Retinal organoid differentiation methods determine organoid cellular composition

Abstract

Aim: We aimed to compare the quantity and quality of aging retinal organoids generated by applying three distinct differentiation protocols for human-derived induced pluripotent stem cells (hiPSC).

Methods: hiPSC were differentiated to retinal organoids using a 3D technique (Method 1) and a 3D-2D-3D technique (Method 2), the latter modified by the addition of BMP4 (Method 3). To investigate the retinal organoid quantity, we counted the number of retinal domains, precursors to organoids during differentiation. The retinal organoid quality was evaluated by immunostaining for markers of different retinal cell types in whole cryosections after days 85, 120, and 200 in culture.

Results: Method 3 produced strikingly more retinal domains per differentiation (65 ± 27) than Methods 1 (12.3 ± 11.2) and 2 (6.3 ± 6.7). Furthermore, retinal organoids differentiated with Method 3 contained significantly more CRX-positive photoreceptors and BRN3A-positive ganglion cells after 85 days in culture, compared to Methods 1 and 2. After 200 days in culture, the retinal organoids differentiated with Method 3 showed proper maturation, as demonstrated by the expression of mature rod and cone photoreceptor markers.

Conclusion: This study demonstrates that the retinal organoid differentiation method can significantly impact the cellular composition of retinal organoids at various time points of development.

Keywords

Retina / retinal organoid / BMP4 / photoreceptor

References

[1] Lancaster MA.Organogenesis in a dish: modeling development and disease using organoid technologies.Science2014;345:1247125

[2] Eiraku M,Matsuo-Takasaki M.Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.Cell Stem Cell2008;3:519-32

[3] Lancaster MA,Wolfinger S.Guided self-organization and cortical plate formation in human brain organoids.Nat Biotechnol2017;35:659-66 PMCID:PMC5824977

[4] Lancaster MA,Martin CA.Cerebral organoids model human brain development and microcephaly.Nature2013;501:373-9 PMCID:PMC3817409

[5] Velasco S,Simmons SK.Individual brain organoids reproducibly form cell diversity of the human cerebral cortex.Nature2019;570:523-7 PMCID:PMC6906116

[6] Schwank G,Sasselli V.Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.Cell Stem Cell2013;13:653-8

[7] Dekkers JF,de Jonge HR.A functional CFTR assay using primary cystic fibrosis intestinal organoids.Nat Med2013;19:939-45

[8] Longmire TA,Hawkins F.Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells.Cell Stem Cell2012;10:398-411 PMCID:PMC3322392

[9] Wong AP,Chin S.Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein.Nat Biotechnol2012;30:876-82 PMCID:PMC3994104

[10] Low JH,Chew EGY.Generation of human PSC-derived kidney organoids with patterned nephron segments and a de novo vascular network.Cell Stem Cell2019;25:373-87.e9 PMCID:PMC6731150

[11] Watanabe K,Nishiyama A.Directed differentiation of telencephalic precursors from embryonic stem cells.Nat Neurosci2005;8:288-96

[12] Eiraku M,Ishibashi H.Self-organizing optic-cup morphogenesis in three-dimensional culture.Nature2011;472:51-6

[13] Nakano T,Takata N.Self-formation of optic cups and storable stratified neural retina from human ESCs.Cell Stem Cell2012;10:771-85

[14] Zhong X,Xue T.Generation of three dimensional retinal tissue with functional photoreceptors from human iPSCs.Nat Commun2014;5:4047 PMCID:PMC4370190

[15] Wahlin KJ,Sripathi SR.Photoreceptor outer segment-like structures in long-term 3D retinas from human pluripotent stem cells.Sci Rep2017;7:766 PMCID:PMC5429674

[16] Kuwahara A,Nakano T,Eiraku M.Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue.Nat Commun2015;6:6286

[17] Sridhar A,Finkbeiner CR.Single-cell transcriptomic comparison of human fetal retina, hPSC-derived retinal organoids, and long-term retinal cultures.Cell Rep2020;30:1644-59.e4 PMCID:PMC7901645

[18] Cowan CS,De Gennaro M.Cell Types of the human retina and its organoids at single-cell resolution.Cell2020;182:1623-40.e34 PMCID:PMC7505495

[19] Lane A,Shortall C.Modelling and rescue of RP2 retinitis pigmentosa using iPSC derived retinal organoids.Stem Cell Reports2020;15:67-79

[20] Huang KC,Chen SJ.Morphological and molecular defects in human three-dimensional retinal organoid model of X-linked juvenile retinoschisis.Stem Cell Reports2019;13:906-23 PMCID:PMC6895767

[21] Kruczek K,Gentry J.Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.Stem Cell Reports2021;16:252-63 PMCID:PMC7878833

[22] Gao ML,Han F.Patient-specific retinal organoids recapitulate disease features of late-onset retinitis pigmentosa.Front Cell Dev Biol2020;8:128 PMCID:PMC7068133

[23] Quinn PM,Mulder AA.Human iPSC-derived retinas recapitulate the fetal CRB1 CRB2 complex formation and demonstrate that photoreceptors and müller glia are targets of AAV5.Stem Cell Reports2019;12:906-19 PMCID:PMC6522954

[24] Kruczek K.Pluripotent stem cell-derived retinal organoids for disease modeling and development of therapies.Stem Cells2020;38:1206-15 PMCID:PMC7586922

[25] Watanabe K,Kamiya D.A ROCK inhibitor permits survival of dissociated human embryonic stem cells.Nat Biotechnol2007;25:681-6

[26] Meyer JS,Capowski EE.Modeling early retinal development with human embryonic and induced pluripotent stem cells.Proc Natl Acad Sci U S A2009;106:16698-703 PMCID:PMC2757802

[27] Zhou S,Abdouh M.Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFβ and Wnt signaling.Development2015;142:3294-306

[28] Bragina O,Serg M.Smoothened agonist augments proliferation and survival of neural cells.Neurosci Lett2010;482:81-5

[29] Osakada F,Mandai M.Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.Nat Biotechnol2008;26:215-24

[30] Capowski EE,Mayerl SJ.Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.Development2019;146:dev171686 PMCID:PMC6340149

[31] Brandl C,Milenkovic VM.In-depth characterisation of Retinal Pigment Epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC).Neuromolecular Med2014;16:551-64 PMCID:PMC4119585

[32] Nachtigal AL,Brandl C.Mutation-dependent pathomechanisms determine the phenotype in the bestrophinopathies.Int J Mol Sci2020;21:1597 PMCID:PMC7084480

[33] Hallam D,Dorgau B.Human-induced pluripotent stem cells generate light responsive retinal organoids with variable and nutrient-dependent efficiency.Stem Cells2018;36:1535-51 PMCID:PMC6392112

[34] Schindelin J,Frise E.Fiji: an open-source platform for biological-image analysis.Nat Methods2012;9:676-82 PMCID:PMC3855844

[35] Huang L.Image thresholding by minimizing the measures of fuzziness.Pattern Recognition1995;28:41-51

[36] Lukowski SW,Sharov AA.A single-cell transcriptome atlas of the adult human retina.EMBO J2019;38:e100811 PMCID:PMC6745503

[37] Soto I,Buckingham BP.Retinal ganglion cells downregulate gene expression and lose their axons within the optic nerve head in a mouse glaucoma model.J Neurosci2008;28:548-61 PMCID:PMC6670511

[38] Kunzevitzky NJ,Goldberg JL.Amacrine cell gene expression and survival signaling: differences from neighboring retinal ganglion cells.Invest Ophthalmol Vis Sci2010;51:3800-12 PMCID:PMC2904021

[39] Yan XX.Early expression of recoverin in a unique population of neurons in the human retina.Anat Embryol (Berl)1997;195:51-63

[40] Furukawa T,Cepko CL.Crx, a Novel otx-like Homeobox Gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation.Cell1997;91:531-41

[41] Chen S,Nie Z.Crx, a Novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes.Neuron1997;19:1017-30

[42] Gonzalez-Cordero A,Naeem A.Recapitulation of human retinal development from human pluripotent stem cells generates transplantable populations of cone photoreceptors.Stem Cell Reports2017;9:820-37 PMCID:PMC5599247

[43] Muranishi Y,Inoue T.Gene expression analysis of embryonic photoreceptor precursor cells using BAC-Crx-EGFP transgenic mouse.Biochem Biophys Res Commun2010;392:317-22

[44] Xiang M,Macke J.The Brn-3 family of POU-domain factors: primary structure, binding specificity, and expression in subsets of retinal ganglion cells and somatosensory neurons.J Neurosci1995;15:4762-85 PMCID:PMC6577904

[45] Sajgo S,Brooks M.Molecular codes for cell type specification in Brn3 retinal ganglion cells.Proc Natl Acad Sci U S A2017;114:E3974-83 PMCID:PMC5441800

[46] Luo Z,Li K.Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid.Stem Cells Int2019;2019:8786396 PMCID:PMC6925930

[47] Cepko CL,Yang X,Ezzeddine D.Cell fate determination in the vertebrate retina.Proc Natl Acad Sci U S A1996;93:589-95 PMCID:PMC40096

[48] Blanks JC.Specific binding of peanut lectin to a class of retinal photoreceptor cells. A species comparison.Invest Ophthalmol Vis Sci1984;25:546-57

[49] Urist MR.Bone: formation by autoinduction.Science1965;150:893-9

[50] Wang RN,Wang Z.Bone Morphogenetic Protein (BMP) signaling in development and human diseases.Genes Dis2014;1:87-105 PMCID:PMC4232216

[51] Winnier G,Labosky PA.Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse.Genes Dev1995;9:2105-16

[52] Huang J,Oltean A.Bmp4 from the optic vesicle specifies murine retina formation.Dev Biol2015;402:119-26 PMCID:PMC4523094

[53] Wagstaff PE,Boon CJF,Ten Asbroek ALMA.The role of small molecules and their effect on the molecular mechanisms of early retinal organoid development.Int J Mol Sci2021;22:7081 PMCID:PMC8268497

[54] Tanaka Y,Xiang Y,Park IH.Synthetic analyses of single-cell transcriptomes from multiple brain organoids and fetal brain.Cell Rep2020;30:1682-9.e3 PMCID:PMC7043376

[55] Chichagova V,Ghareeb A.Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent.Stem Cells2020;38:195-201 PMCID:PMC7383896

[56] Reichman S,Slembrouck A.From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.Proc Natl Acad Sci U S A2014;111:8518-23 PMCID:PMC4060726

[57] Wagstaff PE,Ten Brink JB,Bergen AAB.An alternative approach to produce versatile retinal organoids with accelerated ganglion cell development.Sci Rep2021;11:1101 PMCID:PMC7806597