The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE.
Methods:
This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid, directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE, which were characterized with respect to global gene expression, expression of RPE markers, and cellular function.
Results:
We found that all 5 iPSC lines (iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-12) generated RPE using the directed differentiation protocol; however, 2 of the 5 iPSC lines (iPSC-4 and iPSC-12) did not yield RPE using the SCAC method. Both methods can yield bona fide RPE that expresses signature RPE genes and carry out RPE functions, and are similar, but not identical to fetal RPE. No differences between methods were detected in transcript levels, protein localization, or functional analyses between iPSC-1-RPE, iPSC-2-RPE, and iPSC-3-RPE. Directed iPSC-3-RPE showed enhanced transcript levels of RPE65 compared to directed iPSC-2-RPE and increased BEST1 expression and pigment epithelium-derived factor (PEDF) secretion compared to directed iPSC-1-RPE. In addition, SCAC iPSC-3-RPE secreted more PEDF than SCAC iPSC-1-RPE.
Conclusions:
The directed protocol is a more reliable method for differentiating RPE from various pluripotent sources and some iPSC lines are more amenable to RPE differentiation.
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References
1.
YuJ., VodyanikM.A., Smuga-OttoK., et al.Induced pluripotent stem cell lines derived from human somatic cells. Science. 318:1917–1920, 2007.
2.
TakahashiK., and YamanakaS.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126:663–676, 2006.
3.
TakahashiK., TanabeK., OhnukiM., et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131:861–872, 2007.
4.
WalmsleyG.G., HyunJ., McArdleA., et al.Induced pluripotent stem cells in regenerative medicine and disease modeling. Curr Stem Cell Res Ther., 9:73–81, 2014.
5.
OkanoH., NakamuraM., YoshidaK., et al.Steps toward safe cell therapy using induced pluripotent stem cells. Circ. Res., 112:523–533, 2013.
6.
YuJ., HuK., Smuga-OttoK., et al.Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science. 324:797–801, 2009.
7.
HardingJ., and MirochnitchenkoO.Preclinical studies for induced pluripotent stem cell-based therapeutics. J. Biol.Chem., 289:4585–4593, 2014.
8.
KanemuraH., GoM.J., ShikamuraM., et al.Tumorigenicity studies of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration. PLoS One. 9:e85336, 2014.
9.
KamaoH., MandaiM., OkamotoS., et al.Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Reports. 2:205–218, 2014.
10.
CarrA.-J., VuglerA.A., HikitaS.T., et al.Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PLoS One. 4:e8152, 2009.
11.
CyranoskiD.Stem cells cruise to clinic. Nature. 494:413, 2013.
12.
KleinR., ChouC.-F., KleinB.E.K., ZhangX., MeuerS.M., and SaaddineJ.B.Prevalence of age-related macular degeneration in the US population. Arch Ophthalmol. 129:75–80, 2011.
13.
FriedmanD.S., O'ColmainB.J., MuñozB., et al.Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol. 122:564–572, 2004.
HuQ., FriedrichA.M., JohnsonL.V., and CleggD.O.Memory in induced pluripotent stem cells: reprogrammed human retinal-pigmented epithelial cells show tendency for spontaneous redifferentiation. Stem Cells. 28:1981–1991, 2010.
17.
CrozeR.H., and CleggD.O.Differentiation of pluripotent stem cells into retinal pigmented epithelium. Dev Ophthalmol. 53:81–96, 2014.
18.
KlimanskayaI., HippJ., RezaiK.A., WestM., AtalaA., and LanzaR.Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells. 6:217–245, 2004.
19.
LundR.D., WangS., KlimanskayaI., et al.Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats. Cloning Stem Cells. 8:189–199, 2006.
20.
CarrA.-J., VuglerA., LawrenceJ., et al.Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay. Mol Vis. 15:283–295, 2009.
21.
BuchholzD.E., HikitaS.T., RowlandT.J., et al.Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells. 27:2427–2434, 2009.
22.
VuglerA., CarrA.-J., LawrenceJ., et al.Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation. Exp. Neurol., 214:347–361, 2008.
23.
BuchholzD.E., PenningtonB.O., CrozeR.H., HinmanC.R., CoffeyP.J., and CleggD.O.Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl. Med., 2:384–393, 2013.
24.
LeachL.L., BuchholzD.E., NadarV.P., LowensteinS.E., and CleggD.O.Canonical/ß-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells. Invest. Ophthalmol. Vis. Sci., 56:1002–1013, 2015.
25.
GourraudP.-A., GilsonL., GirardM., and PeschanskiM.The role of human leukocyte antigen matching in the development of multiethnic “haplobank” of induced pluripotent stem cell lines. Stem Cells. 30:180–186, 2012.
26.
TaylorC.J., PeacockS., ChaudhryA.N., BradleyJ.A., and BoltonE.M.Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient HLA types. Cell Stem Cell. 11:147–152, 2012.
27.
HuK., YuJ., SuknunthaK., et al.Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood. 117:e109–e119, 2011.
28.
MeyerJ.S., HowdenS.E., WallaceK.A., et al.Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment. Stem Cells. 29:1206–1218, 2011.
29.
HowdenS.E., GoreA., LiZ., et al.Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc. Natl Acad. Sci. U S A. 108:6537–6542, 2011.
30.
BeersJ., GulbransonD.R., GeorgeN., et al.Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat. Protoc., 7:2029–2040, 2012.
31.
MaminishkisA., ChenS., JalickeeS., et al.Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest. Ophthalmol. Vis. Sci., 47:3612–3624, 2006.
KimD., PerteaG., TrapnellC., PimentelH., KelleyR., and SalzbergS.L.TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14:R36, 2013.
34.
TrapnellC., HendricksonD.G., SauvageauM., GoffL., RinnJ.L., and PachterL.Differential analysis of gene regulation at transcript resolution with rNA-seq. Nat. Biotechnol., 31:46–53, 2012.
35.
ZhangB., KirovS., and SnoddyJ.WebGestalt: an integrated system for exploring gene sets in various biological contexts. Nucleic Acids Res., 33:W741–W748, 2005.
36.
WangJ., DuncanD., ShiZ., and ZhangB.WEB-based GEne SeT AnaLysis Toolkit (WebGestalt): update 2013. Nucleic Acids Res., 41:W77–W83, 2013.
37.
HecquetC., LefevreG., ValtinkM., EngelmannK., and MascarelliF.Activation and role of MAP kinase-dependent pathways in retinal pigment epithelial cells: ERK and RPE cell proliferation. Invest. Ophthalmol. Vis. Sci., 43:3091–3098, 2002.
38.
LeeJ., ZengJ., HughesG., et al.Association of LIPC and advanced age-related macular degeneration. Eye. 27:265–270, 2013; quiz271.
39.
WangY.-F., HanY., ZhangR., QinL., WangM.-X., and MaL.CETP/LPL/LIPC gene polymorphisms and susceptibility to age-related macular degeneration. Sci Rep. 5:15711, 2015.
40.
YuY., ReynoldsR., FagernessJ., RosnerB., DalyM.J., and SeddonJ.M.Association of variants in the LIPC and ABCA1 genes with intermediate and large drusen and advanced age-related macular degeneration. Invest. Ophthalmol. Vis. Sci., 52:4663, 2011.
41.
NealeB.M., FagernessJ., ReynoldsR., et al.Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC). Proc. Natl Acad. Sci. U S A. 107:7395–7400, 2010.
42.
BaileyT.A., KanugaN., RomeroI.A., GreenwoodJ., LuthertP.J., and CheethamM.E.Oxidative stress affects the junctional integrity of retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci., 45:675–684, 2004.
43.
ChenH.-C., ZhuY.-T., ChenS.-Y., and TsengS.C.G.Wnt signaling induces epithelial-mesenchymal transition with proliferation in ARPE-19 cells upon loss of contact inhibition. Lab Invest. 92:676–687, 2012.
44.
LinH., and CleggD.O.Integrin alphavbeta5 participates in the binding of photoreceptor rod outer segments during phagocytosis by cultured human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci., 39:1703–1712, 1998.
45.
ToivonenS., OjalaM., HyysaloA., et al.Comparative analysis of targeted differentiation of human induced pluripotent stem cells (hiPSCs) and Human embryonic stem cells reveals variability associated with incomplete transgene silencing in retrovirally derived hiPSC lines. Stem Cells Transl. Med., 2:83–93, 2013.
46.
Streckfuss-BömekeK., WolfF., AzizianA., et al.Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts. Eur. Heart J., 34:2618–2629, 2013.
47.
TrakarnsangaK., WilsonM.C., GriffithsR.E., et al.Qualitative and quantitative comparison of the proteome of erythroid cells differentiated from human iPSCs and adult erythroid cells by multiplex TMT labelling and nanoLC-MS/MS. PLoS One. 9:e100874, 2014.
48.
MeyerJ.S., ShearerR.L., CapowskiE.E., et al.Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc. Natl Acad. Sci. U S A., 106:16698–16703, 2009.
49.
HuB.Y., WeickJ.P., YuJ., et al.Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc. Natl Acad. Sci. U S A. 107:4335–4340, 2010.
50.
LiuZ., JiangR., YuanS., et al.Integrated analysis of DNA methylation and RNA transcriptome during in vitro differentiation of human pluripotent stem cells into retinal pigment epithelial cells. PLoS One. 9:e91416, 2014.
51.
LiaoJ.-L., YuJ., HuangK., et al.Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells. Hum Mol Genet. 19:4229–4238, 2010.
52.
OsakadaF., JinZ.-B., HiramiY., et al.In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. J Cell Sci. 122(Pt 17):3169–3179, 2009.
53.
ZhuY., CaridoM., MeinhardtA., et al.Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium. LeungY.F., ed. PLoS ONE. 8:e54552, 2013.
54.
HiramiY., OsakadaF., TakahashiK., et al.Generation of retinal cells from mouse and human induced pluripotent stem cells. Neurosci. Lett., 458:126–131, 2009.
55.
PhillipsM.J., WallaceK.A., DickersonS.J., et al.Blood-derived human iPS cells generate optic vesicle-like structures with the capacity to form retinal laminae and develop synapses. Invest. Ophthalmol. Vis. Sci., 53:2007–2019, 2012.
56.
ReichmanS., TerrayA., SlembrouckA., et al.From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium. Proc. Natl Acad. Sci. U S A. 111:8518–8523, 2014.
57.
SinghR., PhillipsM.J., KuaiD., et al.Functional analysis of serially expanded human iPS cell-derived RPE cultures. Invest. Ophthalmol. Vis. Sci., 54:6767–6778, 2013.
58.
YamanakaS.Elite and stochastic models for induced pluripotent stem cell generation. Nature. 460:49–52, 2009.
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