| Part 1 Primary investigation of induced pluripotent stem cell differentiation into retinal progenitor cellsObjective: To investigate the effective way of inducing hiPSCs differentiation into retinal progenitor cells,and provide the basis of the application of seed cells for retinal degeneration diseases in future.Methods: The specific markers of hi PSCs,including Oct4,SSEA4,Sox2,Nanog and alkaline phosphatase(AP)were identified by immunofluorescence staining.The hi PSCs were differentiation into RPCs cultured in DMEM-F12 medium supplemented Dkk-1,Noggin,Lefty A and IGF-1.Pax6,Lhx2 and Chx10 the specific markers of hi PSC-RPC were identified by reverse transcription polymerase chain reaction(RT-PCR),immunofluorescence staining and western blotting.Result: The specific markers of Oct4,SSEA4,Sox2,Nanog for hiPSCs were positively expressed,q PCR showed that the level of Oct4 gene expression decreased and Pax6,Lhx2 and Chx10 gene expression increased in hi PSC-PRC after15 day differentiation.At the same time,eye field related factors(Pax6 and Lhx2)and RPCs specific marker(Chx10 and Nestin)were positively expressed in hi PSC-RPC by immunofluorescence staining.Western blot analysis showed that the protein level of Oct4 decreased in induced RPCs while Lhx2 was increased.Compared with control group,there was astatistical significance(P<0.05)Conclusions: In this study,hi PSCs treated the effect of specific factors could be induced into RPCs.Part2 Protective Effects of hi PSC-RPE in the Degenerating RetinaObjective: Retinitis pigmentosa is an inherited eye disease characterized by irreversible and progressive death of photoreceptor cells,eventually leading to blindness.Cell replacement therapy is one of the most promising treatment methods.hi PSCs derived RPE can provide the theory basis of seed cells for retinal degeneration diseases.Research of the protective effects of human i PSC-RPE to retinal neuronal cells in vitro and sub-retinal transplantation in the rd10 mice retinal degeneration model in vivo are investigated in this study.Methods: 1.Differentiation of hi PSCs towards RPE through cultured in differentiation medium with sequential addition of defined factors [IGF-1,Noggin,DKK-1,Nicotinamide(NIC),b FGF,Activin A,SU5402].The hi PSC-RPE cells were identified by immunofluorescence staining and western blotting.2.The Transwell co-culture of hi PSC-RPE cells with m RGC was performed to study m RGC proliferation and apoptosis.3.The Transwell co-culture of hi PSC-RPE cells with retinal explants from rd10 for 1 days with or without hi PSC-RPE to investigate photoreceptor apoptosis measured by TUNEL assay.4.hi PSC-RPE labeled with the red lipophilic fluorescent dye(PKH26)and spheroid culture 3 days before transplantation.rd10 mice received sub-retinal injections of the dissociated hi PSC-RPE(1×104/eye)on P12.5.Retinal sections were performed to observe the hi PSC-RPE cells whether survived in rd10 retina.ELISA was performed to explore the level of neurotrophic factors from intraocular fliud in hi PSC-RPE-treated rd10 mice.6.Immunocytochemistry,TUNEL assay and Western blot were used to analyze the effects on rd10 ocular condition after sub-retinal transplantation hi PSC-RPE.7.ERG response and thickness of outer nuclear layer used to analyze the hi PSC-RPE-treated rd10 retina structural and visual functioncompared to WT,and untreated rd10 mice.Result: 1.RPE could be generated from hiPSCs through sequential differentiated condition.The hi PSC-RPE could express positive RPE marker proteins,such as RPE-65,CRALB,Mitf,Otx2 and Tyrosinase.2.The m RGC co-cultured with hi PSC-RPE displayed a higher proliferation rate,and lower apoptosis by CCK-8 assay and flow cytometry.Compared with control group,a statistical significance(P<0.05)was observed.3.rd10 retinal explant was co-culture with hi PSC-RPE could rescue photoreceptors measured by TUNEL assay.There was statistical significance(P<0.05)4.Dissociated hi PSC-RPE cells after spheroid treatment hi PSC-RPE cells remained younger,better cell viability and anti-apoptosis detected by SA-β-Gal activity assay and double staining of Calcein AM and Eth D-III.5.hi PSC-RPE survived in the degenerated sub-retina of rd10 mice.The concentration of human PEDF in rd10 hi PSCRPE-transplanted eyes was 1.4ug/ml detected by ELISA.6.The activated microglia were inhibition analyzed by staining after transplantation of hi PSC-RPE into the subretina of rd10 mice.hi PSC-RPE treated rd10 mice downregulated expression levels of apoptosis-related(CD68,Bax)in the retina analyzed by Western blot.hi PSC-RPEtransplanted rd10 mice could reduce photoreceptors from apoptosis measured by TUNEL assay.Compared with control group,a statistical significance(P<0.05)was observed.7.Retinal sections and DAPI stain showed that hi PSC-RPE treated rd10 the degeneration of ONL was delayed.According to the Scotopic and Photopic ERG responses that the visual function of hi PSC-RPE treated rd10 mice was significantly improved.Compared with control group,a statistical significance(P<0.05)was observed.Conclusions: In this study,we are able to differentiate hi PSCs into RPE and transplant these hi PSC-RPE into the eye of rd10 mice.The transplanted hi PSC-RPE cells can well survive in host retina and obviously suppress microglial activation,significantly decrease apoptosis,improve the structural and functional features of photoreceptor degeneration. |
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