Expression of inherited retinal disease related genes in human microglia_Chinese Journal of Ocular Fundus Diseases

Authors：

Xu Jia , Yu Sijian , Wang Jingyi ,  Jin Zibing

Department of Ophthalmology, Beijing Tongren Hospital, Beijing Institute of Ophthalmology, Capital Medical University, Beijing 100730, China;

Corresponding author：

Jin Zibing, Email: jinzibing@foxmail.com

Keywords：

Inherited retinal disease; Microglia; Induced pluripotent stem cell; Cell differentiation; Gene expression

DOI：

10.3760/cma.j.cn511434-20241101-00402

Video：

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Objective To observe the expression of genes related to hereditary retinal diseases (IRD) in human microglia (hMG). Methods A experimental study. Efficient differentiation of human induced pluripotent stem cells (iPSC) into hMG. Identification of octamer-binding transcription factor 4 (OCT4), sex-determining transcription factor 2 (SOX2), Nanog homeobox (NANOG), stage-specific embryonic antigen-4 (SSEA4), alpha-fetoprotein (AFP), α-smooth muscle actin (α-SMA) as markers associated with iPSC dryness and pluripotency by immunofluorescence staining Glial fibrillary acidic protein (GFAP); hMG associated marker transmembrane protein 119 (TMEM119), purinergic receptor P2Y12 (P2RY12), and allograft inflammatory factor 1 (IBA1). The proportion of CD11b⁺ and CD45⁺ cells was detected by flow cytometry. Lipopolysaccharide (LPS) stimulated hMG for 0, 4, 8 and 12 h in group 0 h, group 4 h, group 8 h and group 12 h, respectively. Total RNA samples from the 4 groups were extracted for transcriptome sequencing, and the persistently significant differentially expressed genes (DEG) were screened. Real-time quantitative polymerase chain reaction (qPCR) was used to verify and analyze the expression of DEG mRNA. The two-tailed Student t test was used for comparison between the two groups. Results In this study, the dry and pluripotent properties of iPSC were identified and successfully differentiated into hMG. iPSC expressed the dry related markers OCT4, SOX2, NANOG and SSEA4, and differentiated into endoderm, mesoderm and ectoderm, expressing the corresponding markers AFP, α-SMA and GFAP, respectively. iPSC formed embryoid bodies under specific culture conditions, and then differentiated into hMG, and hMG expressed related markers TMEM119, P2RY12 and IBA1 by immunofluorescence staining. The double positive ratio of CD11b⁺ and CD45⁺ was > 95%. Transcriptomic analysis showed that the expression of 18 genes in hMG stimulated by LPS was changed. qPCR test results showed that compared with group 0 h, mRNA expressions of TLR4, PGK1 and ADAM9 in LPS stimulated group 4 h were significantly increased (t=25.43, 15.54, 6.26; P＜0.01). The mRNA expression levels of MERTK, ABHD12, RDH11 and DRAM2 decreased (t=5.94, 14.14, 8.21, 6.97; P＜0.01), and the differences were statistically significant. Compared with group 0 h, mRNA expressions of RDH11, MERTK, ABHD12, DRAM2 and ADAM9 in group 8 h stimulated by LPS were significantly decreased, with statistical significance (t=25.97, 5.47, 43.97, 38.40, 3.84; P＜0.05). Compared with the group 0 h, the mRNA expressions of TLR4, ADAM9, MERTK, ABHD12, RDH11 and DRAM2 in the 12 h stimulated group were significantly decreased, and the differences were statistically significant (t=6.39, 46.11, 5.34, 14.14, 25.97, 25.65; P＜0.05). Conclusion IRD-related genes may be involved in the occurrence and development of IRD by regulating the function of hMG.

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6.	Rathnasamy G, Foulds WS, Ling EA, et al. Retinal microglia-a key player in healthy and diseased retina[J]. Prog Neurobiol, 2019, 173: 18-40. DOI: 10.1016/j.pneurobio.2018.05.006.
7.	Salter MW, Stevens B. Microglia e合并 as central players in brain disease[J]. Nat Med, 2017, 23(9): 1018-1027. DOI: 10.1038/nm.4397.
8.	Butovsky O, Weiner HL. Microglial signatures and their role in health and disease[J]. Nat Rev Neurosci, 2018, 19(10): 622-635. DOI: 10.1038/s41583-018-0057-5.
9.	Prinz M, Jung S, Priller J. Microglia biology: one century of evolving concepts[J]. Cell, 2019, 179(2): 292-311. DOI: 10.1016/j.cell.2019.08.053.
10.	Nicolás-Ávila JA, Lechuga-Vieco AV, Esteban-Martínez L, et al. A network of macrophages supports mitochondrial homeostasis in the heart[J]. Cell, 2020, 183(1): 94-109. DOI: 10.1016/j.cell.2020.08.031.
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12.	Smith AM, Dragunow M. The human side of microglia[J]. Trends Neurosci, 2014, 37(3): 125-135. DOI: 10.1016/j.tins.2013.12.001.
13.	Yeh H, Ikezu T. Transcriptional and epigenetic regulation of microglia in health and disease[J]. Trends Mol Med, 2019, 25(2): 96-111. DOI: 10.1016/j.molmed.2018.11.004.
14.	Gao ML, Zhang X, Han F, et al. Functional microglia derived from human pluripotent stem cells empower retinal organ[J]. Sci China Life Sci, 2022, 65(6): 1057-1071. DOI: 10.1007/s11427-021-2086-0.
15.	Xu J, Yu SJ, Sun S, et al. Enhanced innate responses in microglia derived from retinoblastoma patient-specific iPSC[J]. Glia, 2024, 72(5): 872-884. DOI: 10.1002/glia.24507.
16.	Deng WL, Gao ML, Lei XL, et al. Gene correction reverses ciliopathy and photoreceptor loss in iPSC-derived retinal organoids from retinitis pigmentosa patients[J]. Stem Cell Reports, 2018, 10(4): 1267-1281. DOI: 10.1016/j.stemcr.2018.02.003.
17.	Xu J, Yu SJ, Jin ZB. Assembling retinal organoids with microglia[J/OL]. J Vis Exp, 2024, (209). 2024, 2024: E1(2024-11-11)[2021-07-26]. https://pubmed.ncbi.nlm.nih.gov/39141532/. DOI: 10.3791/67016. [published online ahead of print].
18.	Li YP, Wang YT, Wang W, et al. Second hit impels oncogenesis of retinoblastoma in patient-induced pluripotent stem cell-derived retinal organoids: direct evidence for Knudson's theory[J/OL]. PNAS nexus, 2022, 1(4): pgac162[2022-08-17]. https://pubmed.ncbi.nlm.nih.gov/36714839/. DOI: 10.1093/pnasnexus/pgac162.
19.	Paolicelli RC, Sierra A, Stevens B, et al. Microglia states and nomenclature: a field at its crossroads[J]. Neuron, 2022, 110(21): 3458-3483. DOI: 10.1016/j.neuron.2022.10.020.
20.	Ramirez AI, de Hoz R, Salobrar-Garcia E, et al. The role of microglia in retinal neurodegeneration: Alzheimer's disease, parkinson, and glaucoma[J/OL]. Front Aging Neurosci, 2017, 9: 214[2017-07-06]. https://pubmed.ncbi.nlm.nih.gov/28729832/. DOI: 10.3389/fnagi.2017.00214.
21.	O'Koren EG, Yu C, Klingeborn M, et al. Microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration[J]. Immol/Lunity, 2019, 50(3): 723-737. DOI: 10.1016/j.immol/Luni.2019.02.007.
22.	Zhou W, Zhou Y, He J, et al. TREM2 deficiency in microglia accelerates photoreceptor cell death and immol/Lune cell infiltration following retinal detachment[J]. Cell Death Dis, 2023, 14(3): 219. DOI: 10.1038/s41419-023-05735-x.
23.	Muffat J, Li Y, Yuan B, et al. Efficient derivation of microglia-like cells from human pluripotent stem cells[J]. Nat Med, 2016, 22(11): 1358-1367. DOI: 10.1038/nm.4189.
24.	Abud EM, Ramirez RN, Martinez ES, et al. iPSC-derived human microglia-like cells to study neurological diseases[J]. Neuron, 2017, 94(2): 278-293. DOI: 10.1016/j.neuron.2017.03.042.
25.	Gu C, Wang F, Zhang YT, et al. Microglial MT1 activation inhibits 脂多糖-induced neuroinflammol/Lation via regulation of metabolic reprogrammol/Ling[J/OL]. Aging cell, 2021, 20(6): e13375.1-05-08]. https://pubmed.ncbi.nlm.nih.gov/33964119/. DOI: 10.1111/acel.13375.Gu C, Wang F, Zhang YT, et al. Microglial MT1 activation inhibits 脂多糖-induced neuroinflammol/Lation via regulation of metabolic reprogrammol/Ling[J/OL]. Aging cell, 2021, 20(6): e13375.1-05-08]. https://pubmed.ncbi.nlm.nih.gov/33964119/. DOI: 10.1111/acel.13375.
26.	Qin L, Wu X, Block ML, et al. Systemic 脂多糖 causes chronic neuroinflammol/Lation and progressive neurodegeneration[J]. Glia, 2007, 55(5): 453-462. DOI: 10.1002/glia.20467.Qin L, Wu X, Block ML, et al. Systemic 脂多糖 causes chronic neuroinflammol/Lation and progressive neurodegeneration[J]. Glia, 2007, 55(5): 453-462.
27.	Catorce MN, Gevorkian G. 脂多糖-induced murine neuroinflammol/Lation model: main features and suitability for pre-clinical assessment of nutraceuticals[J]. Curr Neuropharmacol, 2016, 14(2): 155-164. DOI: 10.2174/1570159x14666151204122017.Catorce MN, Gevorkian G. 脂多糖-induced murine neuroinflammol/Lation model: main features and suitability for pre-clinical assessment of nutraceuticals[J]. Curr Neuropharmacol, 2016, 14(2): 155-164.
28.	Brown GC. The endotoxin hypothesis of neurodegeneration[J]. J Neuroinflammol/Lation, 2019, 16(1): 180. DOI: 10.1186/s12974-019-1564-7.
29.	Xiao X, Liu Z, Su G, et al. A novel uveitis model induced by lipopolysaccharide in zebrafish[J/OL]. Front Immol/Lunol, 2022, 13: 1042849[2022-12-01]. https://pubmed.ncbi.nlm.nih.gov/36532084/. DOI: 10.3389/fimmol/Lu.2022.1042849.
30.	Noailles A, Maneu V, Campello L, et al. Systemic inflammol/Lation induced by lipopolysaccharide aggravates inherited retinal dystrophy[J]. Cell Death Dis, 2018, 9(3): 350. DOI: 10.1038/s41419-018-0355-x.
31.	Olivares-González L, Velasco S, Gallego I, et al. An SPM-enriched marine oil supplement shifted microglia polarization toward M2, ameliorating retinal degeneration in rd10 mice[J]. Antioxidants (Basel), 2022, 12(1): 98. DOI: 10.3390/antiox12010098.
32.	Dorion MF, Yaqubi M, Senkevich K, et al. MerTK is a mediator of alpha-synuclein fibril uptake by human microglia[J]. Brain, 2024, 147(2): 427-443. DOI: 10.1093/brain/awad298.

1. Huang XF, Mao JY, Huang ZQ, et al. Genome-wide detection of copy number variations in unsolved inherited retinal disease[J]. Invest Ophthalmol Vis Sci, 2017, 58(1): 424-429. DOI: 10.1167/iovs.16-20705.
2. 李文生, 郑钦象, 孔繁圣, 等. 遗传性视网膜疾病的基因研究进展[J]. 中华眼科杂志, 2010, 46(2): 186-192. DOI: 10.3760/cma.j.issn.0412-4081.2010.02.020.Li WS, Zheng QX, Kong FS, et al. Progress in gene studies of hereditary retinal diseases[J]. Chin J Ophthalmol, 2010, 46(2): 186-192. DOI: 10.3760/cma.j.issn.0412-4081.2010.02.020.
3. 孙玺皓, 唐仕波, 陈建苏. CRISPR/Cas9基因编辑技术在遗传性视网膜疾病中的应用[J]. 中华实验眼科杂志, 2023, 41(9): 925-930. DOI: 10.3760/cma.j.cn115989-20201120-00787.Sun XH, Tang SB, Chen JS. Application of CRISPR/Cas9 genome editing technology in hereditary retinal diseases[J]. Chin J Exp Ophthalmol, 2023, 41(9): 925-930. DOI: 10.3760/cma.j.cn115989-20201120-00787.
4. 许佳, 金子兵. 小胶质细胞对视网膜及神经系统疾病的监控作用[J]. 中华实验眼科杂志, 2022, 40(8): 758-764. DOI: 10.3760/cma.j.cn115989-20200918-00656.Xu J, Jin ZB. Monitoring function of microglia in retina and nerve system diseases[J]. Chin J Exp Ophthalmol, 2022, 40(8): 758-764. DOI: 10.3760/cma.j.cn115989-20200918-00656.
5. Li Q, Barres BA. Microglia and macrophages in brain homeostasis and disease[J]. Nat Rev Immol/Lunol, 2018, 18(4): 225-242. DOI: 10.1038/nri.2017.125.
6. Rathnasamy G, Foulds WS, Ling EA, et al. Retinal microglia-a key player in healthy and diseased retina[J]. Prog Neurobiol, 2019, 173: 18-40. DOI: 10.1016/j.pneurobio.2018.05.006.
7. Salter MW, Stevens B. Microglia e合并 as central players in brain disease[J]. Nat Med, 2017, 23(9): 1018-1027. DOI: 10.1038/nm.4397.
8. Butovsky O, Weiner HL. Microglial signatures and their role in health and disease[J]. Nat Rev Neurosci, 2018, 19(10): 622-635. DOI: 10.1038/s41583-018-0057-5.
9. Prinz M, Jung S, Priller J. Microglia biology: one century of evolving concepts[J]. Cell, 2019, 179(2): 292-311. DOI: 10.1016/j.cell.2019.08.053.
10. Nicolás-Ávila JA, Lechuga-Vieco AV, Esteban-Martínez L, et al. A network of macrophages supports mitochondrial homeostasis in the heart[J]. Cell, 2020, 183(1): 94-109. DOI: 10.1016/j.cell.2020.08.031.
11. Gosselin D, Skola D, Coufal NG, et al. An environment-dependent transcriptional network specifies human microglia identity[J/OL]. Science, 2017, 356(6344): eaal3222[2017-05-25]. https://pubmed.ncbi.nlm.nih.gov/28546318/. DOI: 10.1126/science.aal3222.
12. Smith AM, Dragunow M. The human side of microglia[J]. Trends Neurosci, 2014, 37(3): 125-135. DOI: 10.1016/j.tins.2013.12.001.
13. Yeh H, Ikezu T. Transcriptional and epigenetic regulation of microglia in health and disease[J]. Trends Mol Med, 2019, 25(2): 96-111. DOI: 10.1016/j.molmed.2018.11.004.
14. Gao ML, Zhang X, Han F, et al. Functional microglia derived from human pluripotent stem cells empower retinal organ[J]. Sci China Life Sci, 2022, 65(6): 1057-1071. DOI: 10.1007/s11427-021-2086-0.
15. Xu J, Yu SJ, Sun S, et al. Enhanced innate responses in microglia derived from retinoblastoma patient-specific iPSC[J]. Glia, 2024, 72(5): 872-884. DOI: 10.1002/glia.24507.
16. Deng WL, Gao ML, Lei XL, et al. Gene correction reverses ciliopathy and photoreceptor loss in iPSC-derived retinal organoids from retinitis pigmentosa patients[J]. Stem Cell Reports, 2018, 10(4): 1267-1281. DOI: 10.1016/j.stemcr.2018.02.003.
17. Xu J, Yu SJ, Jin ZB. Assembling retinal organoids with microglia[J/OL]. J Vis Exp, 2024, (209). 2024, 2024: E1(2024-11-11)[2021-07-26]. https://pubmed.ncbi.nlm.nih.gov/39141532/. DOI: 10.3791/67016. [published online ahead of print].
18. Li YP, Wang YT, Wang W, et al. Second hit impels oncogenesis of retinoblastoma in patient-induced pluripotent stem cell-derived retinal organoids: direct evidence for Knudson's theory[J/OL]. PNAS nexus, 2022, 1(4): pgac162[2022-08-17]. https://pubmed.ncbi.nlm.nih.gov/36714839/. DOI: 10.1093/pnasnexus/pgac162.
19. Paolicelli RC, Sierra A, Stevens B, et al. Microglia states and nomenclature: a field at its crossroads[J]. Neuron, 2022, 110(21): 3458-3483. DOI: 10.1016/j.neuron.2022.10.020.
20. Ramirez AI, de Hoz R, Salobrar-Garcia E, et al. The role of microglia in retinal neurodegeneration: Alzheimer's disease, parkinson, and glaucoma[J/OL]. Front Aging Neurosci, 2017, 9: 214[2017-07-06]. https://pubmed.ncbi.nlm.nih.gov/28729832/. DOI: 10.3389/fnagi.2017.00214.
21. O'Koren EG, Yu C, Klingeborn M, et al. Microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration[J]. Immol/Lunity, 2019, 50(3): 723-737. DOI: 10.1016/j.immol/Luni.2019.02.007.
22. Zhou W, Zhou Y, He J, et al. TREM2 deficiency in microglia accelerates photoreceptor cell death and immol/Lune cell infiltration following retinal detachment[J]. Cell Death Dis, 2023, 14(3): 219. DOI: 10.1038/s41419-023-05735-x.
23. Muffat J, Li Y, Yuan B, et al. Efficient derivation of microglia-like cells from human pluripotent stem cells[J]. Nat Med, 2016, 22(11): 1358-1367. DOI: 10.1038/nm.4189.
24. Abud EM, Ramirez RN, Martinez ES, et al. iPSC-derived human microglia-like cells to study neurological diseases[J]. Neuron, 2017, 94(2): 278-293. DOI: 10.1016/j.neuron.2017.03.042.
25. Gu C, Wang F, Zhang YT, et al. Microglial MT1 activation inhibits 脂多糖-induced neuroinflammol/Lation via regulation of metabolic reprogrammol/Ling[J/OL]. Aging cell, 2021, 20(6): e13375.1-05-08]. https://pubmed.ncbi.nlm.nih.gov/33964119/. DOI: 10.1111/acel.13375.Gu C, Wang F, Zhang YT, et al. Microglial MT1 activation inhibits 脂多糖-induced neuroinflammol/Lation via regulation of metabolic reprogrammol/Ling[J/OL]. Aging cell, 2021, 20(6): e13375.1-05-08]. https://pubmed.ncbi.nlm.nih.gov/33964119/. DOI: 10.1111/acel.13375.
26. Qin L, Wu X, Block ML, et al. Systemic 脂多糖 causes chronic neuroinflammol/Lation and progressive neurodegeneration[J]. Glia, 2007, 55(5): 453-462. DOI: 10.1002/glia.20467.Qin L, Wu X, Block ML, et al. Systemic 脂多糖 causes chronic neuroinflammol/Lation and progressive neurodegeneration[J]. Glia, 2007, 55(5): 453-462.
27. Catorce MN, Gevorkian G. 脂多糖-induced murine neuroinflammol/Lation model: main features and suitability for pre-clinical assessment of nutraceuticals[J]. Curr Neuropharmacol, 2016, 14(2): 155-164. DOI: 10.2174/1570159x14666151204122017.Catorce MN, Gevorkian G. 脂多糖-induced murine neuroinflammol/Lation model: main features and suitability for pre-clinical assessment of nutraceuticals[J]. Curr Neuropharmacol, 2016, 14(2): 155-164.
28. Brown GC. The endotoxin hypothesis of neurodegeneration[J]. J Neuroinflammol/Lation, 2019, 16(1): 180. DOI: 10.1186/s12974-019-1564-7.
29. Xiao X, Liu Z, Su G, et al. A novel uveitis model induced by lipopolysaccharide in zebrafish[J/OL]. Front Immol/Lunol, 2022, 13: 1042849[2022-12-01]. https://pubmed.ncbi.nlm.nih.gov/36532084/. DOI: 10.3389/fimmol/Lu.2022.1042849.
30. Noailles A, Maneu V, Campello L, et al. Systemic inflammol/Lation induced by lipopolysaccharide aggravates inherited retinal dystrophy[J]. Cell Death Dis, 2018, 9(3): 350. DOI: 10.1038/s41419-018-0355-x.
31. Olivares-González L, Velasco S, Gallego I, et al. An SPM-enriched marine oil supplement shifted microglia polarization toward M2, ameliorating retinal degeneration in rd10 mice[J]. Antioxidants (Basel), 2022, 12(1): 98. DOI: 10.3390/antiox12010098.
32. Dorion MF, Yaqubi M, Senkevich K, et al. MerTK is a mediator of alpha-synuclein fibril uptake by human microglia[J]. Brain, 2024, 147(2): 427-443. DOI: 10.1093/brain/awad298.

Chinese Journal of Ocular Fundus Diseases

Latest ArticlesExpression of inherited retinal disease related genes in human microglia

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