Telephone: +34 954 468 004; Fax: +34 954 461 664
Embryonic Stem Cells/induced Pluripotent Stem Cells
Article first published online: 24 APR 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 5, pages 966–978, May 2013
How to Cite
Garita-HernÁndez, M., Diaz-Corrales, F., Lukovic, D., GonzÁlez-Guede, I., Diez-Lloret, A., ValdÉs-SÁnchez, M. L., Massalini, S., Erceg, S. and Bhattacharya, S. S. (2013), Hypoxia Increases the Yield of Photoreceptors Differentiating from Mouse Embryonic Stem Cells and Improves the Modeling of Retinogenesis In Vitro. STEM CELLS, 31: 966–978. doi: 10.1002/stem.1339
Author contributions: M.G.-H.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript, and other (perform experiments and graphic design); S.E.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript; F.D.-C., D.L., I.G.-G., S.M., L.V., and A.D.-L.: other (perform experiments); S.S.B.: principal investigator, conception and design, 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 January 29, 2013.
- Issue published online: 24 APR 2013
- Article first published online: 24 APR 2013
- Accepted manuscript online: 29 JAN 2013 11:01PM EST
- Manuscript Accepted: 23 NOV 2012
- Manuscript Received: 7 AUG 2012
- Junta de Andalucia. Grant Number: PI-0113-2010
- Miguel Servet
- Instituto de Salud Carlos III of Spanish Ministry of Science and Innovation
Additional Supporting Information may be found in the online version of this article.
|sc-12-0730_sm_SupplFigure1.pdf||430K||Supplementary Fig. 1. General overview of the differentiation towards photoreceptors. A. Diagram showing the general approach by which mES cells, Oct4+, are directed towards the differentiation of retinal progenitors, Chx10+, and subsequently towards Rods, Rho1+, cells. B. Schematic diagram of the differentiation protocol used to generate retinal cells. C. Transcription factors defining each major step during retinogenesis.|
|sc-12-0730_sm_SupplFigure2.pdf||448K||Supplementary Fig. 2. Specificity of antibodies used in immunofluorescence analysis of generated cells. A. Immunofluorescence analysis of forebrain marker Otx2 in wild type E14 mouse embryo neural retina. B. Immunofluorescence analysis of RPE progenitor markers ZO-1 (red) and Mitf (green) in wild type P3 mouse retina. C. Immunofluorescence analysis of cone-specific S-Opsin in wild type adult mouse retina. D. Immunofluorescence analysis of photoreceptor-specific marker Recoverin (Rcvn) in wild type adult mouse retina. E. Immunofluorescence analysis of mature RPE-specific marker RPE-65 in wild type adult mouse retina.|
|sc-12-0730_sm_SupplFigure3.pdf||464K||Supplementary Fig. 3. Specificity of antibodies used in immunofluorescence analysis of generated cells. A. Immunofluorescence analysis of rod photoreceptor-specific marker Rhodopsin (monoclonal antibody) in wild type adult mouse retina. B. Immunofluorescence analysis of rod photoreceptor-specific marker Rhodopsin (polyclonal antibody) in wild type adult mouse retina. C. Immunofluorescence analysis of Chx-10 expression in wild type adult mouse retina. D. Immunofluorescence analysis of Rax and Chx-10 expression in wild type E14 mouse neural retina. E. Inset in D. showing detail of nuclear specific staining of Rax and Chx10 transcription factors.|
|sc-12-0730_sm_SupplFigure4.pdf||237K||Supplementary Fig. 4. A. Comparative immunofluorescence analysis showing no colocalization of positive cells for the proliferation marker; Ki67 and Rhodopsin after 28 days of differentiation under hypoxia and normoxia. B. Quantification of the percentage of Ki67+ RPE-1 cells after 3 weeks of culture in normoxia (blue bar) and hypoxia (red bar). C. Cytotoxity assay showing a decrease in toxicity in RPE-1 cells after one week in hypoxia (red bar) when compared to normoxia (blue bar). D. Apoptosis assay showing a decrease in caspase 3/7 activity when RPE-1 cells were cultured for one week in hypoxia (red bar) compared to normoxia (blue bar) E. qPCR analysis showing upregulation of Vegfa gene after 5 and 28 days of differentiation in normoxia (blue bar) and hypoxia (red bar). F. qPCR analysis showing upregulation of Cdkn1a gene after 5 and 28 days of differentiation in normoxia (blue bar) and hypoxia (red bar). G. qPCR analysis showing upregulation of Vegfa gene in D3-mESC after 7 days of culture in presence of LIF in hypoxia (red bar) compared to normoxia (blue bar) and. H. Percentage of Hif1a positive cells in RPE-1 cells after 3 weeks of culture under normoxic (blue bar) and hypoxic (red bar) conditions determined by FACS analysis. Graph corresponds to the mean of 3 independent experiments. I. Percentage of Hif1a positive cells in D3-mESC cells after 1 week of culture in presence of LIF under normoxic (blue bar) and hypoxic (red bar) conditons determined by FACS analysis. Graph corresponds to the mean of 3 independent experiments.* p≤ 0.05 was considered statistically significant.|
|sc-12-0730_sm_SupplFigure5.pdf||207K||Supplementary Fig. 5. A. Diagram showing the details of the subretinal injection used to deliver the transplanted cells. B. Immunohystochemical analysis showing the anatomy of the injection site after 24 hours of transplantation with Rcvn+ cells in green, transplanted PKH26+ cells in red and Dapi in blue.|
|sc-12-0730_sm_SupplFigure6.tif||1034K||Supplementary Fig. 6. Opsin-S positive cells generated under hypoxia. Immunohistochemical analysis showing a few Opsin-S positive cells, after 1 week of transplatation of retinal cells generated under hypoxia. PKH26 staining indicates cells are from the graft and not from the host retina. Control corresponds to an uninjected animal.|
|sc-12-0730_sm_SupplFigure7.pdf||171K||Supplementary Fig. 7. Integration of transplanted cells within the host retina. A. High magnification of immunofluorescence analysis performed in animals transplanted with retinal cells differentiated under hypoxia, showing integration in the outer nuclear layer (ONL) of cells positive for Rcvn and Rhodopsin. B. Integration of transplanted cells was confirmed when analysis for Dapi was performed and the transplanted cells were positive for Recvn and were localized in the ONL among the other host photoreceptors. SS-subretinal space.|
|sc-12-0730_sm_SupplFigure8.pdf||316K||Supplementary Fig. 8. Transplantation of Normoxia-generated cells. Transplantation of the cells cultured under normoxic conditions showed low survival, immature differentiation as cells were not positive for Rcvn and no integration into the host retina was observed.|
|sc-12-0730_sm_SupplFigure9.tif||64K||Supplementary Fig. 9. Karyotype of differentiated mESC under hypoxia. Karyotype (chromosomes arrayed by size) from cells differentiated under hypoxia showing no alteration in chromosome number. ES- D3 cell line used in this study was developed form the inner cell mass of a male blastocyst, which correspond with the XY karyotype.|
|sc-12-0730_sm_SupplTable1.pdf||12K||Supplementary Table 1|
|sc-12-0730_sm_SupplTable2.pdf||11K||Supplementary Table 2|
|sc-12-0730_sm_SupplTable3a.pdf||9K||Supplementary Table 3a|
|sc-12-0730_sm_SupplTable3b.pdf||56K||Supplementary Table 3b|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.