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Article first published online: 22 MAY 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 6, pages 1149–1159, June 2013
How to Cite
Homma, K., Okamoto, S., Mandai, M., Gotoh, N., Rajasimha, H. K., Chang, Y.-S., Chen, S., Li, W., Cogliati, T., Swaroop, A. and Takahashi, M. (2013), Developing Rods Transplanted into the Degenerating Retina of Crx-Knockout Mice Exhibit Neural Activity Similar to Native Photoreceptors. STEM CELLS, 31: 1149–1159. doi: 10.1002/stem.1372
Author contributions: K.H.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; S.O.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; M.M.: collection and/or assembly of data and data analysis and interpretation; N.G., H.K.R., Y.-S.C., S.C., and W.L.: collection and/or assembly of data; T.C.: data analysis and interpretation and manuscript writing; A.S.: financial support, data analysis and interpretation, and manuscript writing; M.T.: conception and design, financial support, administrative support, data analysis and interpretation, and manuscript writing.
Disclosure of potential conflicts of interest is found at the end of this article.
first published online in STEM CELLS EXPRESS February 4, 2013.
- Issue published online: 22 MAY 2013
- Article first published online: 22 MAY 2013
- Accepted manuscript online: 14 MAR 2013 12:02AM EST
- Manuscript Accepted: 29 JAN 2013
- Manuscript Revised: 15 JAN 2013
- Manuscript Received: 16 FEB 2012
- Ministry of Education, Culture, Sports, Science, and Technology (MEXT)
- Ministry of Health Labor and Welfare, a grant-in-aid for Young Scientists (B)
- Japan Society for the Promotion of Science (JSPS)
- JSPS Postdoctoral Fellowships for Research Abroad (Kaitoku-NIH)
- National Eye Institute
- National Institutes of Health, Bethesda, MD, USA
Additional Supporting Information may be found in the online version of this article.
|sc-12-0163_sm_SupplFigure1.tif||264K||Supplemental Figure 1. Calcium imaging of rods at different developmental stages in slice culture preparations. (a) Postnatal day (P)5, (b) P8, and (c) adult retinas were sliced at 150 μm thickness. Retinal slices were stained with Fura-2 and stimulated with 80 mM KCl. (d) Increases in [Ca2+]i (nM) were calculated from each baseline. Data represent mean ± S.D. *p < 0.05.|
|sc-12-0163_sm_SupplFigure2.tif||727K||Supplemental Figure 2. Membrane current recording in rods at different developmental stages in slice culture preparations. Membrane current in Nrlp-eGFP positive cells was recorded at postnatal day (P)5 or P8 (a). Recorded cells were identified by Alexa568 in the electrode solution. (c-f) Membrane current of Nrlp-eGFP-positive cells in (c) P5 and (d) P8 retina were recorded by membrane voltage-clamp from -100 mV to 20 mV, with 10 mV increments. From (c) and (d), I-V curves were plotted in (e) and (f), respectively. (b) Hyperpolarization-activated current (Ih) at -100 mV and outward current at 10 mV in developing rods were compared with those in adult rods. Data represent mean ± S.D. *p < 0.05.|
|sc-12-0163_sm_SupplFigure3.tif||2383K||Supplemental Figure 3. Calcium imaging of grafted rods in Crx-KO mouse retina. (a) Nrlp-eGFP positive cell in flat-mounted Crx-KO mouse retina. Arrows indicate Nrlp-eGFP positive cell clusters. (b, c) Fura-2 ratio imaging of grafted cells in flat mounted Crx-KO mouse retina at 0 sec (b) and 120 sec (c). Red color indicates high concentration of intracellular Ca2+. (d) Nrlp-eGFP positive graft cells showed Ca2+ responses to high-K+ stimulation.|
|sc-12-0163_sm_SupplFigure4.tif||1449K||Supplemental Figure 4. Mouse iPS cells derived from Nrlp-eGFP mice differentiate into all three germ layers in the immunodeficient mice. (a) Colonies of Nrlp-eGFP mouse iPS cells. (b-d) Nrlp-eGFP mouse iPS cells were injected into the testis of immunodeficient (SCID) mice, and produced various types of tissues derived from three germ layers. Scale bars, 200 μm in (a), 100 μm in (b), (c), and (d).|
|sc-12-0163_sm_SupplFigure5.tif||792K||Supplemental Figure 5. Mouse iPS cell-derived Nrlp-eGFP-positive cells show hyperpolarization–activated current. Membrane current in (a) control or (b) ZD7288-treated iPS cell-derived Nrlp-eGFP-positive cells were recorded by membrane voltage-clamp from -100 mV (red line) to 10 mV, with 10 mV increments. (c) I-V curve was plotted from 4 trials. The voltage-dependent inward current recorded at membrane hyperpolarization was suppressed by treatment with ZD7288, an HCN channel blocker. Data represent mean ± S.D. *p < 0.05.|
|sc-12-0163_sm_SupplSvideo1.avi||46086K||Supplemental Online Video 1. Fura-2 Ca2+ imaging of retinal cells in retinal slice preparation. Sliced retinas were stained with Fura-2. 100 μM glutamate (at 0:48 min) and 80 mM KCl (at 2:13 min) were added to the medium. Interval = 5.0 sec, 20 frames/sec, total time = 3.7 min (45 frames).|
|sc-12-0163_sm_SupplSvideo2.avi||51206K||Supplemental Online Video 2. Fura-2 Ca2+ imaging of Nrlp-eGFP-positive developing rods. Retinal cells from postnatal day (P)3 mice were cultured on glass-base dishes for 2-5 days. Retinal cells were stained with Fura-2. 100 μM glutamate (at 1:00 min) and 80 mM KCl (at 5:40 min) were added to the medium. Interval = 12.0 sec, 20 frames/sec, total time = 9.7 min (50 frames).|
|sc-12-0163_sm_SupplSvideo3.avi||115443K||Supplemental Online Video 3. Fura-2 Ca2+ imaging of donor cells in retinal slice preparation. Region of interest (ROI) #1-3 in the video correspond to the same ROI in Figure 3C and indicate donor cells in the wild type retina. 80 mM KCl (at 0:30 min) was added to the medium. Interval = 4.7 sec, 20 frames/sec, total time = 2.0 min (37 frames).|
|sc-12-0163_sm_SupplTable1.pdf||83K||Supplementary Table 1.|
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