Atoh7 promotes the differentiation of retinal stem cells derived from Müller cells into retinal ganglion cells by inhibiting Notch signaling

Stem Cell Research & Therapy

Xiao-bo Xia¹  Xue-yong Zhang¹  Wei-tao Song¹ 

[1] Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, China

关键词: Differentiation;    Stem cells;    Notch;    Atoh7;    Retinal ganglion cells;    Müller cells;   

Others  :  847102
DOI  :  10.1186/scrt305

 received in 2013-05-20, accepted in 2013-08-08,  发布年份 2013

【 摘 要 】

Introduction

Retinal Müller cells exhibit the characteristics of retinal progenitor cells, and differentiate into ganglion cells under certain conditions. However, the number of ganglion cells differentiated from retinal Müller cells falls far short of therapeutic needs. This study aimed to develop a novel protocol to promote the differentiation of retinal Müller cells into ganglion cells and explore the underlying signaling mechanisms.

Methods

Müller cells were isolated and purified from rat retina and induced to dedifferentiate into retinal stem cells. Next the stem cells were transfected with lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP. In addition, the stem cells were transfected with Brn-3b siRNA or Isl-1 siRNA or treated with Notch inhibitor gamma-secretase inhibitor (GSI).

Results

The proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of controls. Knockdown of Brn-3b or Isl-1 inhibited, while GSI promoted, the differentiation into retinal ganglion cells. Atoh7 promoted the expression of Brn-3b and Isl-1 but inhibited the expression of Notch1.

Conclusions

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells by inhibiting Notch signaling, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.

【 授权许可】

   
2013 Song et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Brubaker RF: delayed functional loss in glaucoma. LII Edward Jackson Memorial Lecture. Am J Ophthalmol 1996, 121:473-483.
• [2]Kato K, Sasaki N, Shastry BS: Retinal ganglion cell (RGC) death in glaucomatous beagles is not associated with mutations in p53 and NTF4 genes. Vet Ophthalmol 2012, 15(suppl 2):8-12.
• [3]Baltmr A, Duggan J, Nizari S, Salt TE, Cordeiro MF: Neuroprotection in glaucoma – is there a future role? Exp Eye Res 2010, 91:554-566.
• [4]Bakalash S, Ben-Shlomo G, Aloni E, Shaked I, Wheeler L, Ofri R, Schwartz M: T-cell-based vaccination for morphological and functional neuroprotection in a rat model of chronically elevated intraocular pressure. J Mol Med (Berl) 2005, 83:904-916.
• [5]Fang JH, Wang XH, Xu ZR, Jiang FG: Neuroprotective effects of bis(7)-tacrine against glutamate-induced retinal ganglion cells damage. BMC Neurosci 2010, 11:1-9. BioMed Central Full Text
• [6]Siqueira RC: Stem cell therapy for retinal diseases: update. Stem Cell Res Ther 2011, 2:50. BioMed Central Full Text
• [7]Medina RJ, Archer DB, Stitt AW: Eyes open to stem cells: safety trial may pave the way for cell therapy to treat retinal disease in patients. Stem Cell Res Ther 2011, 2:47. BioMed Central Full Text
• [8]Bull ND, Martin KR: Using stem cells to mend the retina in ocular disease. Regen Med 2009, 4:855-864.
• [9]Das AV, Mallya KB, Zhao X, Ahmad F, Bhattacharya S, Thoreson WB, Hegde GV, Ahmad I: Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling. Dev Biol 2006, 299:283-302.
• [10]Bull ND, Limb GA, Martin KR: Human Müller stem cell (MIO-M1) transplantation in a rat model of glaucoma: survival, differentiation, and integration. Invest Ophthalmol Vis Sci 2008, 49:3449-3456.
• [11]Fischer AJ, McGuire CR, Dierks BD, Reh TA: Insulin and fibroblast growth factor 2 activate a neurogenic program in Müller glia of the chicken retina. J Neurosci 2002, 22:9387-9398.
• [12]Lawrence JM, Singhal S, Bhatia B, Keegan DJ, Reh TA, Luthert PJ, Khaw Peng T, Limb GA: MIO-M1 cells and similar Müller glial cell lines derived from adult human retina exhibit neural stem cell characteristics. Stem Cells 2007, 25:2033-2043.
• [13]Karl MO, Hayes S, Nelson BR, Tan K, Buckingham B, Reh TA: Stimulation of neural regeneration in the mouse retina. Proc Natl Acad Sci U S A 2008, 105:19508-19513.
• [14]Brown NL, Kanekar S, Vetter ML, Tucker PK, Gemza DL, Glaser T: Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis. Development 1998, 125:4821-4833.
• [15]Feng L, Xie ZH, Ding Q, Xie XL, Libby RT, Gan L: MATH5 controls the acquisition of multiple retinal cell fates. Mol Brain 2010, 3:1-16. BioMed Central Full Text
• [16]Yang Z, Ding K, Pan L, Deng M, Gan L: Math5 determines the competence state of retinal ganglion cell progenitors. Dev Biol 2003, 264:240-254.
• [17]Yao J, Sun X, Wang Y, Xu G, Qian J: Math5 promotes retinal ganglion cell expression patterns in retinal progenitor cells. Mol Vis 2007, 13:1066-1072.
• [18]Sapkota D, Wu F, Mu X: Focus on Molecules: Math5 and retinal ganglion cells. Exp Eye Res 2011, 93:796-797.
• [19]Nelson BR, Gumuscu B, Hartman BH, Reh TA: Notch activity is downregulated just prior to retinal ganglion cell differentiation. Dev Neurosci 2006, 28:128-141.
• [20]Zhao X, Das A, Thoreson WB, James J, Wattnem TE, Rodriguez-Sierra J, Ahmad I: Adult corneal limbal epithelium; a model for studying neural potential of non-neural stem cells. Dev Biol 2002, 250:317-331.
• [21]Vardimon L, Fox LE, Moscona AA: Development regulation of glutamine synthetase and carbonic anhydrase II in neural retina. Proc Natl Acad Sci U S A 1986, 83:9060-9064.
• [22]Blackshaw S, Harpavat S, Trimarchi J, Cai L, Huang HY, Kuo WP, Weber G, Lee K, Fraioli RE, Cho SH, Yung R, Asch E, Ohno-Machado L, Wong WH, Cepko CL: Genomic analysis of mouse retinal development. PLoS Biol 2004, 2:1411-1431.
• [23]Giannelli SG, Demontis GC, Pertile G, Rama P, Broccoli V: Adult human Müller glia cells are a highly efficient source of rod photoreceptors. Stem Cells 2011, 29:244-356.
• [24]Cho MS, Kim SJ, Ku SY, Park JH, Lee H, Yoo DH, Park UC, Song SA, Choi YM, Yu HG: Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses. Stem Cell Res 2012, 9:101-109.
• [25]Kubota A, Nishida K, Nakashima K, Tano Y: Conversion of mammalian Müller glia cells into a neuronal lineage by in vitro aggregate. Biochem Biophys Res Commun 2006, 351:514-520.
• [26]Ahmed S, Reynolds BA, Weiss S: BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J Neurosci 1995, 15:5765-5778.
• [27]Guan K, Chang H, Rolletschek A, Wobus AM: Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells. Cell Tissue Res 2001, 305:171-176.
• [28]Cepko CL: The roles of intrinstic and extrinsic cues and bHLH genes in the determination of retinal cell fates. Curr Opin Neurobiol 1999, 9:37-46.