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• W3021856789 abstract "Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs. Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs. Retinal degenerative diseases (RDDs), characterized by degeneration of retinal pigment epithelial (RPE) cells and loss of photoreceptors at their end stage, are the leading causes of irreversible blindness worldwide.1Zarbin M. Cell-based therapy for degenerative retinal disease.Trends Mol. Med. 2016; 22: 115-134Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 2Blond F. Léveillard T. Functional genomics of the retina to elucidate its construction and deconstruction.Int. J. Mol. Sci. 2019; 20: 4922Crossref Scopus (2) Google Scholar, 3Sahel J.A. Léveillard T. Maintaining cone function in rod-cone dystrophies.Adv. Exp. Med. Biol. 2018; 1074: 499-509Crossref PubMed Scopus (12) Google Scholar Previous studies have focused on verifying the safety and efficacy of RPE cells and/or photoreceptor transplantation in treating RDDs.4Lamba D.A. Gust J. Reh T.A. Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice.Cell Stem Cell. 2009; 4: 73-79Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 5Schwartz S.D. Hubschman J.P. Heilwell G. Franco-Cardenas V. Pan C.K. Ostrick R.M. Mickunas E. Gay R. Klimanskaya I. Lanza R. Embryonic stem cell trials for macular degeneration: a preliminary report.Lancet. 2012; 379: 713-720Abstract Full Text Full Text PDF PubMed Scopus (1074) Google Scholar, 6Mandai M. Kurimoto Y. Takahashi M. Autologous induced stem-cell-derived retinal cells for macular degeneration.N. Engl. J. Med. 2017; 377: 792-793Crossref PubMed Scopus (15) Google Scholar Unfortunately, limited evidence of vision restoration has been reported because only a minor subpopulation of donor cells can survive and proliferate in the host subretinal space, and epithelial-mesenchymal transition (EMT) of transplanted RPE cells obstructs their complete differentiation in vivo.7Kole C. Klipfel L. Yang Y. Ferracane V. Blond F. Reichman S. Millet-Puel G. Clérin E. Aït-Ali N. Pagan D. et al.Otx2-genetically modified retinal pigment epithelial cells rescue photoreceptors after transplantation.Mol. Ther. 2018; 26: 219-237Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar,8Kole C. Berdugo N. Da Silva C. Aït-Ali N. Millet-Puel G. Pagan D. Blond F. Poidevin L. Ripp R. Fontaine V. et al.Identification of an alternative splicing product of the Otx2 gene expressed in the neural retina and retinal pigmented epithelial cells.PLoS ONE. 2016; 11: e0150758Crossref PubMed Scopus (9) Google Scholar Human fetal RPE (fRPE) cells as a suitable transplantation cell source demonstrate the full function of native RPE cells and do not induce tumorigenesis.9Liao J.L. Yu J. Huang K. Hu J. Diemer T. Ma Z. Dvash T. Yang X.J. Travis G.H. Williams D.S. et al.Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells.Hum. Mol. Genet. 2010; 19: 4229-4238Crossref PubMed Scopus (141) Google Scholar,10Maminishkis A. Chen S. Jalickee S. Banzon T. Shi G. Wang F.E. Ehalt T. Hammer J.A. Miller S.S. Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.Invest. Ophthalmol. Vis. Sci. 2006; 47: 3612-3624Crossref PubMed Scopus (304) Google Scholar Unfortunately, their limited proliferation ability and EMT tendency restrict their therapeutic efficacy.7Kole C. Klipfel L. Yang Y. Ferracane V. Blond F. Reichman S. Millet-Puel G. Clérin E. Aït-Ali N. Pagan D. et al.Otx2-genetically modified retinal pigment epithelial cells rescue photoreceptors after transplantation.Mol. Ther. 2018; 26: 219-237Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar,11Radeke M.J. Radeke C.M. Shih Y.H. Hu J. Bok D. Johnson L.V. Coffey P.J. Restoration of mesenchymal retinal pigmented epithelial cells by TGFβ pathway inhibitors: implications for age-related macular degeneration.Genome Med. 2015; 7: 58Crossref PubMed Scopus (65) Google Scholar Adult RPE cells were activated to become self-renewing multipotent RPE stem-like cells (RPESCs) by a single-cell nonadherent culture.12Salero E. Blenkinsop T.A. Corneo B. Harris A. Rabin D. Stern J.H. Temple S. Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives.Cell Stem Cell. 2012; 10: 88-95Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar However, they are not actually pluripotent stem cells and cannot generate mature neurons and glia. Furthermore, the mesenchymal fate of RPESCs has not been clarified. Small molecules have advantages in chemical modification of cells because they are nonimmunogenic, cell permeable, cost-effective, and easily synthesized, preserved, and standardized.13Federation A.J. Bradner J.E. Meissner A. The use of small molecules in somatic-cell reprogramming.Trends Cell Biol. 2014; 24: 179-187Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,14Ma X. Kong L. Zhu S. Reprogramming cell fates by small molecules.Protein Cell. 2017; 8: 328-348Crossref PubMed Scopus (60) Google Scholar Small-molecule-modified cells are more technically tractable than cells modified via alternative genetic approaches, and their activity can be restricted spatially and temporally, ensuring an additional level of safety in clinical applications.15Zhang L. Yin J.C. Yeh H. Ma N.X. Lee G. Chen X.A. Wang Y. Lin L. Chen L. Jin P. et al.Small molecules efficiently reprogram human astroglial cells into functional neurons.Cell Stem Cell. 2015; 17: 735-747Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 16Kim Y. Kang K. Lee S.B. Seo D. Yoon S. Kim S.J. Jang K. Jung Y.K. Lee K.G. Factor V.M. et al.Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells.J. Hepatol. 2019; 70: 97-107Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 17Hawkins K.E. Moschidou D. Faccenda D. Wruck W. Martin-Trujillo A. Hau K.L. Ranzoni A.M. Sanchez-Freire V. Tommasini F. Eaton S. et al.Human amniocytes are receptive to chemically induced reprogramming to pluripotency.Mol. Ther. 2017; 25: 427-442Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Previous studies used vitamin C (VC)18Shi Y. Zhao Y. Deng H. Powering reprogramming with vitamin C.Cell Stem Cell. 2010; 6: 1-2Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,19Esteban M.A. Wang T. Qin B. Yang J. Qin D. Cai J. Li W. Weng Z. Chen J. Ni S. et al.Vitamin C enhances the generation of mouse and human induced pluripotent stem cells.Cell Stem Cell. 2010; 6: 71-79Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar and valproic acid (VPA),20Marquez-Curtis L.A. Qiu Y. Xu A. Janowska-Wieczorek A. Migration, proliferation, and differentiation of cord blood mesenchymal stromal cells treated with histone deacetylase inhibitor valproic Acid.Stem Cells Int. 2014; 2014: 610495Crossref PubMed Scopus (21) Google Scholar,21Moschidou D. Mukherjee S. Blundell M.P. Drews K. Jones G.N. Abdulrazzak H. Nowakowska B. Phoolchund A. Lay K. Ramasamy T.S. et al.Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach.Mol. Ther. 2012; 20: 1953-1967Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar respectively, to facilitate stem cell reprogramming and self-renewal. However, the joint or synergistic effects of VC and VPA in regulating retinal progenitor property and mesenchymal-epithelial transition (MET) in fRPE cells has not been studied. In this study, fRPESCs were produced through combined treatment with VV (VC plus VPA). These highly proliferative fRPESCs expressed retinal progenitor markers, underwent MET, and had the ability to differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage cells via upregulation of SOX2 (SRY-box 2). A photoreceptor rescue effect of fRPESC transplantation in a rodent RDD model was evidenced by retinal structural and functional assessments. These findings establish the proof of principle for application of VV to convert fRPE cells into a retinal stem-like cell state that promotes their therapeutic value for RDDs. The primary cultured human fRPE cells showed a cobblestone morphology and pigmentation from passage 0 (P0) to P3. A previous study showed that fRPE cells lose their epithelial morphology and differentiate into fibroblast-like cells with a low proliferation ability at P4 and P5.11Radeke M.J. Radeke C.M. Shih Y.H. Hu J. Bok D. Johnson L.V. Coffey P.J. Restoration of mesenchymal retinal pigmented epithelial cells by TGFβ pathway inhibitors: implications for age-related macular degeneration.Genome Med. 2015; 7: 58Crossref PubMed Scopus (65) Google Scholar In this study, VV was given every 24 h from P3 for 3 weeks. At P5, VV-treated fRPE cells exhibited epithelial morphology without visible pigmentation after reaching confluence (Figure 1A). These highly proliferative cells, which underwent VV treatment for 3 weeks, were noted as fetal RPE stem-like cells (fRPESCs). Before we obtained fRPESCs, the optimal effective concentration of VC and/or VPA was determined by detecting cell cycle and analyzing TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) staining and Ki67 expression on the 7th day after treatment. Our results showed that 50 μg/mL VV treatment induced less TUNEL expression than did 100 μg/mL and increased Ki67 expression more effectively than 25 μg/mL (Figure 1B; Figures S1A and S1B). The highest cell density can be observed in the light image of the 50 μg/mL VV group (Figure S1C). The percentage of cells in the S phase was significantly increased in the 50 μg/mL VV group (15.92%) compared to the PBS group (2.19%), 50 μg/mL VC group (6.22%), and 100 μg/mL VV group (2.44%, Figure S1D). Moreover, combined treatment with VV at 50 μg/mL promoted cell proliferation more effectively than did the use of the two agents separately at the concentration of 50 or 100 μg/mL (Figure 1B; Figure S1). Transmission electron microscopy images showed that fRPE cells lost their pigment granules and apical microvilli and transformed into nonpolarized fRPESCs at P5, with numerous mitochondria and endoplasmic reticulum after 3 weeks of VV treatment (Figure 1C). Our results also indicated a significant elevation of SOX2, OCT4 (organic cation/carnitine transporter 4), and KLF4 (Krüppel-like factor 4), retinal progenitor markers (MITF [microphthalmia-associated transcription factor], OTX2 [orthodenticle homolog 2], and PAX6 [paired box 6]), and mesenchymal stromal markers (CD133, CD73, CD105, and CD90) expression in fRPESCs compared with fRPE cells (Figures 1D–1F). To verify the elevated mesenchymal stromal characteristics of fRPESCs, we exposed fRPE cells and fRPESCs to different media for adipogenic, chondrogenic, and osteogenic differentiation. By staining with cell-specific markers and quantitative real-time PCR, our results showed that fRPESCs were more able to differentiate into adipocytes, chondrocytes, and bone lineage progenies than were fRPE cells (Figures 1G and 1H). To identify the global expression changes in fRPESCs induced by VV, a genome-wide expression analysis was carried out to compare fRPESCs with fRPE cells. RNA sequencing (RNA-seq) results showed that all fRPESC samples were clustered together and clearly distinguishable from fRPE samples, which supported the efficacy of VV treatment (Figures 2A and 2B ). The volcano plot in Figure 2C shows upregulated expression of SOX2, OTX2, PAX6, BEST1 (bestrophin 1), MITF, and E-CADHERIN and downregulated expression of TGFβ1 (transforming growth factor β1) and SNAIL1 in fRPESCs compared with fRPE cells. Gene set enrichment analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Ontology (GO) analysis highlighted pathways that when significantly activated by VV treatment were correlated with neural differentiation (WNT signaling pathway, neurotrophin signaling pathway)22Adachi K. Mirzadeh Z. Sakaguchi M. Yamashita T. Nikolcheva T. Gotoh Y. Peltz G. Gong L. Kawase T. Alvarez-Buylla A. et al.β-Catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone.Stem Cells. 2007; 25: 2827-2836Crossref PubMed Scopus (209) Google Scholar,23Lie D.C. Colamarino S.A. Song H.J. Désiré L. Mira H. Consiglio A. Lein E.S. Jessberger S. Lansford H. Dearie A.R. Gage F.H. Wnt signalling regulates adult hippocampal neurogenesis.Nature. 2005; 437: 1370-1375Crossref PubMed Scopus (1182) Google Scholar, MET (epithelial cell signaling pathway, phosphatidylinositol 3-kinase [PI3K]-AKT signaling pathway, mitogen-activated protein kinase [MAPK] pathway),24Lamouille S. Xu J. Derynck R. Molecular mechanisms of epithelial-mesenchymal transition.Nat. Rev. Mol. Cell Biol. 2014; 15: 178-196Crossref PubMed Scopus (4879) Google Scholar cell proliferation (MAPK pathway, cell cycle),25Zhang W. Liu H.T. MAPK signal pathways in the regulation of cell proliferation in mammalian cells.Cell Res. 2002; 12: 9-18Crossref PubMed Scopus (1474) Google Scholar and retinal development (WNT signaling pathway, Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, Hedgehog signaling pathway, glutamatergic synapse pathway; Figures 2D–2G; Figures S2 and S3).26Flaherty M.S. Salis P. Evans C.J. Ekas L.A. Marouf A. Zavadil J. Banerjee U. Bach E.A. chinmo is a functional effector of the JAK/STAT pathway that regulates eye development, tumor formation, and stem cell self-renewal in Drosophila.Dev. Cell. 2010; 18: 556-568Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 27Borday C. Cabochette P. Parain K. Mazurier N. Janssens S. Tran H.T. Sekkali B. Bronchain O. Vleminckx K. Locker M. Perron M. Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation.Development. 2012; 139: 3499-3509Crossref PubMed Scopus (54) Google Scholar, 28Amato M.A. Boy S. Perron M. Hedgehog signaling in vertebrate eye development: a growing puzzle.Cell. Mol. Life Sci. 2004; 61: 899-910Crossref PubMed Scopus (67) Google Scholar, 29Hack I. Koulen P. Peichl L. Brandstätter J.H. Development of glutamatergic synapses in the rat retina: the postnatal expression of ionotropic glutamate receptor subunits.Vis. Neurosci. 2002; 19: 1-13Crossref PubMed Scopus (24) Google Scholar, 30Huang T.S. Li L. Moalim-Nour L. Jia D. Bai J. Yao Z. Bennett S.A. Figeys D. Wang L. A regulatory network involving β-catenin, E-cadherin, PI3k/Akt, and slug balances self-renewal and differentiation of human pluripotent stem cells in response to Wnt signaling.Stem Cells. 2015; 33: 1419-1433Crossref PubMed Scopus (59) Google Scholar Heatmaps demonstrated that VV dramatically elevated SOX2 expression and generally suppressed photoreceptor marker expression (Figures S2C and S3C). Therefore, our RNA-seq results indicated that fRPESCs are a type of highly proliferating mesenchymal-to-epithelial-transitioned retinal stem-like cells and that SOX2 may act as a master regulator in VV treatment. Then, the regulatory role of SOX2 was preliminarily explored by knocking down SOX2 (SOX2KD) in fRPESCs before adipogenic, chondrogenic, and osteogenic differentiation. By staining with cell-specific markers and quantitative real-time PCR, our results showed that fRPESCs lost mesenchymal differentiation potency after SOX2KD (Figures 2H and 2I). Next, we tested our hypothesis that SOX2 plays a central role in promoting retinal progenitor markers and mesenchymal stromal markers expressions in fRPESCs. The expression of OCT4 and KLF4 was significantly reduced in fRPESCs-SOX2KD compared with fRPESCs (Figure 3A). By analyzing transcriptome sequencing data and researching the protein-protein interaction network (PPI), SOX2 was predicted to play a key role in upregulating retinal progenitor markers (OTX2, PAX6, and MITF) and BEST1 expression in fRPESCs (Figures 3B and 3C). Furthermore, the percentage of CD73+, CD133+, CD105+, and CD90+ cells was dramatically downregulated in fRPESCs after SOX2KD (Figures 3D–3G). According to protein analysis results, the expression of PAX6, OTX2, MITF, and BEST1 was significantly downregulated in fRPESCs after SOX2KD (Figure 3H). By knocking down PAX6, OTX2, BEST1, and MITF independently, our results suggested that VV-SOX2 elevated the expression of BEST1 by upregulating OTX2 and elevated the expression of MITF by upregulating PAX6 in fRPESCs (Figures 3I and 3J). During our primary culture of fRPE cells, we observed an obvious EMT tendency in fRPE cells from P3: fRPE cells differentiated into spindle-shaped cells resembling fibroblasts and failed to reach confluency at P4. However, after treatment with VV, we observed a change in the epithelial morphology of fRPESCs at P5 (Figure 1A). Consistent with our observations and RNA-seq results, western blotting (WB) results demonstrated that the expression of TGF-β1 and SNAIL1 is significantly downregulated while that of E-CADHERIN is significantly upregulated in fRPESCs compared with fRPE cells and fRPESCs-SOX2KD (Figure 4A). The MET phenomenon in fRPESCs and the regulatory role of SOX2 were further confirmed by a mRNA level test of mesenchymal markers (SNAIL1, ZEB1 [zinc finger E-box binding homeobox 1], SNAIL2, ZEB2 [zinc finger E-box binding homeobox 2]) and epithelial markers (KSA [EPCAM, epithelial cell adhesion molecule], E-CADHERIN, OCLN [occludin]; Figures 4B and 4C). RNA-seq analysis confirmed activated MET in fRPESCs, and PPI predicted the regulatory role of SOX2 and its candidate targets (TGF-β1, SNAIL1, and E-CADHERIN), as shown in Figures 4D and 4E. To investigate the downstream SOX2 regulatory pathway, TGF-β1, SNAIL1, and E-CADHERIN were knocked down with small interfering RNA (siRNA). As shown by the WB assay outcomes, TGF-β1KD did not significantly change expressions of SNAIL1 and E-CADHERIN in fRPESCs (Figure 4F). SNAIL1KD did not significantly change the protein expression level of TGF-β1 and E-CADHERIN in fRPESCs (Figure 4G). E-CADHERINKD did not change the expression level of TGF-β1 and SNAIL1 significantly in either fRPESCs or fRPE cells (Figure 4H). Moreover, the expression levels of mesenchymal markers were not significantly changed in fRPESCs-TGF-β1KD compared with fRPESCs (Figure 4I), and the expression of ZEB2 was significantly downregulated in fRPESCs-SNAIL1KD compared with fRPESCs (Figure 4K). The expression of epithelial markers was significantly downregulated in fRPESCs-TGF-β1KD compared with fRPESCs (Figure 4J), and epithelial marker expression showed no significant differences between fRPESCs-SNAIL1KD and fRPESCs (Figure 4L). These findings indicate the presence of a TGF-β1-independent pathway of SNAIL1 in MET of fRPESCs. Therefore, our results illustrated that VV-SOX2 suppressed the expression of SNAIL1 by downregulating TGF-β1 and elevated the expression of E-CADHERIN by downregulating SNAIL1 in fRPESCs. The entire rod photoreceptor (RP) differentiation protocol was generally divided into three steps by using three different medium culture systems. During rod photoreceptor differentiation, fRPESCs first formed a round shape and then stretched out several synapsis-like structures to finally form a tubular, rod-like structure resembling the outer segment (Figure 5A). In the first stage, retinal progenitor cells (RPCs) were generated after differentiation from fRPESCs for 1 week. Our results showed that the expression of RPC markers (PAX6 and VSX2 [visual system homeobox 2]) was significantly elevated in fRPESCs compared with fRPE cells and fRPESCs-SOX2KD (Figure S4). The percentages of PAX6+ and VSX2+ cells in fRPESCs were approximately 64.5% and 45.8% (Figures S4C and S4D). In the second stage, RPCs were differentiated into photoreceptor precursor cells (PPCs) after differentiation for another week. Our outcomes showed significantly elevated PPC markers (NRL [neural retina leucine zipper] and CRX [cone-rod homeobox]) expression in fRPESCs compared with fRPE cells and fRPESCs-SOX2KD (Figure S5). The percentages of NRL+ and CRX+ cells in fRPESCs were approximately 36.5% and 49.3%, respectively (Figures S5C and S5D). In the terminal stage, rod photoreceptor cells expressing rod-specific phototransduction and outer segment structural markers (RHODOPSIN, rod ARRESTIN, RECOVERIN, GNAT1 [G protein subunit alpha transducin 1]) were obtained from PPCs after 2 weeks of differentiation. Our results revealed a significantly higher expression of rod photoreceptor markers in fRPESCs than in fRPE cells and fRPESCs-SOX2KD (Figures 5B–5E; Figure S6). Confocal images of fRPESC-derived rod photoreceptors showed a round head with a tubular outer segment-like structure (Figures 5C–5D). The percentages of RECOVERIN+, RHODOPSIN+, ARRESTIN+, and GNAT1+ cells in fRPESCs were approximately 61.2%, 38.8%, 43%, and 33.8%, respectively (Figures 5C and 5D; Figure S6). To determine RPE differentiation of fRPESCs in vitro, VV treatment was stopped, and fRPESCs were cultured in RPE medium for 3 weeks. The fRPESCs gradually reached confluency and showed pigmentation. Confocal images of fRPESC-derived RPE cells showed a typical cobblestone-like epithelial morphology (Figures S7A and S7B). An immunofluorescence assay showed a significantly increased percentage of CRALBP (cellular retinaldehyde-binding protein)+ and ZO1+ cells in fRPESC-derived RPE cells compared with fRPE cells and fRPESCs-SOX2KD, as shown in Figures S7A and S7B. To elucidate the therapeutic potential of fRPESCs in RDDs, a sodium iodate (NaIO3)-induced retinal degeneration mouse model was established, and subretinal transplantation of fRPE cells, fRPESCs, or fRPESCs-SOX2KD cells was performed. The successful transplantation and survival of fRPE cells, fRPESCs, and fRPESCs-SOX2KD was confirmed by staining of mouse retina sections with the human-specific marker STEM121. STEM121+ cells were observed lying in the subretinal space of the RDD mouse model at 1 month posttransplantation (Figure S7C). To verify retinal structural preservation after fRPESC transplantation, optical coherence tomography (OCT) and outer nuclear layer (ONL) nuclei count methods were employed at 1 month after transplantation. As shown in 3D maps, the thicknesses of the retina (internal limiting membrane [ILM] to RPE) and ONL were significantly attenuated after NaIO3 treatment. The retinal and ONL thickness in the RDD mouse model was significantly recovered after fRPESC transplantation, and the average retinal and ONL thickness was significantly higher in the fRPESC treatment group than in the fRPE and fRPESC-SOX2KD treatment groups. Additionally, a gradual increase in retinal and ONL thickness toward the dorsal-temporal quarter, where the cells were injected, was observed (Figures 6A–6D). To verify retinal function restoration after fRPESC transplantation, an electroretinogram (ERG) was used and scotopic visual acuity (VA) was tested. After NaIO3 treatment, the a- and b-wave scotopic ERG amplitude (rod response) and head-tracing response underwent a rapid and continuous decline. Transplantation was conducted at 1 week after NaIO3 treatment (0 week posttransplantation). The a- and b-wave amplitude was significantly preserved during the 12 weeks of follow-up, and VA was significantly elevated at 1 month in the fRPESC treatment group. Moreover, significantly greater increases in b-wave amplitude and VA were detected in the fRPESC treatment group than in the fRPE and fRPESC-SOX2KD treatment groups (Figures 6E and 6F; Figure S7D). To determine the rod photoreceptor differentiation potential of grafts, we stained mouse retina sections with rod photoreceptor markers for human CRX and RHODOPSIN. CRX+/STEM121+ cells were found in both the subretinal space and ONL after fRPESC transplantation. However, RHODOPSIN+/STEM121+ cells were only found in the ONL layer after fRPESC transplantation. The percentages of CRX+ and RHODOPSIN+ cells among the STEM121+ cells in the fRPESC treatment group were about 67.8% and 28.3%, respectively, at 1 month (Figures 7A and 7C ). The expression of human rod photoreceptor markers in the fRPESC treatment group increased from 2 weeks to 1 month and decreased from 1 month to 3 months (Figures 7B and 7D). To verify the RPE differentiation potential of grafts, we stained mouse retinal sections with human RPE65 (RPE-specific 65-kDa protein) and MERTK (MER proto-oncogene tyrosine kinase). A consistently and significantly larger number of RPE65+/MERTK+ RPE cells were detected in the subretinal space of fRPESC-transplanted eyes when compared with fRPE-transplanted eyes from 2 weeks to 3 months (Figures 8A–8C). The expression level of human RPE65 and MERTK continuously declined from 2 weeks to 1 month and to 3 months (Figures 8B and 8C). Additionally, SOX2KD repressed both photoreceptor and RPE differentiation potential of fRPESCs in vivo.Figure 8RPE Differentiation of fRPESCs in the Subretinal Space in the RDD Mouse ModelShow full caption(A) Immunofluorescence staining of human RPE65 (red) and human MERTK (green) indicating that the number of graft-derived RPE cells was significantly higher in the fRPESC group than in the fRPE and fRPESC-SOX2KD groups at 2 weeks and 1 month. Scale bars represent 20 μm (above), 10 μm (middle), and 5 μm (below). (B and C) Quantitative real-time PCR analysis demonstrating significantly higher expression of human (C) RPE65 and (B) MERTK in the fRPESC group than in the fRPE and fRPESC-SOX2KD groups at 2 weeks and 1 month (n = 6). (D) Pairwise correlation analysis showing no significant correlation between scotopic ERG and STEM121+ cell numbers in fRPESC treatment eyes from 2 weeks to 3 months posttransplantation. R2 = 0.2872, p > 0.05. (E) Pairwise correlation analysis showing a significant correlation between scotopic ERG and MERTK+ cell numbers in fRPESC treatment eyes from 2 weeks to 3 months posttransplantation. R2 = 0.5090, p < 0.05. (F) Pairwise correlation analysis showing a significant correlation between scotopic ERG and RPE65+ cell numbers in fRPESC treatment eyes from 2 weeks to 3 months posttransplantation. R2 = 0.4232, p < 0.05. (G) Pairwise correlation analysis showing a strong correlation between scotopic ERG and RHODOPSIN+ cell numbers in fRPESC treatment eyes from 2 weeks to 3 months posttransplantation. R2 = 0.5928, p < 0.01. (H) Pairwise correlation analysis showing a significant correlation between scotopic ERG and CRX+ cell numbers in fRPESC treatment eyes from 2 weeks to 3 months posttransplantation. R2 = 0.5118, p < 0.05. 1M, 1 month; 3M, 3 months; 2W, 2 weeks. ∗p < 0.05, ∗∗p < 0.01, versus fRPE group; #p < 0.05, ##p < 0.01, ###p < 0.001, versus fRPESC-SOX2KD group. All experiments were repeated at least three times; error bars indicate SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Immunofluorescence staining of human RPE65 (red) and human MERTK (green) indicating that the number of graft-derived RPE cells was significantly higher in the fRPESC group than in the fRPE and fRPESC-SOX2KD groups at 2 weeks and 1 month. Scale bars represent 20 μm (above), 10 μm (middle), and 5 μm (below). (B and C) Quantit" @default.
• W3021856789 created "2020-05-13" @default.
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• W3021856789 date "2020-07-01" @default.
• W3021856789 modified "2023-09-24" @default.
• W3021856789 title "Vitamin C- and Valproic Acid-Induced Fetal RPE Stem-like Cells Recover Retinal Degeneration via Regulating SOX2" @default.
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