Rapid and efficient generation of a transplantable population of functional retinal ganglion cells from fibroblasts

Abstract

Glaucoma and other optic neuropathies lead to progressive and irreversible vision loss by damaging retinal ganglion cells (RGCs) and their axons. Cell replacement therapy is a potential promising treatment. However, current methods to obtain RGCs have inherent limitations, including time-consuming procedures, inefficient yields and complex protocols, which hinder their practical application. Here, we have developed a straightforward, rapid and efficient approach for directly inducing RGCs from mouse embryonic fibroblasts (MEFs) using a combination of triple transcription factors (TFs): ASCL1, BRN3B and PAX6 (ABP). We showed that on the 6th day following ABP induction, neurons with molecular characteristics of RGCs were observed, and more than 60% of induced neurons became iRGCs (induced retinal ganglion cells) in the end. Transplanted iRGCs had the ability to survive and appropriately integrate into the RGC layer of mouse retinal explants and N-methyl-D-aspartic acid (NMDA)-damaged retinas. Moreover, they exhibited electrophysiological properties typical of RGCs, and were able to regrow dendrites and axons and form synaptic connections with host retinal cells. Together, we have established a rapid and efficient approach to acquire functional RGCs for potential cell replacement therapy to treat glaucoma and other optic neuropathies.

References

[1] Bassett EA, Wallace VA. Cell fate determination in the vertebrate retina. Trends Neurosci. 2012;35(9):565-573.

[2] Zhang KY, Aguzzi EA, Johnson TV. Retinal ganglion cell transplantation: approaches for overcoming challenges to functional integration. Cells. 2021;10(6):1426.

[3] Sleath B, Robin AL, Covert D, Byrd JE, Tudor G, Svarstad B. Patient-reported behavior and problems in using glaucoma medications. Ophthalmology. 2006;113(3):431-436.

[4] Yook E, Vinod K, Panarelli JF. Complications of micro-invasive glaucoma surgery. Curr Opin Ophthalmol. 2018;29(2):147-154.

[5] Venugopalan P, Wang Y, Nguyen T, Huang A, Muller KJ, Goldberg JL. Transplanted neurons integrate into adult retinas and respond to light. Nat Commun. 2016;7:10472.

[6] Hertz J, Qu B, Hu Y, Patel RD, Valenzuela DA, Goldberg JL. Survival and integration of developing and progenitor-derived retinal ganglion cells following transplantation. Cell Transplant. 2014;23(7):855-872.

[7] Ohlemacher SK, Sridhar A, Xiao Y, et al. Stepwise differentiation of retinal ganglion cells from human pluripotent stem cells enables analysis of glaucomatous neurodegeneration. Stem Cells. 2016;34(6):1553-1562.

[8] Teotia P, Chopra DA, Dravid SM, et al. Generation of functional human retinal ganglion cells with target specificity from pluripotent stem cells by chemically defined recapitulation of developmental mechanism. Stem Cells. 2017;35(3):572-585.

[9] Riazifar H, Jia Y, Chen J, Lynch G, Huang T. Chemically induced specification of retinal ganglion cells from human embryonic and induced pluripotent stem cells. Stem Cells Transl Med. 2014;3(4):424-432.

[10] Chen M, Chen Q, Sun X, et al. Generation of retinal ganglion-like cells from reprogrammed mouse fibroblasts. Invest Ophthalmol Vis Sci. 2010;51(11):5970-5978.

[11] Lamba DA, Karl MO, Ware CB, Reh TA. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006;103(34):12769-12774.

[12] Deng F, Chen M, Liu Y, et al. Stage-specific differentiation of iPSCs toward retinal ganglion cell lineage. Mol Vis. 2016;22:536-547.

[13] Tanaka T, Yokoi T, Tamalu F, Watanabe SI, Nishina S, Azuma N. Generation of retinal ganglion cells with functional axons from mouse embryonic stem cells and induced pluripotent stem cells. Invest Ophthalmol Vis Sci. 2016;57(7):3348-3359.

[14] Vrathasha V, Nikonov S, Bell BA, et al. Transplanted human induced pluripotent stem cells- derived retinal ganglion cells embed within mouse retinas and are electrophysiologically functional. iScience. 2022;25(11):105308.

[15] Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 2010;463(7284):1035-1041.

[16] Liu S, Xiang K, Yuan F, Xiang M. Generation of self-organized autonomic ganglion organoids from fibroblasts. iScience. 2023;26(3):106241.

[17] Ieda M, Fu JD, Delgado-Olguin P, et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 2010;142(3):375-386.

[18] Xiao D, Liu X, Zhang M, et al. Direct reprogramming of fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a. Nat Commun. 2018;9(1):2865.

[19] Mahato B, Kaya KD, Fan Y, et al. Pharmacologic fibroblast reprogramming into photoreceptors restores vision. Nature. 2020;581(7806):83-88.

[20] Pan SH, Zhao N, Feng X, Jie Y, Jin ZB. Conversion of mouse embryonic fibroblasts into neural crest cells and functional corneal endothelia by defined small molecules. Sci Adv. 2021;7(23):eabg5749.

[21] Xiao D, Deng Q, Guo Y, et al. Generation of self-organized sensory ganglion organoids and retinal ganglion cells from fibroblasts. Sci Adv. 2020;6(22):eaaz5858.

[22] Todd L, Jenkins W, Finkbeiner C, et al. Reprogramming Müller glia to regenerate ganglion-like cells in adult mouse retina with developmental transcription factors. Sci Adv. 2022;8(47):eabq7219.

[23] Xiang M. Intrinsic control of mammalian retinogenesis. Cell Mol Life Sci. 2013;70(14):2519-2532.

[24] Lyu J, Mu X. Genetic control of retinal ganglion cell genesis. Cell Mol Life Sci. 2021;78(9):4417-4433.

[25] Nadal-Nicolás FM, Jiménez-López M, Sobrado-Calvo P, et al. Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci. 2009;50(8):3860-3868.

[26] Lee J, Choi SH, Kim YB, et al. Defined conditions for differentiation of functional retinal ganglion cells from human pluripotent stem cells. Invest Ophthalmol Vis Sci. 2018;59(8):3531-3542.

[27] Gan L, Xiang M, Zhou L, Wagner DS, Klein WH, Nathans J. POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells. Proc Natl Acad Sci U S A. 1996;93(9):3920-3925.

[28] Wu F, Kaczynski TJ, Sethuramanujam S, et al. Two transcription factors, Pou4f2 and Isl1, are sufficient to specify the retinal ganglion cell fate. Proc Natl Acad Sci U S A. 2015;112(13):E1559-E1568.

[29] Rheaume BA, Jereen A, Bolisetty M, et al. Single cell transcriptome profiling of retinal ganglion cells identifies cellular subtypes. Nat Commun. 2018;9(1):2759.

[30] Gong J, Jellali A, Mutterer J, Sahel JA, Rendon A, Picaud S. Distribution of vesicular glutamate transporters in rat and human retina. Brain Res. 2006;1082(1):73-85.

[31] Stella SL, Li S, Sabatini A, Vila A, Brecha NC. Comparison of the ontogeny of the vesicular glutamate transporter 3 (VGLUT3) with VGLUT1 and VGLUT2 in the rat retina. Brain Res. 2008;1215:20-29.

[32] Zheng GX, Terry JM, Belgrader P, et al. Massively parallel digital transcriptional profiling of single cells. Nat Commun. 2017;8:14049.

[33] Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36(5):411-420.

[34] Chiasseu M, Cueva Vargas JL, Destroismaisons L, Vande Velde C, Leclerc N, di Polo A. Tau accumulation, altered phosphorylation, and Missorting promote neurodegeneration in glaucoma. J Neurosci. 2016;36(21):5785-5798.

[35] Chiasseu M, Alarcon-Martinez L, Belforte N, et al. Tau accumulation in the retina promotes early neuronal dysfunction and precedes brain pathology in a mouse model of Alzheimer's disease. Mol Neurodegener. 2017;12(1):58.

[36] Pattamatta U, McPherson Z, White A. A mouse retinal explant model for use in studying neuroprotection in glaucoma. Exp Eye Res. 2016;151:38-44.

[37] Xiang M, Zhou L, Macke JP, et al. The Brn-3 family of POU-domain factors: primary structure, binding specificity, and expression in subsets of retinal ganglion cells and somatosensory neurons. J Neurosci. 1995;15(7 Pt 1):4762-4785.

[38] Wang SW, Kim BS, Ding K, et al. Requirement for math5 in the development of retinal ganglion cells. Genes Dev. 2001;15(1):24-29.

[39] Jiang Y, Ding Q, Xie X, Libby RT, Lefebvre V, Gan L. Transcription factors SOX4 and SOX11 function redundantly to regulate the development of mouse retinal ganglion cells. J Biol Chem. 2013;288(25):18429-18438.

[40] Jin K, Jiang H, Mo Z, Xiang M. Early B-cell factors are required for specifying multiple retinal cell types and subtypes from postmitotic precursors. J Neurosci. 2010;30(36):11902-11916.

[41] Zhang Q, Zagozewski J, Cheng S, et al. Regulation of Brn3b by DLX1 and DLX2 is required for retinal ganglion cell differentiation in the vertebrate retina. Development. 2017;144(9):1698-1711.

[42] Riesenberg AN, le TT, Willardsen MI, Blackburn DC, Vetter ML, Brown NL. Pax6 regulation of Math5 during mouse retinal neurogenesis. Genesis. 2009;47(3):175-187.

[43] Lalitha S, Basu B, Surya S, et al. Pax6 modulates intra-retinal axon guidance and fasciculation of retinal ganglion cells during retinogenesis. Sci Rep. 2020;10(1):16075.

[44] Wapinski OL, Vierbuchen T, Qu K, et al. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell. 2013;155(3):621-635.

[45] Mu X, Fu X, Beremand PD, Thomas TL, Klein WH. Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2. Proc Natl Acad Sci U S A. 2008;105(19):6942-6947.

[46] Sun Y, Dykes IM, Liang X, Eng SR, Evans SM, Turner EE. A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs. Nat Neurosci. 2008;11(11):1283-1293.

[47] Dykes IM, Tempest L, Lee SI, Turner EE. Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation. J Neurosci. 2011;31(27):9789-9799.

[48] Tsunemoto R, Lee S, Szűcs A, et al. Diverse reprogramming codes for neuronal identity. Nature. 2018;557(7705):375-380.

[49] de Melo J, Qiu X, du G, Cristante L, Eisenstat DD. Dlx1, Dlx2, Pax6, Brn3b, and Chx10 homeobox gene expression defines the retinal ganglion and inner nuclear layers of the developing and adult mouse retina. J Comp Neurol. 2003;461(2):187-204.

[50] Wu YR, Hashiguchi T, Sho J, Chiou SH, Takahashi M, Mandai M. Transplanted mouse embryonic stem cell-derived retinal ganglion cells integrate and form synapses in a retinal ganglion cell-depleted mouse model. Invest Ophthalmol Vis Sci. 2021;62(13):26.

[51] Lieven O, Rüther U. The Dkk1 dose is critical for eye development. Dev Biol. 2011;355(1):124-137.

[52] Messina A, Incitti T, Bozza A, Bozzi Y, Casarosa S. Noggin expression in the adult retina suggests a conserved role during vertebrate evolution. J Histochem Cytochem. 2014;62(7):532-540.

[53] Tucker KL, Meyer M, Barde Y-A. Neurotrophins are required for nerve growth during development. Nat Neurosci. 2001;4(1):29-37.

[54] Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002;295(5556):868-872.

[55] Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998;72(11):8463-8471.

[56] Mo Z, Li S, Yang X, Xiang M. Role of the Barhl2 homeobox gene in the specification of glycinergic amacrine cells. Development. 2004;131(7):1607-1618.

[57] Stuart T, Butler A, Hoffman P, et al. Comprehensive integration of single-cell data. Cell. 2019;177(7):1888-1902.

[58] Wu T, Hu E, Xu S, et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation (Camb). 2021;2(3):100141.

[59] Jin K, Xiang M. In vitro explant culture and related protocols for the study of mouse retinal development. Methods Mol Biol. 2012;884:155-165.

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2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.