells expressed a specific marker for stem cells, Bcrp1. This is consistent with previous reports and the characteristics of SP cells in salivary glands are similar to those of other organs. Furthermore, SP cells formed sheet-like structure from single cell by culture on plates, suggesting that some of the SP cells are derived from salivary epithelium and proliferative potentials (data not shown). However, it remains still unclear which factors can induce expansion of stem cells. Therefore, we have to investigate the candidate genes for stem cell expansion by evaluating the gene expression profile of SP cells for cell therapy using salivary stem cell. Acknowledgement This study was in part supported by a grant from the Japan Ministry of Health, Labour and Welfare (No 14165001). References [1] M.A. Goodell, et al., Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species, Nat. Med. 3 (12) (1997) 1337 – 1345. [2] E. Gussoni, et al., Dystrophin expression in the mdx mouse restored by stem cell transplantation, Nature 401 (6751) (1999) 390 – 394. [3] R. Summer, et al., Side population cells and Bcrp1 expression in lung, Am. J. Physiol. Lung Cell Mol. Physiol. 285 (1) (2003) L97 – L104. [4] K.A. Jackson, T. Mi, M.A. Goodell, Hematopoietic potential of stem cells isolated from murine skeletal muscle, Proc. Natl. Acad. Sci. U. S. A. 96 (25) (1999) 14482 – 14486.
www.ics-elsevier.com
Identification of side population cells of salivary glands Kenji Mishima, Hiroko Inoue, Ichiro Saito * Tsurumi University School of Dental Medicine, Yokohama, Japan
Abstract. Side population cells (SP cells) have a highly enriched stem cell activity and can proliferate to regenerate damaged tissues. In this study, we identified SP cells in mouse salivary glands to apply them for the cell therapy of hypofunction in damaged salivary glands. SP cells were isolated from mouse salivary glands by flow cytometry, based on the efflux of fluorescent dye, Hoechst 33342. The frequency of the SP cells was approximately 0.5% and showed epithelium-like sheet formation on culture dishes. Isolated SP cells expressed Bcrp1, which is a marker molecule for SP cells from diverse sources. Here we identified and characterized SP cells in salivary glands for clinical uses. D 2005 Elsevier B.V. All rights reserved. Keywords: Tissue stem cell; SP cells; Salivary glands
Destruction of salivary glands was caused by Sjogren syndrome (SS) and radiation therapy for head and neck cancer. This leads to loss of secretory function in salivary glands, with which patients suffer from considerable morbidity. As a novel strategy to treat this condition, we have chosen to apply cell transfer therapy with stem cells. A technique based on the exclusion of the DNA binding dye Hoechst 33342 has been used to identify and purify, using a fluorescence-activated cell sorter (FACS), a subpopulation of cells, termed SP cells. Hoechst dye exclusion of SP cells is due to Bcrp1 [1]. Here, we identified SP cells in mouse salivary glands and examined the expression of Bcrp1. Salivary tissues obtained from C57BL/6J (6-week-old male) were digested for 1 h at 37 8C with collagenase and hyaluronidase. Following enzyme digestion, the salivary organoids were isolated and disaggregated in trypsin–EDTA. Isolated epithelial cells were incubated in 5 Ag/ml of Hoechst 33342, and subsequently, reserpine was added to * Corresponding author. E-mail address: [email protected] (I. Saito). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.014
84
K. Mishima et al. / International Congress Series 1284 (2005) 83–84
Fig. 1. Flow-cytometric analysis (A) and RT-PCR analyses for Bcrp1 expression in SP and MP cells (B). (A) SP cells present in salivary glands and addition of reserpine prevented Hoechst exclusion from SP cells. (B) Bcrp1 expression of SP cells was higher than that of MP cells. Bone marrow was used for positive control.
samples. Cells were incubated for 90 min at 37 8C and SP cells and non-SP cells (main population, MP cells) were sorted on a BD FACS Vantage SE cell sorter. RT-PCR analysis of Bcrp1 in SP and MP cells were carried out. Density dot-plot analysis (Hoechst red vs. Hoechst blue) confirmed the presence of SP cells in salivary glands, (Fig. 1A). The formation of the SP population was blocked by reserpine, a potent functional inhibitor of several ABC transporters. SP cells comprised about 0.5% (range 0.2–1.0%) in salivary glands (Fig. 1A). The higher expression of Bcrp1 mRNA in SP cells was detected by RT-PCR analysis as compared with MP cells (see Fig. 1B). SP cells have been identified from various types of adult tissues [2–4]. In this study, we identified SP cells in salivary glands of adult mice and showed that these cells expressed a specific marker for stem cells, Bcrp1. This is consistent with previous reports and the characteristics of SP cells in salivary glands are similar to those of other organs. Furthermore, SP cells formed sheet-like structure from single cell by culture on plates, suggesting that some of the SP cells are derived from salivary epithelium and proliferative potentials (data not shown). However, it remains still unclear which factors can induce expansion of stem cells. Therefore, we have to investigate the candidate genes for stem cell expansion by evaluating the gene expression profile of SP cells for cell therapy using salivary stem cell. Acknowledgement This study was in part supported by a grant from the Japan Ministry of Health, Labour and Welfare (No 14165001). References [1] M.A. Goodell, et al., Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species, Nat. Med. 3 (12) (1997) 1337 – 1345. [2] E. Gussoni, et al., Dystrophin expression in the mdx mouse restored by stem cell transplantation, Nature 401 (6751) (1999) 390 – 394. [3] R. Summer, et al., Side population cells and Bcrp1 expression in lung, Am. J. Physiol. Lung Cell Mol. Physiol. 285 (1) (2003) L97 – L104. [4] K.A. Jackson, T. Mi, M.A. Goodell, Hematopoietic potential of stem cells isolated from murine skeletal muscle, Proc. Natl. Acad. Sci. U. S. A. 96 (25) (1999) 14482 – 14486.