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Version of Record online: 20 AUG 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 9, pages 1925–1937, September 2012
How to Cite
Mishima, K., Inoue, H., Nishiyama, T., Mabuchi, Y., Amano, Y., Ide, F., Matsui, M., Yamada, H., Yamamoto, G., Tanaka, J., Yasuhara, R., Sakurai, T., Lee, M.-C.-i., Chiba, K., Sumimoto, H., Kawakami, Y., Matsuzaki, Y., Tsubota, K. and Saito, I. (2012), Transplantation of side population cells restores the function of damaged exocrine glands through clusterin. STEM CELLS, 30: 1925–1937. doi: 10.1002/stem.1173
Author contributions: K.M. and I.S.: conception and design, Collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; H.I., T.N., Y.M., Y.A., F.I., M.M., H.Y., G.Y., J.T., R.Y., T.S., M.-C.-i.L., K.C., and H.S.: collection and assembly of data and final approval of manuscript; Y.K. and K.T.: data analysis and interpretation and final approval of manuscript; Y.M.: collection and assembly of data, data analysis and interpretation, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS July 10, 2012.
- Issue online: 20 AUG 2012
- Version of Record online: 20 AUG 2012
- Accepted manuscript online: 10 JUL 2012 04:16PM EST
- Manuscript Accepted: 14 JUN 2012
- Manuscript Received: 16 NOV 2011
- Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan
- Grants-in-Aid for Scientific Research
- Ministry of Health, Labor, and Welfare
Additional Supporting Information may be found in the online version of this article.
|SC-11-1087_sm_SupplTable1.pdf||10K||Supplemetary Table 1|
|SC-11-1087_sm_SupplTable2.pdf||13K||Supplemetary Table 2|
|SC-11-1087_sm_SupplFigure1.tif||2268K||Figure S1. BrdU-retaining cells in lacrimal and salivary glands of BrdU-injected mice 10 weeks after injection. (A) Immunofluorescence staining for BrdU. BrdU-positive stainings (green) were mainly found on the nuclei of ductal cells in both glands. (Scale bar, 10μm) (B) BrdU-positive cells were detected in cytospun specimens of SP and MP cells from lacrimal and salivary glands. Slides were counterstained with hematoxyline. HSY cells cultured for 24 hours with and without 20mM of BrdU were used as a positive control and a negative control, respectively. Positive stainings (arrow heads) were sparsely found in cytospin of SP and MP cells from both glands. Scale bar, 10μm.|
|SC-11-1087_sm_SupplFigure2.tif||563K||Figure S2. The ability of SP cells to produce microvessels in vitro and in vivo. (A) The SP cells isolated from the lacrimal and salivary glands by flow cytometry were cultured for 1 week. (B) Tube formation by SP cells isolated from lacrimal and salivary glands 1 week after seeding the cells on Matrigel. MVEC; mouse vascular endothelial cells. Scale bar, 20 μm. (C) Isolated SP cells, MP cells (10,000 cells), or PBS was directly injected into each gland 2 weeks after irradiation. Mice were sacrificed for analysis 8 weeks after the injection of SP cells, MP cells, or PBS. Sections of lacrimal and salivary glands were immunostained with anti-CD31 antibody. Capillary density is expressed as the number of CD31-positive features per high power field (×400). (D) Gene expression of CD31 was examined in SP cell- and PBS –injected salivary glands by real-time RT-PCR (normalised to GAPDH expression).|
|SC-11-1087_sm_SupplFigure3.tif||1557K||Figure S3. Histology of the glands in clusterin−/− mouse. There was no remarkable difference of histologies in lacrimal, submandibular, parotid, and sublingual glands between clusterin+/+ and clusterin−/− 12-week old male mice. Scale bar, 50 μm.|
|SC-11-1087_sm_SupplFigure4.tif||1309K||Figure S4. SP-cell characterization of salivary glands in clusterin−/− mice (n = 5). (A) The SP and MP regions isolated from salivary glands of clusterin-deficient mice are indicated by a quadrilateral and an oval, respectively. Clusterin-deficient mice have normal populations of SP and MP cells. (B) SP cells isolated from salivary glands of clusterin-deficient mice and of wild-type mice were labeled with CM-H2DCFDA, respectively. SP cells isolated from salivary glands of clusterin-deficient mice increased levels of CM-H2DCFDA fluorescecnce as compared with those of wild-type mice. (C) Flow cytometry of SP cells isolated from clusterin-deficient mice and wild-type mice, stained with anti-CD31 or anti-Sca-1 antibody. Surface expression of Sca-1 was weaker in SP cells isolated from salivary glands of clusterin-deficient mice than those of wild-type mice, while that of CD31 was not apparently different between them.|
|SC-11-1087_sm_SupplFigure5.tif||1437K||Figure S5. Lentivirus-mediated gene transfer into a mouse salivary gland cell line (MSG) and salivary glands. (A) Western blotting analysis of clusterin expression in Lenti-U6i and Lenti-Clu-infected MSGs. (B) Mice were sacrificed for analysis 1 week after injection of Lenti-U6i into salivary glands, and infected cells were identified by immunofluorescence staining using an anti-GFP antibody. Confocal microscopy revealed that the percentage of GFP-positive cells (red) was 16.4%. In addition, GFP-positive cells were mainly distributed in ductal cells. Scale bar, 50 μm. (C) Clusterin gene expression was compared between 1×106 TU Lenti-Clu- and 1×106 TU Lenti-U6i-injected salivary glands by real-time RT-PCR. Salivary glands were taken 1 week after injection of viruses, and clusterin gene expression was determined. All values are expressed relative to GAPDH expression. *P<0.01|
|SC-11-1087_sm_SupplFigure6.tif||835K||Figure S6. Recombinant clusterin production. His-tagged recombinant clusterin was produced in a baculovirus system and purified from Sf9 cell culture supernatant using nickel affinity resin (Ni-NTA). (A) The purified clusterin was stained with Coomassie blue. (B) The recombinant clusterin was confirmed by western blot analysis using an anti-clusterin antibody, which is a polyclonal antibody against the C-terminus of clusterin β-chains. sClu: secreted form of clusterin.|
|SC-11-1087_sm_SupplFigure7.tif||213K||Figure S7. Clusterin gene expressions of MP and SP cells in mice bone marrow, lung, liver, and Kidney. MP and SP cells were isolated from each organ by flow cytometry. Total RNA was extracted from MP and SP cells, respectively. After DNaseI treatment, cDNAs were synthesized with hexanucleotide random primers by M-MLV reverse transcriptase. Samples were then subjected to real-time PCR (SYBR Green, LightCycler; Roche) using specific primers.|
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