Photoreceptor loss is the final common endpoint in most retinopathies that lead to irreversible blindness, and there are no effective treatments to restore vision1,2. Chemical reprogramming of fibroblasts offers an opportunity to reverse vision loss; however, the generation of sensory neuronal subtypes such as photoreceptors remains a challenge. Here we report that the administration of a set of five small molecules can chemically induce the transformation of fibroblasts into rod photoreceptor-like cells. The transplantation of these chemically induced photoreceptor-like cells (CiPCs) into the subretinal space of rod degeneration mice (homozygous for rd1, also known as Pde6b) leads to partial restoration of the pupil reflex and visual function. We show that mitonuclear communication is a key determining factor for the reprogramming of fibroblasts into CiPCs. Specifically, treatment with these five compounds leads to the translocation of AXIN2 to the mitochondria, which results in the production of reactive oxygen species, the activation of NF-κB and the upregulation of Ascl1. We anticipate that CiPCs could have therapeutic potential for restoring vision.
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Wright, A. F., Chakarova, C. F., Abd El-Aziz, M. M. & Bhattacharya, S. S. Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait. Nat. Rev. Genet. 11, 273–284 (2010).
Bramall, A. N., Wright, A. F., Jacobson, S. G. & McInnes, R. R. The genomic, biochemical, and cellular responses of the retina in inherited photoreceptor degenerations and prospects for the treatment of these disorders. Annu. Rev. Neurosci. 33, 441–472 (2010).
Schwartz, S. D. et al. Human embryonic-stem cell derived retinal pigment eputhellium in patients with age-related macular degeneration and Stagardt’s macular dystrophy: follow up of two open-label phase 1/2 studies. Lancet 385, 509–516 (2015).
Dyer, M. A. An eye on retinal recovery. Nature 540, 350–351 (2016).
Mellough, C. B., Sernagor, E., Moreno-Gimeno, I., Steel, D. H. & Lako, M. Efficient stage-specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells. Stem Cells 30, 673–686 (2012).
Zhong, X. et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat. Commun. 5, 4047 (2014).
Gamm, D. M. & Wright, L. S. From embryonic stem cells to mature photoreceptors. Nat. Biotechnol. 31, 712–713 (2013).
Völkner, M. et al. Retinal organoids from pluripotent stem cells efficiently recapitulate retinogenesis. Stem Cell Reports 6, 525–538 (2016).
Fu, Y. et al. Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails. Cell Res. 25, 1013–1024 (2015).
Tian, E. et al. Small-molecule-based lineage reprogramming creates functional astrocytes. Cell Rep. 16, 781–792 (2016).
Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010).
Babos, K. & Ichida, J. K. Small molecules take a big step by converting fibroblasts into neurons. Cell Stem Cell 17, 127–129 (2015).
Zhang, L. et al. Small molecules efficiently reprogram human astroglial cells into functional neurons. Cell Stem Cell 17, 735–747 (2015).
Ueki, Y. et al. Transgenic expression of the proneural transcription factor Ascl1 in Müller glia stimulates retinal regeneration in young mice. Proc. Natl Acad. Sci. USA 112, 13717–13722 (2015).
Jorstad, N. L. et al. Stimulation of functional neuronal regeneration from Müller glia in adult mice. Nature 548, 103–107 (2017).
Pollak, J. et al. ASCL1 reprograms mouse Müller glia into neurogenic retinal progenitors. Development 140, 2619–2631 (2013).
Akimoto, M. et al. Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors. Proc. Natl Acad. Sci. USA 103, 3890–3895 (2006).
Kim, J. W. et al. NRL-regulated transcriptome dynamics of developing rod photoreceptors. Cell Rep. 17, 2460–2473 (2016).
Osakada, F. et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat. Biotechnol. 26, 215–224 (2008).
Khanna, H. et al. Retinoic acid regulates the expression of photoreceptor transcription factor NRL. J. Biol. Chem. 281, 27327–27334 (2006).
Onishi, A. et al. Pias3-dependent SUMOylation directs rod photoreceptor development. Neuron 61, 234–246 (2009).
Fu, Y. et al. Feedback induction of a photoreceptor-specific isoform of retinoid-related orphan nuclear receptor β by the rod transcription factor NRL. J. Biol. Chem. 289, 32469–32480 (2014).
Kim, J. W. et al. Recruitment of rod photoreceptors from short-wavelength-sensitive cones during the evolution of nocturnal vision in mammals. Dev. Cell 37, 520–532 (2016).
Jia, L. et al. Retinoid-related orphan nuclear receptor ROR is an early-acting factor in rod photoreceptor development. Proc. Natl Acad. Sci. USA 106, 17534–17539 (2009).
Brzezinski, J. A., IV, Kim, E. J., Johnson, J. E. & Reh, T. A. Ascl1 expression defines a subpopulation of lineage-restricted progenitors in the mammalian retina. Development 138, 3519–3531 (2011).
Zhu, J., Cifuentes, H., Reynolds, J. & Lamba, D. A. Immunosuppression via loss of IL2rγ enhances long-term functional integration of hESC-derived photoreceptors in the mouse retina. Cell Stem Cell 20, 374–384.e5 (2017).
Lin, B., Koizumi, A., Tanaka, N., Panda, S. & Masland, R. H. Restoration of visual function in retinal degeneration mice by ectopic expression of melanopsin. Proc. Natl Acad. Sci. USA 105, 16009–16014 (2008).
Nishiguchi, K. M. et al. Gene therapy restores vision in rd1 mice after removal of a confounding mutation in Gpr179. Nat. Commun. 6, 6006 (2015).
Pang, Z. P. et al. Induction of human neuronal cells by defined transcription factors. Nature 476, 220–223 (2011).
Swaroop, A., Kim, D. & Forrest, D. Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina. Nat. Rev. Neurosci. 11, 563–576 (2010).
Zhang, Y. et al. Nuclear factor kappa B signaling initiates early differentiation of neural stem cells. Stem Cells 30, 510–524 (2012).
Formentini, L., Sánchez-Aragó, M., Sánchez-Cenizo, L. & Cuezva, J. M. The mitochondrial ATPase inhibitory factor 1 triggers a ROS-mediated retrograde prosurvival and proliferative response. Mol. Cell 45, 731–742 (2012).
Andreakos, E. et al. Distinct pathways of LPS-induced NF-kappa B activation and cytokine production in human myeloid and nonmyeloid cells defined by selective utilization of MyD88 and Mal/TIRAP. Blood 103, 2229–2237 (2004).
Vernochet, C. et al. Adipose-specific deletion of TFAM increases mitochondrial oxidation and protects mice against obesity and insulin resistance. Cell Metab. 16, 765–776 (2012).
Kim, H. et al. Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nat. Commun. 4, 2403 (2013).
Shin, J. H., Kim, H. W., Rhyu, I. J. & Kee, S. H. Axin is expressed in mitochondria and suppresses mitochondrial ATP synthesis in HeLa cells. Exp. Cell Res. 340, 12–21 (2016).
Rehemtulla, A. et al. The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression. Proc. Natl Acad. Sci. USA 93, 191–195 (1996).
Morrow, E. M., Furukawa, T., Lee, J. E. & Cepko, C. L. NeuroD regulates multiple functions in the developing neural retina in rodent. Development 126, 23–36 (1999).
Ortin-Martinez, A. et al. A reinterpretation of cell transplantation: GFP transfer from donor to host photoreceptors. Stem Cells 35, 932–939 (2017).
Singh, M. S. et al. Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation. Proc. Natl Acad. Sci. USA 110, 1101–1106 (2013).
Zhou, G., Meng, S., Li, Y., Ghebre, Y. T. & Cooke, J. P. Optimal ROS signaling is critical for nuclear reprogramming. Cell Rep. 15, 919–925 (2016).
Shadel, G. S. & Horvath, T. L. Mitochondrial ROS signaling in organismal homeostasis. Cell 163, 560–569 (2015).
Cagin. U., Duncan, O. F., Gatt, A. P., Dionne, M. S., Sweeney, S. T. & Bateman, J. M. Mitochondrial retrograde signaling regulates neuronal function. Proc. Natl Acad. Sci. USA 112, E6000–E6009 (2015).
Jozefczuk, J., Drews, K. & Adjaye, J. Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. J. Vis. Exp. 3854 (2012).
Chen, H. Y. et al. Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation. Mol. Vis. 22, 1077–1094 (2016).
Kaya, K. D. et al. Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal. Mol. Vis. 25, 663–678 (2019).
Wojtala, A. et al. Methods to monitor ROS production by fluorescence microscopy and fluorometry. Methods Enzymol. 542, 243–262 (2014).
Tormos, K. V. et al. Mitochondrial complex III ROS regulate adipocyte differentiation. Cell Metab. 14, 537–544 (2011).
S.H.C. is supported by the Nancy Lee and Perry R. Bass Endowment, Foundation Fighting Blindness, and NEI awards EY021171 and EY025667. A.S. is supported by NEI Intramural Research Program (ZIAEY000450, ZIAEY000474 and ZIAEY000546). S.M. and S.B. are supported by NEI awards EY025905 and EY025717. T.M. is supported by 2T32AG020494-16A1. We thank A. Quiambao for performing the in vivo rodent injection studies, A. Ganguly for pupillometry assistance, the Histology Research Core at the University of North Carolina at Chapel Hill, and L. Gieser of NNRL, NEI for performing RNA-seq.
B.M. and S.H.C. are listed as inventors on a pending patent application (PCT/US2018/037784), filed by the University of North Texas, that covers the chemical reprogramming method reported in this paper. S.H.C. owns equity in and is a founder of CIRC Therapeutics, Inc., a start-up company that plans to commercialize novel treatments for ocular disease related to this technology. All other authors declare no competing interests.
Peer review information Nature thanks Jayakrishna Ambati, Pete Coffey and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, DNA gel shows cloned Nrl and DsRed fragments digested out from the pENTR2B vector. This experiment was performed once. b, Expression of Nrl–DsRed in human CiPCs on day 8. c, The number of Nrl–GFP+ cells upon subtraction of the indicated small molecules from the mixture. d, The number of Nrl–GFP+ cells after conversion (on day 11) upon treatment with each small molecule alone. e, Scheme showing the generation of the mouse model for lineage tracing. f, Flow sorting of FSP1Cre–tdTomato+ MEFs. Left, scatter plot from FACS; middle, MEFs before cell sorting; right, MEFs after cell sorting. Scale bars, 25 μm. This experiment was performed once. g, Cells expressing NRL are FSP1–tdTomato-positive on day 11, suggesting that they originate from fibroblasts. h, Percentage of cells that are positive for both NRL and tdTomato, compared with those that are just positive for NRL. In c, d, h data are presented as mean ± s.e.m. of n = 3 independently treated wells. Statistical significance was assessed using a two-tailed Student’s t-test.
a, Images of BrdU-stained CiPCs on day 11. Scale bars, 10 μm. b, Heat map analysis of RNA-seq data for the indicated cell-type-specific genes. c, Timeline for subretinal transplantation of CiPCs and functional analysis. d, Scotopic b-wave after transplantation of converted CiPCs. n denotes the number of biologically independent animals.
a–c, Analysis for the scotopic a-wave of ERG after transplantation of CiPCs at day 45 (a), day 59 (b) and day 77 (c). d, Scatter plot of pupil constriction measurements for CiPC-injected rd1 mice taken three months after transplantation. e, f, Measurement of visual acuity (e) and contrast sensitivity (f) for CiPC-injected mice that demonstrated improved pupil response after transplantation and control mice. OR, optokinetic response. For the CiPC-injected rd1 experiment, repeated values are plotted (for n = 1 mouse). In a–e, n denotes biologically independent animals and the experiment in f was performed twice.
Extended Data Fig. 4 Transplanted CiPCs express photoreceptor-specific markers and connected to the inner retina three months after transplantation.
a, Expression of recoverin (Recov, red) in CiPCs (green). b, Expression of rhodopsin (Rho, red) in GFP-expressing CiPCs. c, Expression of rod ribbon-synapse protein ribeye (red) in transplanted CiPCs (green). The bottom panel shows a magnified version of the image (arrowhead). d, Close apposition of transplanted CiPCs (green) with PKC-positive (red) rod bipolar cells (white arrowhead). e, Expression of synaptic marker protein synaptophysin (red, arrowhead) in transplanted CiPCs (green) (co-localization, yellow). d, e, Left, images at 20× magnification; right, images at higher digital magnification. In a–e, experiments were repeated twice with similar results. Scale bars, 10 μm.
Extended Data Fig. 5 Effect of NF-κB knockdown, H2O2, TNFα and the overexpression of Ascl1 on the reprogramming of fibroblasts to CiPCs.
a, qPCR analysis following shRNA-mediated knockdown (kd) of RelA (which encodes NF-κB) in MEFs. b, qPCR analysis of Ascl1 expression after the reprogramming of RelA-depleted MEFs on day 11. c, Number of Nrl–GFP cells after the conversion of RelA-knockdown MEFs on day 11. d, Effect of different concentrations of H2O2, generated by galactose and galactose oxidase (GAO), on the reprogramming of fibroblasts to CiPCs. e, Effect of cytokine (TNF, 50 ng ml−1) on CiPC conversion. f, qPCR analysis showing the overexpression (OE) of Ascl1 in MEFs. g, Phase contrast micrographs of MEFs overexpressing Ascl1 before and after reprogramming in inducing medium. No Nrl–GFP+ cells were evident up to day 15. h, Fluorometric measurement of mROS in Ascl1-overexpressing cells on day 8 of reprogramming. Data are presented as mean ± s.e.m. of n = 3 independently treated wells. Statistical significance was assessed using a two-tailed Student’s t-test.
Extended Data Fig. 6 NF-κB–luciferase activity and generation of mROS during the reprogramming of fibroblasts to CiPCs.
a, A luciferase activity assay reveals decreased activation of NF-κB upon treatment with MitoTEMPO and increased NF-κB activity upon depletion of TFAM on day 11 of reprogramming to CiPCs. b, Luciferase activity shows decreased NF-κB activation in Axin2-knockdown MEFs on day 8 and day 11 of reprogramming. SM, small molecules. c, MEFs stained with MitoSOX showed low levels of mROS production (scale bar, 33 μm). d, Plot of mROS accumulation on day 8, as visualized using fluorescence microscopy after MitoSOX staining, in wild-type (WT) Nrl–GFP MEFs treated with all compounds (top), Axin2-knockdown Nrl–GFP MEFs treated with all compounds (middle) and control Nrl–GFP MEFs (bottom). e, qPCR analysis following Tfam depletion in MEFs using shRNA. f, Fluorometric measurement of mROS (after MitoSOX staining) in MitoTEMPO-treated and TFAM-knockdown cells on day 11. g, ChIP assay shows reduced binding of activated NF-κB near the Ascl1 locus upon treatment with MitoSOX. In a, b, e–g, data are presented as mean ± s.e.m. of n = 3 independently treated wells. Statistical significance was assessed using a two-tailed Student’s t-test.
Extended Data Fig. 7 Mitochondrial localization of AXIN2 in converted cells and a mito-stress test for CiPCs.
a, Western blot after subcellular fractionation of CiPCs showed that AXIN2 was localized in the mitochondria. C, cytoplasm; M, mitochondria. For gel source data, see Supplementary Fig. 1e, f. b, Micrograph of AXIN2 (red)- and GFP (green)-stained CiPCs after z-stack and 3D reconstruction, also showing that AXIN2 localizes in the mitochondria. In a, b experiments were performed once. c, CiPCs were found to have a reduced oxygen consumption rate (OCR) compared with MEFs and day-8 reprogramming intermediates, as measured using a Seahorse assay. Oligo, oligomycin; FCCP, carbonyl cyanide-4-phenylhydrazone. d, The indicated mitochondrial parameters of CiPCs, quantified in terms of OCR using a Seahorse assay, compared with those of MEFs and day-8 reprogramming intermediates. e, The extracellular acidification rate (ECAR) of CiPCs, MEFs and day-8 reprogramming intermediates was measured using a Seahorse assay. f, The indicated mitochondrial parameters of CiPCs, quantified in terms of ECAR using a Seahorse assay, compared with those of MEFs and day-8 reprogramming intermediates. In c–f, data are presented as mean ± s.e.m. of n = 3 independently treated wells.
Extended Data Fig. 8 Conversion of human fibroblasts into CiPCs and in vivo functional testing of mouse CiPCs.
a, qPCR analysis shows expression of the indicated photoreceptor-specific genes in CiPCs. b, Fluorescence microscopy image demonstrates the expression of NRL in CiPCs. a, b, HADF from patients with retinitis pigmentosa. This experiment was performed three times with similar results. c, Confocal microscopy image of the retina of an rd1 mouse, immunostained with GFP (red) and PNA (peanutagglutinin, green), three months after transplantation of CiPCs. d, Left, confocal microscopy image of a retinal section showing GFP-positive CiPCs three months after transplantation. This experiment was repeated twice with similar results. Right, conversion of human HFL1s (original protocol, see Methods) and a modified protocol using HADFs (Fig. 1f) to CiPCs. e, The correlation between the number of transplanted CiPCs and the extent of pupil constriction. n = 6 eyes classified as pupil responders and n = 8 eyes classified as pupil non-responders. f, CiPC survival in the eyes of rd1 mice classified as pupil responders and pupil non-responders. The n values denote the number of eyes. In a, d, e, data are presented as mean ± s.e.m. of n = 3 independently treated wells.
a, Pairwise gene expression comparison of RNA-seq data demonstrates that neuronal markers are increased, glial markers are not activated and fibroblast genes are silenced. Numbers on both axes represent logarithmic base 2-transformed counts per million values for each gene. b, Heat map analysis (from RNA-seq data) of photoreceptor genes along with other neuronal and fibroblast genes. c, Fibroblast-specific gene expression as determined by RNA-seq during reprogramming. The expression of Col1a1, Col2a1, Thy1, Ctgf, S100a4 (also known as Fsp1) and Slc17a5 is reduced in reprogramming intermediates as well as in CiPCs. d, Pie charts of results of ATAC–seq analysis show increased open chromatin corresponding to the proximal promoter region in reprogramming intermediates. e, Principal component analysis of ATAC–seq data shows open chromatin regions for the indicated samples. f, ATAC–seq analysis shows opening of specific chromatin regions for the indicated photoreceptor-specific loci (Ascl1, Rho, Gngt1 and Thrb). All data are representative of three independently treated samples.
Extended Data Fig. 10 Schematic of the transplantation study and the mechanism of reprogramming fibroblasts to CiPCs.
a, Schematic of reprogramming to CiPCs and functional analysis. b, Schematic of the mechanism of reprogramming fibroblasts to CiPCs.
This file contains Supplementary Figures 1 and 2. Supplementary Figure 1: Uncropped scans of source data for immunoblots and DNA gels. Results from immunoblots indicated the role of axin2 in ROS generation from mitochondria. DNA gels show the expression of indicated photoreceptor specific genes in CiPC. Supplementary Figure 2: Gating strategy. Representative example of gating strategy used for flow cytometry studies.
Composition of various cell culture media used for this study. IMR90 medium was used for the growth of mouse embryonic fibroblasts (MEFs). PIM and PDM medium were used during CiPC reprogramming with small molecules.
Description of all the small molecules used for CiPC reprogramming. Concentration and source of all small molecules are detailed.
Sequence of all the primers including RT-PCR used for the study.
Details of all the antibodies used in this study. Source and antibody dilutions are indicated.
Outline for the screening of various small molecules used for this study including the rationale for the use of each small molecule.
List of all the human fibroblasts tested for the chemical reprogramming process. Vendors and catalog number for each cell source is also indicated.
Motif enrichment in ATACSeq for indicated transcription factors during CiPC reprogramming.
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Mahato, B., Kaya, K.D., Fan, Y. et al. Pharmacologic fibroblast reprogramming into photoreceptors restores vision. Nature 581, 83–88 (2020). https://doi.org/10.1038/s41586-020-2201-4
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