Comparison Of The Biological Characteristics Of 2D And 3D Induced Human Embryonic Stem Cell-derived Retinal Pigment Epithelium

Background and purposeRetinitis pigmentosa(RP)is an inherited blindness causing disease which mainly conducted by degeneration of retinal pigment epithelium(RPE)and retinal photoreceptors.At present,there is no effective therapy in treating RP.Transplantation of RPE cells seems to be the most promising therapy.Cells used for transplantation majorly came from early autogenic or homologous RPE cells and embryonic RPE cells1-5.Restricted by source problem,low cell viability of RPE cells,heavy immune rejection and complication of surgery,clinical application is pushed slowly.Recently,because of the abundant source and adorable biological features,pluripotent stem cell derived RPE cells had gradually become the main source of RPE-cell transplantation.On 2012 and 2015,Schwartz6-7 reported using human embryonic stem cells(h ESCs)derived RPE to treat two retinal degenerative diseases: age-related macular degeneration(AMD)and Stargardt's disease twice.All results showed that h ESCs-derived RPE cells is safe and effective.These researches are the first clinical report of h ESCs-derived RPE cell transplantation.There were many ways to induce h ESCs to become RPE cells.However,different methods showed total difference in purity,viability,function as well as efficiency of RPE cells.With serum-free culture methods,mouse or human embryonic stem cells could be aggregated quickly to form embryonic bodies(EBs)in V-bottom culture dishes.Low-concentration growth factor could effectively promote ESCs to become organoids8-9,in which therapeutic cells including RPE cells could be harvested.For new 3D inducements simulating the normal organogenesis process,therapeutic cells which were generated under those methods could have better biological characteristics and be closer to primary therapeutic cells.Nevertheless,there is no particular studies compared the biological characteristics of classical 2D and novel 3D generated RPE cells.Current work firstly set up 3D induced h ESCs to generated optic cups,from which RPE cells were dissociated.Then,to verify the feature of 3D induced RPE cells,the maturity,cellular function and protective effect after transplantation of RPE cells were compared between 2D and 3D methods.All these results will provide solid evidence to further clinical application.Methods1.2 D inducement of RPE cells from h ESCs(2D RPE cells)1.1 culture and identification of h ESCs cell line H11.2 Morphological study of the inducing process of 2D-RPE cells.1.3 Immunofluorescence identification of 2D-RPE cells via RPE specific markers including Bestrophin,CRALBP,MITF and PAX6.2.3 D inducement of RPE cells from h ESCs(3D RPE cells)2.1 Morphological study of the inducing process of 3D-RPE cells.2.2 Immunofluorescence identification of 3D-RPE cells via RPE specific markers including Bestrophin,CRALBP,MITF and PAX6.3.Comparison of biological characteristics of 2D RPE cells and 3D RPE cells3.1 Comparison of the expression level of PAX6 in 2D RPE cells and 3D RPE cellson day 40,day 60 and day 100 via immunofluorescence to show the difference of PRE progenitor cells3.2 Comparison of the expression level of mature RPE cell protein,RPE65,in 2D RPE cells and 3D RPE cellson day 40,day 60 and day 100 via immunofluorescence and western-blot to show the difference of mature RPE cells4.Observation of the safety and effect of 2D RPE cells and 3D RPE cells transplanted into the chronic retinal degeneration model,Royal college of surgeon(RCS)4.1 Counter staining of Ki67,mitochondrial and Dil on 4 weeks after transplantation to verify the proliferation of two RPE cells.Injection of 2D RPE cells and 3D RPE cells into the inguinal region of immunodeficient mouse,SCID mouse,to observe theformation of teratoma weekly till 4 months.4.2 Counter staining of Ki67,mitochondrial and Dil on 4 weeks after transplantation to verify the viability of two RPE cells.The thickness of out nuclear layer of RCS rats were measured according to Rhodopsin staining on 4 weeks,8 weeks and 12 weeks after transplantation of 2D RPE cells and 3D RPE cells.The amplitudes of b-wave in electroretinograms were tested to analyze the protective effect of transplantation of 2D RPE cells and 3D RPE cells.Results1.Identification of h ESCs cell line H11.1 Cells were single or multiple with ramified shape on 1 day after passaging.After replacing culture medium containing Y-27632,h ESCs cells were showed small round shape.Several days later,single clones enlarged and gradually fused.1.2 h ESCs express embryonic stem cell marker including Nanog,SSEA-4,Sox2 and Oct4.Only SSEA-4 was expressed at the cell membrane,all other cell markers were expressed within nuclear with over 99% positive rate.2.Comparison of 2D and 3D inducement process and morphology of two-source RPE cells2.1 h ESCs were both 2D and 3D induced to form RPE cells.Inducement duration were last identically and finally generated RPE cells had same morphologies.Several brown pigment could be observed on day 20 in both 2D and 3D inducement.Dissociated RPE cells showed single-layer and pave stone-like morphology on day 60 with abundant pigment.Cells were arranged orderly and tightly.2.2 Both 2D RPE cells and 3D RPE cells expressed RPE specific marker including Bestrophin,CRALBP,MIT and PAX6 on day 60.Bestrophin and CRALBP were mainly expressed at the cell manbrane.Whole RPE cells presented a regular reticulate structure.MITF and PAX6 were expressed within nuclear.The positive ratio of all these four markers were over 99%.After 40-60-and 100-day inducement,PAX6 was continuously expressed within 3D RPE cells,but not within 2D RPE cells.On the other hand,RPE65 was emerged later in 3D RPE cells compared with 2D RPE cells.Both of these results indicated that 3D RPE cells were less mature than 2D RPE cells.2.3 The immunofluorescence of optic cup showed the neural retina marker,PAX6,and RPE cell marker,CRALBP,double staining,where single layered RPE cell could be observed.The inner part of this pigment layer was multilayered neural retina,which indicated that h ESCs could differentiated into optic cup like structure.This process is identical to normal retinal development.3.The retinal protective effect of2 D RPE cells and 3D RPE cells transplanted into RCS rats.3.1 Both 2D RPE cells and 3D RPE cells were injected into inguinal region of SCID mice.After 4 month follow up,no subcutaneous enclosed mass was observed.However,positive control was treated with h ESCs which were injected at the same site in other SCID mice and showed teratoma formation.These results showed that both 2D RPE cells and 3D RPE cells had no tumorigenicity.3.2 Both 2D RPE cells and 3D RPE cells were injected into the subretinal space in RCS rats.4 weeks later,single layered transplanted RPE cells could be observed.These cells were conterstained with human mitochondrial marker and red fluorescence marker DIL,which indicated that transplanted cells were viable and no obvious immune rejection could be seen.3.3 4 weeks after transplantation,Ki67 staining was negative in both 2D RPE cells and 3D RPE cells,which indicated that transplanted cells were remain silent rather than further proliferating.3.4 After 60-day culture of RPE cells in vitro,2D RPE cells expressed mature RPE cell marker RPE65 but 3D RPE cells didn't.However,after transplantation of2 D RPE cells and 3D RPE cells into subretinal space of RCS rats,4 weeks later,both of these RPE cells could express RPE65,which indicated that 3D RPE cells could gradually become mature in vivo.3.5 After 4-week,8-week and 12-week transplantation of 3D RPE cells into subretinal space of RCS rats,electroretinograms showed that the amplitude of b-wave was better than controls.Specially,the protection of photoreceptor is the strongest at 4 weeks,which declined at both 8 weeks and 12 weeks.Both the thickness of out nuclear layer and electroretinograms showed that there were no obvious differences between 2D RPE cells and 3D RPE cells.Conclusion1.RPE cells generated from 2D and 3D inducement of h ESCs could form pigment.The passaging efficiency and RPE specific markers were identical to normal RPE cells.However,3D RPE cells expressed PAX6 longer but showed pigment and expressed RPE65 later,compared with 2D RPE cells,which indicated that 3D RPE cells were younger than 2D RPE cells.2.Teratoma formation test showed that after injection of both 2D RPE and 3D RPE cells into the inguinal region of SCID mice,there was no teratoma formation could be observed.3.Subretinal space transplantation of two RPE cells showed protection of photoreceptor via out nuclear thickness analysis and electroretinograms.There is no obvious difference between 2D and 3D RPE cells.