L-Phenylalanine high efficiently induces differentiation of human embryonic stem cells into retinal pigment epithelial cell_Chinese Journal of Ocular Fundus Diseases

Authors：

WuYixiang , YangChunbo , YuMin ,  李筱荣

Eye Research Institute, Tianjin Medical University Hospital, Tianjin Medical University Eye Institute, School of Optometry and Ophthalmology, Tianjin Medical University, Tianjin 300384, China;

Corresponding author：

李筱荣, Email: xiaorli@163.com

Keywords：

Phenylalanine/drug effects; Embryonic stem cells; Cell differentiation; Retinal pigment epithelium

DOI：

10.3760/cma.j.issn.1005-1015.2015.01.015

Video：

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Objective To investigate the impact of L-Phenylalanine on the efficiency of retinal pigment epithelial (RPE) cell derivation from human embryonic stem cells (hESCs) and explore the underlying mechanisms. Methods H1 hESCs were routinely cultured with mTeSR medium and divided into control and experimental groups. When cells reached over-confluence, spontaneous differentiation was triggered using 10% KSR differentiation medium without bFGF. L-Phenylalanine (0.2 mmol/L) was supplemented in the experimental group from the 3rd week. The expression of RPE markers and Wnt signaling components in the two groups was detected by Real time-RCR, Western blot and Flow cytometry analyses. Purified hESC-RPE cells and PBS were injected into the subretinal space of sodium iodine-induced retinal degeneration rats separately. Retinal function was assessed by ERG 6 weeks after the transplantation. Results On the 7th week, much more pigment cell clumps appeared in the experimental group compared to the control group. Within these areas there were monolayer hexagonal RPE cells full of pigment granules. The experimental group showed significantly higher expression of Pax6, MITF, Tyrosinase, RPE65, Wnt3a, Lef1 and Tcf7 genes than the control group (P < 0.01). Higher expression level of MITF and RPE65 proteins and higher percentage of RPE65 (+) cells (P < 0.01) were detected in the experimental group. 6 weeks after sub-retinal transplantation of hESC-RPE cells, the amplitudes of a-b wave in the transplanted eyes were significantly higher than those in the control eyes (P < 0.01) at the stimulus intensity of 3.0 cd·s/m². Conclusions L-Phenylalanine effectively promoted the differentiation of embryonic stem cells into retinal pigment epithelial cells, and its impacts on the Wnt/β-catenin signaling pathway may partially explain the underlying mechanisms. Subretinal transplantation of hESC-RPE remarkably improved the retinal functions of retinal degenerative animal models.

Citation： WuYixiang, YangChunbo, YuMin. L-Phenylalanine high efficiently induces differentiation of human embryonic stem cells into retinal pigment epithelial cell. Chinese Journal of Ocular Fundus Diseases, 2015, 31(1): 57-61. doi: 10.3760/cma.j.issn.1005-1015.2015.01.015 Copy

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1. Sowden JC. ESC-derived retinal pigmented epithelial cell transplants in patients:so far, so good[J]. Cell Stem Cell, 2014, 15(5):537-538.
2. 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(3):217-245.
3. Osakada F, Jin ZB, Hirami Y, et al. In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction[J]. J Cell Sci, 2009, 122(17):3169-3179.
4. Idelson M, Alper R, Obolensky A, et al. Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells[J]. Cell Stem Cell, 2009, 5(4):396-408.
5. Hanley WB, Lee A W, Hanley AJG, et al."Hypotyrosinemia" in phenylketonuria[J]. Mol Genet Metab, 2000, 69(4):286-294.
6. Liao JL, Yu J, Huang K, et al. Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells[J]. Hum Mol Genet, 2010, 19(21):4229-4238.
7. Mallon BS, Hamilton RS, Kozhich OA, et al. Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin[J]. Stem Cell Res, 2014, 12(2):376-386.
8. Scott CT, McCormick JB, Owen-Smith J. And then there were two:use of hESC lines[J]. Nat Biotechnol, 2009, 27(8):696-697.
9. Yang Y, Ng TK, Ye C, et al. Assessing sodium iodate-induced outer retinal changes in rats using confocal scanning laser ophthalmoscopy and optical coherence tomography[J].Invest Ophthalmol Vis Sci, 2014, 55(3):1696-1705.
10. Osakada F, Ikeda H, Sasai Y, et al. Stepwise differentiation of pluripotent stem cells into retinal cells[J]. Nat Protoc, 2009, 4(6):811-824.
11. Ripamonti C, Henning GB, Ali RR, et al. Nature of the visual loss in observers with leber's congenital amaurosis caused by specific mutations in RPE65[J]. Invest Ophthalmol Vis Sci, 2014, 55(10):6817-6828.
12. Slominski A, Tobin DJ, Shibahara S, et al. Melanin pigmentation in mammalian skin and its hormonal regulation[J]. Physiol Rev, 2004, 84(4):1155-1228.
13. Schallreuter KU, Wood JM. The importance of L-phenylalanine transport and its autocrine turnover to L-tyrosine for melanogenesis in human epidermal melanocytes[J]. Biochem Biophys Res Commun, 1999, 262(2):423-428.
14. Slominski A, Costantino R. L-tyrosine induces tyrosinase expression via a posttranscriptional mechanism[J]. Experientia, 1991, 47(7):721-724.
15. Schwahn DJ, Xu W, Herrin AB, et al. Tyrosine levels regulate the melanogenic response toα-melanocyte-stimulating hormone in human melanocytes:implications for pigmentation and proliferation[J]. Pigment Cell Research, 2001, 14(1):32-39.
16. Fuhrmann S. Wnt signaling in eye organogenesis[J]. Organogenesis, 2008, 4(2):60-67.
17. Zhou CJ, Molotkov A, Song L, et al. Ocular coloboma and dorsoventral neuroretinal patterning defects in Lrp6 mutant eyes[J]. Dev Dyn, 2008, 237(12):3681-3689.
18. Fujimura N, Taketo MM, Mori M, et al. Spatial and temporal regulation of Wnt/β-catenin signaling is essential for development of the retinal pigment epithelium[J]. Dev Biol, 2009, 334(1):31-45.
19. Westenskow P, Piccolo S, Fuhrmann S. Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression[J].Development, 2009, 136(15):2505-2510.
20. Guo H, Yang K, Deng F, et al. Wnt3a inhibits proliferation but promotes melanogenesis of melan-a cells[J]. Int J Mol Med, 2012, 30(3):636-642.
21. Guo H, Yang K, Deng F, et al. Wnt3a promotes melanin synthesis of mouse hair follicle melanocytes[J]. Biochem Biophys Res Commun, 2012, 420(4):799-804.

Chinese Journal of Ocular Fundus Diseases

L-Phenylalanine high efficiently induces differentiation of human embryonic stem cells into retinal pigment epithelial cell

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