Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors

Abstract

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases, including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells, direct cell reprogramming driven by lineage-determining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter, we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover, they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination, but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling, drug screening, and cell therapy of retinal degeneration.

Key words： retinal pigment epithelium; fibroblasts; direct conversion

References

1 AclandGM, AguirreGD, BennettJ, AlemanTS, CideciyanAV, BennicelliJ, DejnekaNS,Pearce-KellingSE, MaguireAM, PalczewskiK (2005) Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. Mol Ther12: 1072-1082

2 Ben-DavidU, BenvenistyN (2011) The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat Rev Cancer11: 268-277

3 BhartiK, NguyenMT, SkuntzS, BertuzziS, ArnheiterH (2006) The other pigment cell: specification and development of the pigmented epithelium of the vertebrate eye. Pigment Cell Res19: 380-394

4 BoucherieC, MukherjeeS, HenckaertsE, ThrasherAJ, SowdenJC, AliRR (2013) Brief report: self-organizing neuroepithelium from human pluripotent stem cells facilitates derivation of photoreceptors. Stem Cells31: 408-414

5 BoulangerA, LiuS, HenningsgaardAA, YuS, RedmondTM (2000) The upstream region of the Rpe65 gene confers retinal pigment epithelium-specific expression in vivo and in vitro and contains critical octamer and E-box binding sites. J Biol Chem275: 31274-31282

6 BuchholzDE, HikitaST, RowlandTJ, FriedrichAM, HinmanCR, JohnsonLV, CleggDO (2009) Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells27: 2427-2434

7 CarrAJ, VuglerAA, HikitaST, LawrenceJM, GiasC, ChenLL, BuchholzDE, AhmadoA, SemoM, SmartMJ (2009) Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PLoS ONE4: e8152

8 EfeJA, HilcoveS, KimJ, ZhouH, OuyangK, WangG, ChenJ, DingS (2011) Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol13: 215-222

9 EsumiN, OshimaY, LiY, CampochiaroPA, ZackDJ (2004) Analysis of the VMD2 promoter and implication of E-box binding factors in its regulation. J Biol Chem279: 19064-19073

10 EsumiN, KachiS, HacklerL Jr, MasudaT, YangZ, CampochiaroPA, ZackDJ (2009) BEST1 expression in the retinal pigment epithelium is modulated by OTX family members. Hum Mol Genet18: 128-141

11 GiorgettiA, MarchettoMC, LiM, YuD, FazzinaR, MuY, AdamoA, ParamonovI, CardosoJC, MonasterioMB (2012) Cord blood-derived neuronal cells by ectopic expression of Sox2 and c-Myc. Proc Natl Acad Sci USA109: 12556-12561

12 HuangP, HeZ, JiS, SunH, XiangD, LiuC, HuY, WangX, HuiL (2011) Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature475: 386-389

13 KhandhadiaS, CherryJ, LoteryAJ (2012) Age-related macular degeneration. Adv Exp Med Biol724: 15-36

14 KimJ, EfeJA, ZhuS, TalantovaM, YuanX, WangS, LiptonSA, ZhangK, DingS (2011) Direct reprogramming of mouse fibroblasts to neural progenitors. Proc Natl Acad Sci USA108: 7838-7843

15 KurianL, Sancho-MartinezI, NivetE, AguirreA, MoonK, PendariesC, Volle-ChallierC, BonoF, HerbertJM, PulecioJ (2013) Conversion of human fibroblasts to angioblast-like progenitor cells. Nat Methods10: 77-83

16 LiuGH, YiF, SuzukiK, QuJ, Izpisua BelmonteJC (2012) Induced neural stem cells: a new tool for studying neural development and neurological disorders. Cell Res22: 1087-1091

17 LuB, MalcuitC, WangS, GirmanS, FrancisP, LemieuxL, LanzaR, LundR (2009) Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells27: 2126-2135

18 Martinez-MoralesJR, RodrigoI, BovolentaP (2004) Eye development: a view from the retina pigmented epithelium. BioEssays26: 766-777

19 MeyerJS, ShearerRL, CapowskiEE, WrightLS, WallaceKA, McMillanEL, ZhangSC, GammDM (2009) Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci USA106: 16698-16703

20 NakanoT, AndoS, TakataN, KawadaM, MugurumaK, SekiguchiK, SaitoK, YonemuraS, EirakuM, SasaiY (2012) Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell10: 771-785

21 OkadaY, ShimazakiT, SobueG, OkanoH (2004) Retinoic-acidconcentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev Biol275: 124-142

22 OsakadaF, IkedaH, SasaiY, TakahashiM (2009) Stepwise differentiation of pluripotent stem cells into retinal cells. Nat Protoc4: 811-824

23 PangZP, YangN, VierbuchenT, OstermeierA, FuentesDR, YangTQ, CitriA, SebastianoV, MarroS, SudhofTC (2011) Induction of human neuronal cells by defined transcription factors. Nature476: 220-223

24 PanopoulosAD, RuizS, Izpisua BelmonteJC (2011) iPSCs: induced back to controversy. Cell Stem Cell8: 347-348

25 SaleroE, BlenkinsopTA, CorneoB, HarrisA, RabinD, SternJH, TempleS (2012) Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives. Cell Stem Cell10: 88-95

26 Sancho-MartinezI, BaekSH, Izpisua BelmonteJC (2012) Lineage conversion methodologies meet the reprogramming toolbox. Nat Cell Biol14: 892-899

27 SchwartzSD, HubschmanJP, HeilwellG, Franco-CardenasV, PanCK, OstrickRM, MickunasE, GayR, KlimanskayaI, LanzaR (2012) Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet379: 713-720

28 SekiyaS, SuzukiA (2011) Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature475: 390-393

29 SteingrimssonE, CopelandNG, JenkinsNA (2004) Melanocytes and the microphthalmia transcription factor network. Annu Rev Genet38: 365-411

30 StraussO (2005) The retinal pigment epithelium in visual function. Physiol Rev85: 845-881

31 TakahashiK, YamanakaS (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell126: 663-676

32 TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK, YamanakaS (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell131: 861-872

33 VierbuchenT, OstermeierA, PangZP, KokubuY, SudhofTC, WernigM (2010) Direct conversion of fibroblasts to functional neurons by defined factors. Nature463: 1035-1041

34 YiF, LiuGH, Izpisua BelmonteJC (2012) Rejuvenating liver and pancreas through cell transdifferentiation. Cell Res22: 616-619

35 ZahabiA, ShahbaziE, AhmadiehH, HassaniSN, TotonchiM, TaeiA, MasoudiN, EbrahimiM, AghdamiN, SeifinejadA (2012) A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. Stem Cells Dev21: 2262-2272

36 ZhangW, DuanS, LiY, XuX, QuJ, ZhangW, LiuGH (2012) Converted neural cells: induced to a cure? Protein Cell3: 91-97

37 ZhangK, YiF, LiuGH, Izpisua BelmonteJC (2013) New march towards the regeneration of sensation and cognition: hear more, see more and learn more. J Mol Cell Biol5: 151-153

38 ZhuY, CaridoM, MeinhardtA, KurthT, KarlMO, AderM, TanakaEM (2013) Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium. PLoS ONE8: e54552

39 ZuberME, GestriG, ViczianAS, BarsacchiG, HarrisWA (2003) Specification of the vertebrate eye by a network of eye field transcription factors. Development130: 5155-5167