TGF-β receptor inhibitor Compound C promotes the directed differentiation of human embryonic stem cells into retinal pigment epithelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

YangBoyu , YangChunbo , YuMin ,  李筱荣

Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, Tianjin 300384, China;

Corresponding author：

李筱荣, Email: xiaorli@163.com

Keywords：

TGF-beta superfamily proteins/antagonists & inhibitors; Embryonic stem cells/transplantation; Retinal pigment epithelium/cytology

DOI：

10.3760/cma.j.issn.1005-1015.2014.01.019

Video：

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Objective To observe the effect of TGF-β receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells (hESC) into retinal pigment epithelial (RPE) cells. Methods H1 hESC were divided into control group and experimental group. When the hESC reached over confluence, the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation. The experimental group was supplemented with 1 μmol/L TGF-β receptor inhibitor Compound C at the first six days of induction. Real-time PCR was carried out to examine the expression of paired-box gene 6 (PAX6), microphthalmia-associated transcription factor (MITF), cellular retinaldehyde blinding protein (CRALBP), and RPE65 in both groups at the 1, 3, 5 weeks of the induction process. hESC-derived RPE (hESC-RPE) cells were isolated mechanically and purified. Real-time PCR, Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells. Results Pigmented colonies were observed in experimental group at the 4 weeks of the induction process, while no pigmented colony could be detected in the control group. All the purified pigmented cells from experimental group showed polygons morphology. Experimental group showed significantly higher expression of RPE marker genes PAX6, MITF, CRALBP and RPE65 than the control group(P<0.05). Compared with the hESC and ARPE-19 cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65(P<0.05).High expression level of PAX6 and RPE65 proteins were observed in hESC-RPE cells. Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE cells. Conclusion TGF-β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE.

Citation： YangBoyu, YangChunbo, YuMin. TGF-β receptor inhibitor Compound C promotes the directed differentiation of human embryonic stem cells into retinal pigment epithelial cells. Chinese Journal of Ocular Fundus Diseases, 2014, 30(1): 75-79. doi: 10.3760/cma.j.issn.1005-1015.2014.01.019 Copy

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1. Uygun BE, Sharma N, Yarmush M. Retinal pigment epithelium differentiation of stem cells:current status and challenges[J]. Crit Rev Biomed Eng, 2009,37:355-375.
2. Clegg DO, Buchholz D, Hikita S, et al. Retinal pigment epithelial cells development in vivo and derivation from human embryonic stem cells in vitro for treatment of age-related macular degeneration[J]. Stem Cell Res Ther, 2008, 1:1-24.
3. Dahlmann-Noor A, Vijay S, Jayaram H, et al. Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve[J]. Can J Ophthalmol,2010,45:333-341.
4. Gaillard F, Sauve Y. Cell-based therapy for retina degeneration:the promise of a cure[J]. Vision Res,2007,47:2815-2824.
5. Da CL, Chen FK, Ahmado A, et al. RPE transplantation and its role in retinal disease[J]. Prog Retin Eye Res,2007, 26:598-635.
6. Zhou J, Su P, Li D, et al. High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors[J]. Stem Cells,2010,28:1741-1750.
7. Lund RD, Wang S, Klimanskaya I, et al. Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats[J]. Cloning Stem Cells,2006,8:189-199.
8. Haruta M. Embryonic stem cells:potential source for ocular repair[J]. Semin Ophthalmol,2005,20:17-23.
9. Schwartz SD, Hubschman JP, Heilwell G, et al. Embryonic stem cell trials for macular degeneration:a preliminary report[J]. Lancet,2012,379:713-720.
10. Graw J. Eye development[J]. Curr Top Dev Biol,2010,90:343-386.
11. Kozmik Z. Pax genes in eye development and evolution[J]. Curr Opin Genet Dev,2005,15:430-438.
12. Osakada F, Ikeda H, Sasai Y, et al. Stepwise differentiation of pluripotent stem cells into retinal cells[J]. Nat Protoc,2009,4:811-824.
13. Klimanskaya I, Hipp J, Rezai KA, et al. Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics[J]. Cloning Stem Cells,2004,6:217-245.
14. Osakada F, Ikeda H, Mandai M, et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells[J]. Nat Biotechnol,2008,26:215-224.
15. Mcgill TJ, Lund RD, Douglas RM, et al. Preservation of vision following cell-based therapies in a model of retinal degenerative disease[J]. Vision Res,2004,44:2559-2566.
16. Wang S, Lu B, Girman S, et al. Morphological and functional rescue in RCS rats after RPE cell line transplantation at a later stage of degeneration[J]. Invest Ophthalmol Vis Sci,2008,49:416-421.

Chinese Journal of Ocular Fundus Diseases

TGF-β receptor inhibitor Compound C promotes the directed differentiation of human embryonic stem cells into retinal pigment epithelial cells

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