Stage-specific differentiation of iPSCs toward retinal ganglion cell lineage.
OBJECTIVE
As an alternative and desirable approach for regenerative medicine, human induced pluripotent stem cell (hiPSC) technology raises the possibility of developing patient-tailored cell therapies to treat intractable degenerative diseases in the future. This study was undertaken to guide human Tenon's capsule fibroblasts-derived iPSCs (TiPSCs) to differentiate along the retinal ganglion cell (RGC) lineage, aiming at producing appropriate cellular material for RGC regeneration.


METHODS
By mimicking RGC genesis, we deliberately administered the whole differentiation process and directed the stage-specific differentiation of human TiPSCs toward an RGC fate via manipulation of the retinal inducers (DKK1+Noggin+Lefty A) alongside master gene (Atoh7) sequentially. Throughout this stepwise differentiation process, changes in primitive neuroectodermal, eye field, and RGC marker expression were monitored with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and/or flow cytometry.


RESULTS
Upon retinal differentiation, a large fraction of the cells developed characteristics of retinal progenitor cells (RPCs) in response to simulated environment signaling (DKK1+Noggin+Lefty A), which was selectively recovered with manual isolation approaches and then maintained in the presence of mitogen for multiple passages. Thereafter, overexpression of ATOH7 further promoted RGC specification in TiPSC-derived RPCs. A subset of transfected cells displayed RGC-specific expression patterns, including Brn3b, iSlet1, calretinin, and Tuj, and approximately 23% of Brn3b-positive RGC-like cells were obtained finally.


CONCLUSIONS
Our DKK1+Noggin+Lefty A/Atoh7-based RGC-induction regime could efficiently direct TiPSCs to differentiate along RGC lineage in a stage-specific manner, which may provide a benefit to develop possible cell therapies to treat retinal degenerative diseases such as glaucoma.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Fei
F
Deng
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Mengfei
M
Chen
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Ying
Y
Liu
Shengzhen Ophthalmic Center of Jinan University, Shenzhen Eye Hospital, Shenzhen, China.
Huiling
H
Hu
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Yunfan
Y
Xiong
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Chaochao
C
Xu
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Yuchun
Y
Liu
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Kangjun
K
Li
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Jing
J
Zhuang
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Jian
J
Ge
D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage / Q000502:physiology*; D005136:Eye Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D039904:Pluripotent Stem Cells / Q000166:cytology*; D060888:Real-Time Polymerase Chain Reaction; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000378:metabolism; D058475:Tenon Capsule / Q000166:cytology; D014162:Transfection
D015415:Biomarkers; D005136:Eye Proteins