Spheroid transplantable and functional retinal pigment epithelium derived from non-colony dissociated human induced pluripotent stem cells*

GUO Xiao-ling，ZHU De-liang，LIAN Rui-ling，ZENG Qiao-lang，Sanjana MATHEW，TANG Shi-bo，CHEN Jian-su，，△

（1Key Laboratory for Regenerative Medicine，Ministry of Education，Jinan University，Guangzhou 510632，China；2Center of Scientific Research，The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University，Wenzhou 325027，China；3Eye Institute，School of Medicine，Jinan University，Guangzhou 510632，China；4Aier School of Ophthalmology，Central South University，Changsha 410015，China.E-mail：chenjiansu2000@163.com）

［ABSTRACT］AIM：To explore the feasibility of non-colony dissociated human induced pluripotent stem cells（hiPSCs）to differentiate into functional retinal pigment epithelial（RPE）cells（hiPSC-RPE cells），and offer an alterna?tive transplantation method based on cell spheroids.METHODS：The hiPSC-RPE cells were identified by RT-PCR，im?munofluorescence assay，and flow cytometry.The in vitro and in vivo functions of hiPSC-RPE were assessed by fluorescein leakage test，transepithelial electrical resistance assay，atomic force microscopic observation，photoreceptor outer segment（POS）phagocytosis assay，frozen tissue sections，live/dead cell staining assay，senescence-associated β-galactosidase（SA-β-Gal）staining，and immunocytochemistry.RESULTS：The hiPSC-RPE cells positively expressed biomarkers of RPE cells but not iPSCs，such as CRALBP（97.4%），EMMPRIN（93.8%），Oct4（2.1%），and Sox2（2.0%）.These cells displayed RPE cell-like characteristics including barrier function，phagocytic activity，and polarized membrane.The cells derived from hiPSC-RPE cell spheroids positively expressed nestin and exhibited reduced SA-β-Gal staining（P＜0.01）.The hiPSC-RPE cell spheroids were able to form monolayer on decellularized corneal matrices（DCM）.After 1 month of subretinal transplantation，the hiPSC-RPE cell spheroids survived and maintained segmental sheet growth in Chinchilla rabbits with sodium iodate-induced RPE degeneration.CONCLUSION：Non-colony dissociated hiPSCs can effectively differentiate into functional RPE cells，and hiPSC-RPE spheroids maintain segmental sheet growth in the sub?retinal space of RPE-degenerative Chinchilla rabbits in vivo，which may offer cell spheroid transplantation as an alternative method for the treatment of RPE degenerative diseases in the future.

［KEY WORDS］Human induced pluripotent stem cells；Retinal pigment epithelial cells；Cell differentiation；Cellular spheroids；Transplantation

Retinal pigment epithelial（RPE）cells are po?larized monolayer cells distributed between the chorio?capillaris and neural retina，playing a crucial role in maintaining the function of photoreceptors and the reti?na.Degeneration and dysfunction of RPE cells re?sulting in photoreceptor loss are the main causes of reti?nal diseases including Stargardt's macular dystrophy and dry age-related macular degeneration（AMD）［1］.Since there is no cure for most patients affected by these diseases，RPE transplantation represents an at?tractive therapeutic alternative.Various cell types have been examined for their utility in RPE replacement in?cluding ARPE19 cells，adult RPE cells，fetal RPE cells，and other non-RPE cell lines［2］.However，the sources of donor cells are still very limited，and some?times the cells cause immune rejection.

Human pluripotent stem cells（hPSCs）including human embryonic stem cells（hESCs）and human in?duced pluripotent stem cells（hiPSCs）are charac?terized by unlimited self-renewal and their ability to dif?ferentiate into any cell type.In 2004，Klimanskaya et al［3］firstly reported that putative RPE cells were de?rived from spontaneous differentiation of ESCs.How?ever，the application of ESCs has ethical and immune rejection problems.The iPSCs without ethical issue de?rived from the patient's own cells，and HLA-matched or CRISPR-engineered iPSCs made based on the cur?rent gene editing technique，can be used to avoid the problem of immune rejection.So far，a number of re?searchers have reported that iPSCs can be differentiated into RPE-like cells（iPS-RPE cells）［4］，but these iPSCs were based on traditional colony culture for differentia?tion.

Recently，scientists developed a non-colony but monolayer culture method for dissociated cells.The key element in this technique is seeding dissociated cells at high density on Matrigel coating plates in the presence of ROCK inhibitor Y-27632 to facilitate disso?ciated-cell plating efficiency.One of the advantages of non-colony dissociated culture is the generation of ho?mogeneous hiPSCs.Researchers found that the cells under non-colony dissociated culture showed a more ho?mogeneous response to BMP-4 signaling than WA01 cells in colony.Our previous study also demonstrated that a half-exchange mTeSR1 medium culture system that combined Y-27632 and high-density dissociatedcell seeding facilitated the homogeneous growth of hiPSCs［5］.So we try to use this method of non-colony dissociated culture to harvest homogeneous hiPSCs for RPE cell differentiation in this study.

So far，two approaches for RPE cell transplanta?tionin vivoare predominant：the subretinal injection of cell suspension and the subretinal insertion of RPE cell sheet grown in monolayer.Subretinal transplantation of pluripotent stem cell（PSC）-derived RPE cell suspen?sions or RPE-containing scaffold sheets was found to be safe and rescued vision in animal models and even in patients with retinal degeneration［6］.Polarized cell monolayer transplantation improves cell survival com?pared with cell suspension transplantation，but the pro?cess of cell sheet transplantation is not easy.So explo?ration of more alternative methods for RPE cell trans?plantationin vivois necessary.

Huang et al［7］reported that use of the appropriate size of dermal papilla（DP）spheroids effectively in?duced hair follicle regeneration and maintained high cell viability when transplantedin vivo.Our previous studies plus other work also confirmed that spheroid culture enhanced the stemness and viability of bovine corneal endothelial cells（B-CECs），corneal stromal cells（CSCs），and human adipose-derived stem cells（ADSCs）［8-9］.These research foundations inspired us to generate spheroids of RPE cells derived from human iPSCs（hiPSC-RPE cells）for transplantationin vivo.In this study，hiPSC-RPE cell spheroids were generated based on commercially available silicone micro-wells，which was used to upscale the production of cell spheroids with a controllable size.Moriguchi et al［10］ex?plored the mechanism causing degeneration of RPE in mice after an intravenous injection of sodium iodate（NaIO3）.Therefore，we would also use NaIO3treat?ment in this study to establish RPE degeneration model in Chinchilla rabbits for cell transplantationin vivo.

In this study，we would examine the feasibility of non-colony dissociated hiPSCs instead of clonal hiPSCs to differentiate into transplantable and functional RPE cells.Then，we seeded hiPSC-RPE cell spheroids onto decellularized corneal matrices（DCM）for simulation experimentsin vitro.Lastly，we injected hiPSC-RPE cell spheroids into the subretinal space of Chinchilla rabbits with RPE degeneration induced by NaIO3treat?mentin vivo.Our study will lay the foundation for the use of hiPSC-RPE cell spheroids as an alternative method of cell transplantation for treatment of RPE de?generative diseases.

MATERIALS AND METHODS

1 Cells and culture of hiPSCs

hiPSCs were a gift from the Guangzhou Institutes of Biomedicine and Health，Chinese Academy of Sciences.These hiPSCs were reprogrammed from hu?man umbilical cord mesenchymal cells using methods

described in a previous report.hiPSCs were cultured in 1% Matrigel（BD Biosciences）-coated dishes at 37℃in 5% CO2and refreshed daily with mTeSR1 medium（Stem Cell Technologies Inc.）.The cells were pas?saged once every 6 d with 0.25% EDTA（Sigma）and were then seeded into 6-well dishes at a ratio of 1∶6.They were supplemented with 10 μmol/L Y-27632（Sig?ma）on the first day of passaging.For generating disso?ciated hiPSCs，the harvested clonal hiPSCs were pipet?ted approximately 30～50 times and then were filtered through a 40 μm cell strainer（BD）.The dissociated hiPSCs（1×105/cm2）were plated into 6-well dishes.Half-exchange mTeSR1 medium was used for further culture of non-colony dissociated hiPSCs.

2 Experimental methods

2.1 Isolation and culture of adult RPE cellsHu?man eyes were obtained from 6 male donors without ocu?lar disease after informed consent at the age of（45±5）years old.The study was approved by the Human Re?search and Ethical Committee of Jinan University，and the procurement and use of human tissues were in com?pliance with the Declaration of Helsinki.Human adult RPE cells were isolated and cultured as described in a previous report［11］.Briefly，RPE cells were isolated from the posterior section of the eyeballs using 0.25%EDTA-trypsin（Gibco）and harvested by centrifugation at 400×gfor 5 min.The hRPE cells were cultured in RPE medium that consisted of high-glucose Dulbecco's modified Eagle medium（HG-DMEM，Gibco），10%fe?tal bovine serum（FBS，Gibco），1×105U/L penicillin and 100 mg/L streptomycin（P/S，Gibco）at 37℃in 5% CO2.After reaching 100% confluence，the RPE cells were passaged and seeded in 6-well plates with 1% Matrigel coating.The medium containing 100 μg/L activin A（R&D Systems）was changed every 3 d.

2.2 Differentiation of non-colony dissociated hiPSCs into RPE cellsBecause a prerequisite for hiPSC dif?ferentiation is shutdown of the self-renewal machinery，dissociated hiPSCs were pretreated in E7 medium with?out FGF2 for 2 d to encourage spontaneous differentia?tion.FGF2 withdrawal from the culture medium may promote neuro-ectoderm induction，and RPE belongs to neuro-ectoderm lineages.Therefore，the dissociated hiPSCs were switched into proneural medium with the sequential addition of defined factors at specific time points.The point at which dissociated hiPSCs ex?panded to 100% confluence in half-exchange mTeSR1 medium was defined as day 2，and from day -2 to 0，hiPSCs were cultured in E7 medium without FGF2 but with 10 μmol/L Y-27632.Prior to the beginning of dif?ferentiation，dissociated hiPSCs were cultured in pro?neural medium containing DMEM/F12（Gibco），1%nonessential amino acids（NEAA，Invitrogen），and 1% N-2 supplement（Invitrogen）.From 0 to 2 d，10 μg/L IGF-1（R&D Systems），50 μg/L noggin（R&D Systems），10 μg/L Dkk-1（R&D Systems）and 10 mmol/L nicotinamide（NIC，Sigma）were added into the proneural medium.From 2 to 4 d，10 μg/L IGF-1，10 μg/L noggin，10 μg/mL Dkk-1，10 mmol/L NIC and 5 μg/L bFGF were added to the proneural medium.From 4 to 6 d，10 μg/L IGF-1，10 μg/L Dkk-1 and 100 μg/L activin A were added to the proneural medium.From 6 to 14 d，100 μg/L activin A and 10 μmol/L SU5402（EMD Millipore）were added to the proneural medium.Then，the differentiated cells were mechani?cally enriched by scraping away non-RPE-like（spin?dle）cells，and the remaining RPE-like（epithelioid）cells were passaged using 0.25% EDTA and seeded in?to 1% Matrigel-coating dishes.From 14 to 20 d，the enriched cells were cultured in enrichment medium con?taining HG-DMEM（Gibco），1% FBS（Gibco），100 ng/L activin A，1× sodium pyruvate，and 1× Gluta?MAX（Invitrogen）.From 20 to 30 d，these enriched cells were passaged again as passage 1（P1）and cul?tured in RPE medium.

2.3 Gene expression analysisTotal RNAs from the cells were isolated using a Tissue RNA Miniprep Kit（Biomega）.These RNAs were reversely tran?scribed into cDNA using the Superscript II kit（Invitro?gen），and the DNAs were used for reverse transcription polymerase chain reaction（RT-PCR）to identify the mRNA expression of GADPH，Nanog，Oct4，Sox2，Klf4，RPE-65，CRALBP，EMMPRIN，Otx2，and Lin28.The RT-PCR products were examined after elec?trophoresis on 2% agarose gels.The gels were scanned for further analysis.The primer sequences were shown in Table 1.

2.4 Flow cytometryFlow cytometry was used to analyze the interested cell purity according to a previous report with small modifications［12］.Samples includinghiPSC，hiPSC-RPE cells and hRPE cells were fixed type control antibodies，such as Oct4，Sox2，RPE-65，CRALBP，Mitf and EMMPRIN，were labeled with fluo?rophore-conjugated secondary antibodies at 4℃for 30 min.The labeled samples were detected by a flow cy?tometry analyzer（BD）.using 4% paraformaldehyde and permeabilized by 0.1% Triton X-100（Sigma）.Then，the cells were in?cubated with isotype control or primary antibodies as shown in Table 2 at 4℃for 30 min.Primary and iso?

Table 1.Primer information

Table 2.Antibodies

2.5 Immunofluorescence assayImmunofluores?cence staining was performed to identify hiPSC-RPE cells as described in a previous report［13］.Briefly，4%paraformaldehyde-fixed cells were permeabilized with 0.1% Triton X-100 and incubated with 3%（w/v）bo?vine serum albumin（BSA；Sigma）for blocking.Cells were then incubated with primary antibodies such as CRALBP，Mitf，tyrosinase，Otx2，EMMPRIN，RPE-65，ZO-1，occludin，Nanog，AHNA，Nanog，Oct4，Sox2，SSEA4 and TRA-1-60 as shown in Table 2 over?night at 4℃.On the second day，the cells were washed twice with PBS and then incubated with FITC-conju?gated anti-mouse，F(xiàn)ITC-conjugated anti-rabbit，Cy3-conjugated anti-mouse，or Cy3-conjugated anti-rabbit IgG secondary antibodies（1∶1 000，concentration of 1 g/L；Bioword）at room temperature for 60 min.Cells were rinsed 3 times with PBS and stained with DAPI（Sigma）before examination by a fluorescence micro?scope（OLYMPUS）.

2.6 Transepithelial electrical resistance（TEER）assayThe TEER assay was used to assess the dy?namic barrier function of the epithelioid cells［14］.Cells were seeded into 24-Transwell inserts at 1×105/cm2.Af?ter reaching 100% confluence on day 7，the dynamic barrier of the cells was determined through measuring TEER across the cell monolayer using Millicell-ERS-2 Voltohmmeter（EMD Millipore）.The value of TEER was calculated according to the following equation：TEER（Ω·cm2）=（Rtotal-Rinsertw/oMatrigel）×A，where Rtotal（Ω）was the resistance measured，Rinsertw/oMatrigel（Ω）was the resistance of the insert without 1% Matrigel coating，and A was the membrane area（cm2）of the in?sert.

2.7 Observation by atomic force microscopy（AFM）AFM was used to observe the ultrastructure of the cells as described in a previous report.The cells were fixed with 4% paraformaldehyde for 10 min and dried at room temperature before imaging.The curva?ture radius of the AFM tips was 10 nm.The spring con?stant was 20―50 N/m with a resonance frequency of 278―317 kHz.The scanning speed was kept at 0.5 Hz.The ultrastructure of the cells was measured in con?tact mode.The data analysis was performed using Na?noScope Analysis 2.1（Thermo）.

2.8 Photoreceptor outer segment（POS）phagocy?tosis assayThe POS was isolated as described in a previous report.Briefly，the retinas of porcine eyeballs were collected and agitated in KCl buffer（0.5 mmol/L CaCl2，1 mmol/L MgCl2，0.3 mol/L KCl，and 10 mmol/L HEPES）with 48%（w/v）sucrose at pH 7.0，and then were centrifuged at 5 000×gfor 5 min.The super?natant containing the POS was filtered using sterile gauze，diluted 1∶1 with KCl buffer without sucrose，and centrifuged at 4 000×gfor 10 min.The isolated POS was then resuspended in 1 mL of PBS and were la?beled with FITC（Sigma-Aldrich）at room temperature for 1 h.The labeled FITC-POS was then rinsed and re?suspended using HG-DMEM with 5% sucrose.The cells were incubated with FITC-POS at 37℃in 5% CO2for 2 h.Last，immunofluorescence was performed using Cy3-conjugated mouse monoclonal ZO-1 antibody and DAPI，and then the cells were examined under an in?verted fluorescence microscope（OLYMPUS）.

2.9 Observation of polarized membrane by Zstack confocal microscopyZ-stack confocal micros?copy was used to observe the polarized membrane of the cells.First，200 μL of the cells（1×105/cm2）were seeded into 6-well dishes and incubated at 37℃in 5%CO2for 5 d.Immunofluorescence was conducted with Cy3-conjugated mouse monoclonal ZO-1 antibody and DAPI，and the stained cells were examined under a confocal microscope（LSM 510 META；Zeiss）.

2.10 Western blotThe cells were washed using cold PBS and lysed using RIPA（Beyotime Biotechnolo?gy）.A total of 50μg protein was electrophoresed on 10% SDS-PAGE gels，and then transferred to polyvi?nylidene difluoride（PVDF）membranes（Sigma）and blocked using 5% fat-free milk.Then，the membranes were incubated with primary anti-nestin and anti-β-actin antibodies as shown in Table 2 at 4℃overnight.The membranes were washed 5 times with TBST and incu?bated with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies（1∶3 000，concentration of 1 g/L；Bioword）at room temperature for 2 h.The bands were visualized with enhanced chemiluminescence（ECL；Pierce）.

2.11 Production of agarose micro-multiwell dishes and hiPSC-RPE cell spheroidsA volume of 500 μL liquid solution of 2%（g/mL）agarose was pipetted into a commercially available silicone micro-mold with eighty-one wells（Micro Tissues Inc.）.After solidifica?tion，the microwell agarose mold was removed using sterilized forceps.The agarose molds were placed in 6-well dishes.Then，200 μL of cell suspension con?taining approximately 2×105cells was carefully pipetted into the microwell plate and incubated at 37℃in 5%CO2.The medium was changed every 2 d.The cell spheroids（approximately 80 μm in diameter，5×103cells per spheroid）were formed after 3 d.

2.12 Preparation of DCMThe lamellar corneal matrix（100 μm thickness）was excised from porcine eyeballs using a microkeratome（Kangming）and was rinsed 3 times with PBS.The excised lamellar corneal matrix was treated with 0.25% EDTA-trypsin（Invitro?gen）at 37℃for 30 min and then was fixed with 4%paraformaldehyde for 1 d at 4℃.It was treated with 0.8% SDS（Sigma）solution at -80℃for 30 min and then was transferred to a 37℃shaking table（350 r/min）for 1 h.It was rinsed 3 times with PBS and pre?served in 100% glycerol at 4℃as DCM.Before the seeding of cells，the DCM was washed 3 times with PBS containing P/S solution and sterilized under ultra?violet light for 2 h.

2.13 Seeding the cell spheroids on the DCMTo test whether cell spheroids in the biomimetic microenvi?ronment could grow well，anin vitrosimulation experi?ment of seeding cell spheroids onto DCM was con?ducted as described previously.Briefly，cell spheroids were seeded on the DCM.The medium containing 10 μmol/L Y-27632 was changed every 3 d.Viable cell staining with Calcein AM was used for better observa?tion of the cell spheroids on the DCM under a fluores?cent microscope.The adherent growing area of the cell spheroid periphery stained by a Live-Dead Cell Staining Kit（Biotium）was measured using ImageJ on days 7 and 14.

2.14 Frozen tissue sectionsTissue samples were firstly fixed with 4% paraformaldehyde for 15 min，and then were mounted using tissue freezing medium（SAKURA Tissue-Tek）and placed at -80℃refrigera?tor for at least 2 h until frozen.The frozen tissues were sectioned at a thickness of 15 μm using a cryo-micro?tome（Thermo Fisher）as described in a previous re?port.Sections were placed on one side of microscope slides（SAKURA Tissue-Tek）.Some sections were in?cubated with DAPI for 15 min，washed with PBS for 3 times，and examined under an inverted fluorescence microscope（OLYMPUS）.Other sections were used for hematoxylin-eosin（HE）staining and were imaged using an inverted microscope（Nikon）.

2.15 Verification of live/dead cells by Calcein AM and EthD-III double stainingCalcein AM and EthD-III double staining（Molecular Probes）was per?formed as described in a previous report［15］.Briefly，a standard working solution containing 2 μmol/L Calcein AM and 4 μmol/L EthD-III was prepared.Cells were incubated with the standard working solution at room temperature for 40 min，and were then imaged underan inverted fluorescence microscope.

2.16 Senescence-associated β-galactosidase（SAβ-Gal）stainingSA-β-Gal staining was performed using a Cellular Senescence Assay Kit（Beyotime Bio?technology）following the manufacturer's instructions.We used spheroid or monolayer adherent cells on days 7 and 14 to conduct SA-β-Gal staining.Briefly，after reaching 100% confluence，the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and were then incubated in a staining solution overnight at 37℃.On the next day，the stained cells were washed with PBS and observed under an inverted micro?scope.A blue color indicated the presence of SA-β-Gal.The intensity of the SA-β-Gal staining was calcu?lated using ImageJ software.

2.17 Tagging hiPSC-RPE with PKH26The stan?dard protocol was performed as described on the PKH26 Product Information Sheet（MINI2）.Briefly，a suspension containing 2×107cells was centrifuged（400×g，5 min）and washed once using fresh medium without serum.After centrifuging，the cells were resus?pended in 1 mL of Diluent C.Dye Solution（4×10-6mol/L）was prepared by adding 4 μL of PKH26 ethano?lic dye solution into 1 mL of Diluent C.Then，1 mL of Dye Solution was rapidly added to the cell suspension.The final concentration after mixing was 2×10-6mol/L PKH26 with 1×105cells/cm2.The mixing suspension was incubated with periodic mixing at room temperature for 5 min.The staining was stopped by adding an equal volume（2 mL）of serum.Then，the suspension was centrifuged at 400×gfor 10 min and washed 3 times.Finally，cells tagged with PKH26 were used for injec?tion.

2.18 RPE degeneration Chinchilla rabbit modelAmirpour et al［16］transplanted retinal cells derived from hESCs into the subretinal space of NaIO3-injected pig?mented rabbits to restored a small but significant Bwave.Therefore，we would also use NaIO3treatment to establish RPE degeneration model in Chinchilla pig?mented rabbits for cell transplantationin vivo.The rab?bits were weighed on an electronic scale and then in?jected with 1% NaIO3（40 mg/kg；Sigma）via the ear marginal vein.After 1 week，the rabbits were injected with 1% NaIO3（40 mg/kg）again.The successful le?sioned model was assessed by electroretinogram（ERG）assay using the Roland Retiport（RETI-Port/Scan 21）system，which showed no distinctly functional ERG re?sponse compared with normal rabbits under similar ex?citation.Then，the successful model was used for fol?lowing cell transplantation.

2.19 Preliminary test of hiPSC-RPE cell spheroids in vivoAnimal experiments were approved by the In?stitutional Animal Care and Use Committee of Jinan University，and animal procedures were conducted fol?lowing the guidelines of the US National Institutes of Health.Six-month-old Chinchilla rabbits（n=12）weighing 1―2 kg were raised in a 12 h dark/light cycle，temperature at（23±2）℃，and relative humidity of 45% to 55%.Water and food were changed every day.These Chinchilla rabbits were randomly divided into 4 groups：control group（n=3，6 eyes），which received no treatment；NaIO3group（n=3，6 eyes），which re?ceived NaIO3treatment；NaIO3+PBS group（n=6，6 right eyes），which received NaIO3treatment and PBS injection；and NaIO3+hiPSC-RPE group（n=6，6 left eyes），which received NaIO3treatment and hiPSC-RPE cell injection.The leakage or bystander effects were ex?cluded through the inspection of different people.Wa?ter containing 210 mg/L cyclosporin A（Sigma）and prednisone were given to these rabbits throughout the experiment to reduce allograft rejection.For PBS or hiPSC-PRE cell injection，the model rabbits were anes?thetized with pentobarbital sodium（25 mg/kg；Sigma）and chlorpromazine（5 mg/kg；Sigma）.The pupil was dilated using tropicamide（Alcon）and the eye lid was kept open using a lid speculum.Cell transplantation was performed under a surgical microscope（Ocular In?struments）.For subretinal injection，the peritomy was made 2.0 mm posterior to the limbus in the superotem?poral quadrant of each eyeball.A sideport knife Beaver blade（BD）was used to make a longitudinal triangular scleral incision starting 2 mm away from the limbus at about the 5° axis toward the choroid until minimal blood reflux appeared.At this point an additional tract through the choroid toward the RPE layer was created using a 30-gauge needle.The hiPSC-RPE cell spheroids labeled with PKH26 were suspended in PBS containing 10 μmol/L Y-27632，and then 10 μL of cell spheroids（approximately 1×105cells）at a density of 20 cell spheroids per μL were slowly injected through the scleral tunnel using a 50 μL Hamilton blunt syringe with a 30-gauge needle（BD）.The syringe was immedi?ately pulled back.

2.20 ImmunocytochemistryRabbits were sacri?ficed with an overdose of sodium pentobarbital.Their eyeballs were removed，punched using a 23-gauge nee?dle（BD），and fixed in 4% paraformaldehyde overnight at 4℃.Then，these eyeballs were dehydrated with graded series of ethanol and xylene，and subsequently were embedded using paraffin wax.The paraffin sec?tions（5 mm）were cut and dewaxed in water.After an?tigen repair，these sections were fixed with 4% parafor?maldehyde for 15 min and washed 3 times with PBS.They were permeabilized with 0.1% Triton X-100 for 15 min at room temperature and were blocked with 3%（w/v）BSA at room temperature for 1 h.Then，they were incubated with primary antibodies as shown in Ta?ble 2 for 2 h at room temperature followed by FITC-con?jugated secondary antibody（1∶1 000，concentration of 1 g/L；Bioword）for 1 h at room temperature.These sections were rinsed 3 times with PBS and incubated with DAPI（10 mg/L）for 15 min.Then，they were ex?amined under an inverted fluorescence microscope.

3 Statistical analysis

All of the data were presented as the mean±SEM（standard error of mean）of at least 3 separated experi?ments，and statistical significance was evaluated using one-way ANOVA followed by Tukey's multiple compari?son test.Student's unpairedt-test was used to compare 2 different groups.P＜0.05 was considered statistically significant.

RESULTS

1 Differentiation and identification of hiPSC-RPE cells

A schematic illustration for differentiation was dis?played in Figure 1A.Both clonal and dissociated hiPSCs grew very well.The nucleoplasmic relation of the dissociated hiPSCs became lower on day 0，and the epithelioid morphological change of the cells began to appear on day 8.The epithelium-like cell monolayer was formed on day 14.The RPE-like（epithelioid）cells were mechanically enriched，passaged，and reached 100% confluence on day 20（Figure 1B）.Im?munofluorescence staining was used to characterize the expression of RPE-specific biomarkers in the enriched hiPSC-RPE cells（P1）.The results showed that the hiPSC-RPE cells strongly expressed CRALBP，Mitf，Tyrosinase，Otx2，EMMPRIN，and RPE-65（Figure 1C）.RT-PCR showed the mRNA expression of RPE-65，EMMPRIN，Otx2 and CRALBP in both hiPSCRPE and hRPE cells，but not Nanog，Oct4，Sox2，Klf4 or Lin 28.In contrast，the hiPSCs expressed Nanog，Oct4，Sox2 and Klf4，but not RPE-65，EMMPRIN，Otx2 or CRALBP（Figure 1D）.

Figure 1.Differentiation and identification of hiPSC-RPE cells.A：a schematic illustration of the differentiation protocol of the hiPSC-RPE cells；B：morphological changes in the differentiation of cells under an inverted microscope（scale bar=100 μm）；C：identification of hiPSC-RPE cells（P1）using immunofluorescence assay（scale bar=100 μm）；D：identification of hiPSC-RPE cells（P1）using RT-PCR.

Meanwhile，the results of flow cytometry revealed that hiPSC-RPE cells expressed low levels of Oct4（2.1%）and Sox2（2.0%），but expressed high levels of RPE-65（74.4%），CRALBP（97.4%），Mitf（86.8%）and EMMPRIN（93.8%）.These properties were simi?lar to those of hRPE cells，which expressed Oct4（1.9%），Sox2（1.7%），RPE-65（84.2%），CRALBP（100%），Mitf（94.2%），and EMMRPIN（95.2%）.hiPSCs showed high levels of Oct4（99.9%）and Sox2（99.9%），but low levels of RPE-65（4.5%），CRAL?BP（1.7%），Mitf（0.4%），and EMMRPIN（4.1%）.See Figure 2A.

AFM images of hiPSCs in the cells displayed a vol?cano-like distribution.The surface of the hiPSCs was relative rough，and the cell nucleus showed an oval shape.AFM image of hiPSC-RPE cells revealed a pan?cake-like configuration，which was similar to that of the hRPE cells.The cell surface of the hiPSC-RPE cells was relatively smooth，and the cell nucleus displayed a cobblestone-like appearance.The nucleo-cytoplasmic ratio of the hiPSCs was higher than that of hiPSC-RPE cells and hRPE cells（Figure 2B and Figure 3A）.Sta?tistical analysis showed that cell length，width and height parameters of hiPSC-RPE cells were similar to those of hRPE cells（P＞0.05）but were different from those of hiPSCs（P＜0.05）.See Figure 3B.At the same time，the cell nuclear length，width and height parameters of hiPSC-RPE cells were similar to those of hRPE cells（P＞0.05）but were different from those of hiPSCs（P＜0.05）.See Figure 3C.

Figure 2.Further identification of hiPSC-RPE cells.A：representative flow cytometry histograms of hiPSC，hiPSC-RPE cells（P1），and hRPE cells；B：the images of 2D and 3D topography for hiPSC，hiPSC-RPE cells（P1）and hRPE cells under atomic force microscope.The scale bar=60 μm or 100 μm.

2 Maintenance of native RPE cell function in hiPSCRPE cells

Cytoplasmic anchor protein ZO-1 is often used to establish tight junctions.Z-stack confocal microscopy revealed that ZO-1 showed strong expression on the api?cal side of both hRPE cells and hiPSC-RPE cells（Figure 4A）.An essential function of RPE cells is phagocytosis of outer segments shed from photorecep?tors.Our results showed that FITC-POS particles were specifically engulfed by cells，and extensively dis?tributed into the cytoplasm of hiPSC-RPE cells and hRPE cells（Figure 4B），indicating that these cells were capable of phagocytosis.In addition，a TEER as?say was conducted to assess the barrier behavior of the hRPE cells and hiPSC-RPE cells（Figure 4C）.The da?ta showed that the value of TEER in hRPE cells was（129.3±1.4）Ω·cm2，and that in hiPSC-RPE cells was（127.1±1.1）Ω·cm2，without significant dif?ference between them（P＞0.05）.See Figure 4D.

3 Seeding of hiPSC-RPE cell spheroids in vitro

Figure 3.The morphological observation of hiPSCs，hiPSC-RPE cells and hRPE cells by atomic force microscopy（AFM）.A：the cell，cell surface and cell nucleus images of 2D and 3D AFM for hiPSCs，hiPSC-RPE cells and hRPE cells（scale bar=5×5 μm，10×10 μm，20×20 μm，25×25 μm or 60×60 μm）；B：statistical comparison of the cell length，width and height in hiPSCs，hiPSC-RPE cells and hRPE cells；C：statistical comparison of the nuclear length，width and height in hiPSCs，hiPSC-RPE cells and hRPE cells.Mean±SEM. n=3.*P＜0.05 vs hiPSCs.

To determine whether hiPSC-RPE cell spheroids remained viable afterin vivotransplantation on DCM，the porcine lamellar corneal matrix with the thickness of 100 μm and the diameter of（9.29±0.24）mm was pro?duced using a microkeratome（Figure 5A）.The results of DAPI and HE staining showed that the DCM had al?most no cells in it，but the control cornea had many cells in the epithelial and stroma layers（Figure 5B）.

Figure 4.Identification of RPE-like function in hiPSC-RPE cells.A：a Z-stack confocal micrograph showing typical polarized expression of ZO-1 demonstrated apical localization in hRPE and hiPSC-RPE cells（scale bar=20 μm）；B：the phagocytosis of FITC-POS and ZO-1 staining in hRPE and hiPSC-RPE cells（scale bar=50 μm）；C：typical recording in transepithelial electrical resistance（TEER）［19］mode；D：TEER assay assessed the dynamic barrier function in hRPE and hiPSC-RPE cells.Mean±SEM. n=3.

Figure 5.Injectability of the hiPSC-RPE cell spheroids.A：preparation of porcine lamellar corneal matrix（100 μm thickness）using a microkeratome；B：the frozen sections of cellularized and decellularized corneal matrix with DAPI and HE staining（scale bar=50 μm or 100 μm）；C：the process of hiPSC-RPE cell spheroid generation（scale bar=50 μm or 100 μm）.

3D agarose molds containing 81 wells were used to generate hiPSC-RPE cell spheroids（Figure 5C）.The results of inverted light microscopic observation showed that proliferating epithelium-like hiPSC-RPE cells mi?grated from the periphery of adherent cell spheroids.On day 14，hiPSC-RPE cell spheroids disappeared and instead formed an epithelium-like monolayer that com?pletely covered the DCM（Figure 6A）.On day 0，the cells in hiPSC-RPE cell spheroids were viable，with green fluorescence by Calcein AM staining.Red fluo?rescence indicated dead cells by EthD-III staining.Af?ter 7 d，the majority of the cells showed green fluores?cence，and a limited number of dead cells were located mainly in the center of hiPSC-RPE cell spheroids，showing red fluorescence.On day 14，cell spheroids disappeared，and the remaining monolayer cells were only showed green fluorescence（Figure 6B）.Mean?while，double immunofluorescence staining showed that the monolayer hiPSC-RPE cells cultured on the DCM expressed occludin，Mitf，ZO-1，and RPE-65（Figure 6C）.

In addition，positive staining of SA-β-Gal was de?tected in spheroids or conventionally cultured hiPSCRPE cells on days 7 and 14（Figure 6D）.Statistical analysis of SA-β-Gal staining revealed that the per?centages of positive staining cells in spheroids and con?ventionally cultured hiPSC-RPE cells were（8.01±0.44）% and（22.47±0.75）% on day 7，respectively.The senescent cells in the spheroid cultured hiPSCRPE were lower at（14.46±0.32）% than those in the conventionally cultured hiPSC-RPE cells（P＜0.01）.See Figure 6E.After 14 d，the cell spheroids disap?peared，and there were（15.23±0.61）% senescent cells in the spheroid cultured hiPSC-RPE and（68.68±1.34）% in the conventionally cultured hiPSC-RPE cells.There was a（53.38±0.95）% increase in senes?cence in the conventionally cultured hiPSC-RPE cells compared with the spheroid cultured hiPSC-RPE cells（P＜0.01）.See Figure 6E.Furthermore，Western blot showed that hiPSC-RPE cells in a spheroid culture ex?pressed nestin，which was not expressed in hiPSC-RPE cells in conventional culture（Figure 6F）.

4 Preliminary test of hiPSC-RPE cell spheroids in vivo

To evaluate the states of transplanted cellsin vivo，hiPSC-RPE cell spheroids were labeled with lipophilic red dye（PKH26），which displayed red fluorescence（Figure 7A）.The classical external approach for RPE cell transplantation was conducted to inject hiPSC-RPE cell spheroids into the upper temporal region of the equator in rabbit eyeballs（Figure 7B）.A schematic of the external approach cell transplantation was shown in Figure 7C.

One month after transplantation，HE staining showed that the eyes in control group had a complete RPE layer and these RPE cells had rich pigmentation，but the RPE layers in NaIO3and NaIO3+PBS groups showed serious damage and the rest of most RPE cells showed less pigmentation.Meanwhile，a segmental sheet growth of RPE layer having obvious RPE cells with pigmentation was observed in NaIO3+hiPSC-RPE group（Figure 8A）.The immunocytochemical staining showed that a specific marker of human cells（AHNA）was not detected in control group，NaIO3group and NaIO3+PBS group，and was positive in NaIO3+hiPSCRPE group.Meanwhile，the cells tagged with PKH26 were not observed in control group，NaIO3group and NaIO3+PBS group，and were detected in NaIO3+hiPSCRPE group（Figure 8B）.

DISCUSSION

The retina is a sensor and processor of visual infor?mation.Retinal damage causes permanent loss of vi?sion.However，recent studies in regenerative medicine have aroused hope for rescuing visual function.One of the strategies for treatment of retinal diseases is the transplantation of retinal cells，especially RPE cells.Partial recovery of visual function by replacement of RPE has been reported［16］.

Due to the limited resources of donor cells，RPE cells derived from stem cells，especially ESCs and iPSCs，are required.In the field of ophthalmology，great progress has been made in research on iPSC dif?ferentiation.Currently，iPSCs are successfully differen?tiated into corneal epithelial cells（CECs）［17］，eye tra?becular reticular cells，rod cells，retinal progenitor cells，RPE，and so on.Researchers documented that adult dermal fibroblasts serve as a valuable resource for iPSC-RPE cells with characteristics highly reminiscent of RPE cells.

Usually，the process of differentiation of iPSCs in?to RPE cells is time consuming and has a low efficien?cy.The survival rate of transplanted iPSC-RPE cellsin vivois not high.Recently，Buchholz et al［18］reported that iPSCs induced with a series of small molecules such as IGF-1，Noggin，DKK-1，nicotinamide，bF?GF，activin A，SU5402 and VIP were rapidly differen?tiated into RPE phenotype cells after only 14 d.In our study，we modified the differentiation method proposed by Buchholz et al.Non-colony dissociated hiPSCs in?stead of clonal hiPSCs were pretreated with E7 iPS cul?ture medium without bFGF for 2 d to inhibit prolifera?tion and promote differentiation.RPE-like cells began to appear on day 8 and formed a cell monolayer after re?moval of the dead cells through changing the medium on day 14.Our results showed that the percentage of CRALBP+cells among the hiPSC-RPE cells reached 97.4%.

Figure 6.Seeding hiPSC-RPE cell spheroids on decellularized corneal matrices（DCM）.A：bright-field images of hiPSC-RPE cell spheroids on DCM on days 0，7 and 14（scale bar=200 μm）；B：Calcein AM and EthD-III double staining of hiPSCRPE cell spheroids on DCM on days 0，7 and 14（scale bar=200 μm）；C：the double staining of occludin+Mitf or ZO-1+RPE-65 in hiPSC-RPE cell spheroids on DCM on day 14（scale bar=100 μm）；D：detection of senescence-associated βgalactosidase（SA-β-Gal）staining in spheroid or conventionally cultured hiPSC-RPE cells on days 7 and 14（scale bar=100 μm）；E：quantification of SA-β-Gal staining with ImageJ software；F：the expression of nestin in spheroid or conven?tionally cultured hiPSC-RPE on day 14.Mean±SEM. n=3.**P＜0.01 vs conventional hiPSC-RPE.

Figure 7.Injectability of hiPSC-RPE cell spheroids in vivo.A：hiPSC-RPE cell spheroids were labeled with PKH26（scale bar=100 μm）；B：the external approach to transplantation of hiPSC-RPE cell spheroids into the subretinal space of Chinchilla rabbits（arrow represents the transplantation position）；C：a schematic diagram of the external approach to transplantation of hiPSC-RPE cell spheroids into the location between Bruch's membrane and original RPE cells.

Usually，an RPE cell subpopulation with diver?gent morphology is partially separated by selective tryp?sinization of calcium-dependent adhesion.In our study，RPE-like cells were digested using 0.25%EDTA instead of trypsin to reduce any damage to their calcium-dependent adhesion.Generally，RPE cells tends to undergo morphological changes after passaging due to epithelial-mesenchymal transition（EMT）［19］.The tight junction of cells is vital to maintain epithelial morphology and restrain EMT occurrence.One of the most important properties of RPE cells is the barrier function created by tight junctions.Tight junctions en?circle each cell to form the occluding seal monolayers that retard diffusion across the paracellular space.In this study，hiPSC-RPE cells retained their epithelial morphology and barrier function as shown by Matrigel and activin-A treatment based on our previous study.Their morphological features showed that the hiPSCRPE cells were similar to hRPE cells but not hiPSCs.In addition，the hiPSC-RPE cells expressed ZO-1 in re?lation to tight junctions.This polarized expression of ZO-1 was in accordance with previous reports.The hiPSC-RPE cells had similar functions to hRPE cells in term of barrier and phagocytosis activity.Taken to?gether，the hiPSC-RPE cells in our study possessed normal morphological features and functions like RPE cells.Other researchers also demonstrated that iPSRPE cells exhibited ion transport，membrane potential and gene expression patterns similar to native RPEs cells.

Recent advances in cell culture research show that 3D cultures bridge the gap between cell cultureex vivoand live tissuein vivo.Cells cultured in the 3D environ?ment generate many differences in cell behaviors and characteristics compared with those in the conventional 2D environment［20］.Cell-to-cell and cell-to-matrix inter?actions are maintained，and the stemness of progenitor cells is enhanced in the 3D spheroid microenviron?ment.The 3D spheroid cultures form distinct extracel?lular matrix（ECM）and establish new ECM interac?tions to improve cell viability and influence cell fate［21］.Bayoussef et al［8］documented that spheroid muscle cells exhibited higher proliferation capacity.

Figure 8.Preliminary test of hiPSC-RPE cell spheroids in vivo. A：HE staining of paraffin sections after 1 month of hiPSC-RPE cell spheroid injection in vivo（arrows represent RPE layer；scale bar=50 μm）；B：immunohistochemistry of paraffin sec?tions after 1 month of hiPSC-RPE cell spheroid injection in vivo（white arrow represents RPE cells layer in control group，NaIO3group and NaIO3+PBS group；green arrow represents AHNA positive of partial hiPSC-RPE sheets in NaIO3+hiPSCRPE group；red arrow represents partial hiPSC-RPE sheets labeled PKH26 in NaIO3+hiPSC-RPE group；scale bar=100 μm）.

There are many approaches to generating cell spheroids，including centrifugation，pellet culture，hanging drop culture，spinner or rotary dynamic culture systems，and so on.Hanging drop or pipetting cells in?to multiwell plates are time consuming and laborious ap?proaches.The generation of cell spheroids in a suspen?sion culture in non-adhesive culture vessels results in lack of homogeneity.Here，we reported another method to produce hiPSC-RPE cell spheroids using commercial?ly available agarose multiwell dishes.Cell spheroids with the uniform size were quickly formed at the bottom of the non-adhesive agarose microwells.The agarose multiwell dishes easily allowed for the simultaneous generation of hundreds of homogenous hiPSC-RPE cell spheroids.

The progenies derived from cell spheroids showed only limited staining for the senescence marker SA-β-Gal，had a regular shape，and grew at a higher density compared with passaged CECs［22］.Short-term spheroid formation of adipose-derived stem cells（ASCs）before monolayer culture enhanced their properties of stem?ness，angiogenesis，and chemotaxis.The ASC-derived cell spheroids exhibited higher expansion efficiency with less senescence including SA-β-Gal staining and p21 expression［23］.In our study，SA-β-Gal staining as?says showed that spheroid cultures reduced cell senes?cence in hiPSC-RPE cells，and hiPSC-RPE cell spheroids positively expressed nestin，a major interme?diate filament protein of embryonic central nervous sys?tem progenitor cells.Expression of "progenitor" marker nestin revealed a distinct molecular reprogramming of the cells into a stem cell state.

The polarized monolayer RPE cell has a basal side adherent to the Bruch's membrane（BM），which con?sists of 2―4 μm acellular ECM complexes among which collagen forms a major component.The collagenous layer is composed of collagen type I，III，and V fibrils.The collagen grid is embedded in a mass of interacting biomolecules，such as glycosaminoglycans（chondroi?tin sulfate，dermatan sulfate，and hyaluronic acid）and components of the coagulation and complement system.To better observe the monolayer growth of hiPSC-RPE cell spheroidsin vitro，we used DCM as a BM substi?tute.DCM has similar structure to BM，but the former is more convenient to manipulate and observe.DCM is also comprised of aligned arrays of hydrated collagen type I/V fibrils，glycosaminoglycan，keratin sulfate，and dermatan sulfate.An experiment of cell spheroids seeded on the DCMin vitrowas used to assess the state of cells，which might offer some useful experiences for transplantationin vivo.

Huang et al［7］reported that cells from DP spheroids were able to maintain their structural integri?ty and cellular viability after anin vitrosimulation ex?periment of the injectability of DP spheroids on culture plates.As a natural ECM biomaterial，DCM is easier to obtain and is more suitable for observing cell attach?ment and migration.Our results showed that hiPSCRPE cells migrated from the periphery of adherent cell spheroids and grew into the confluent monolayer on the DCMin vitroover 14 d when incubated with the ROCK inhibitor Y-27632.The hiPSC-RPE cell spheroids maintained high cell viability for transplantation.Y-27632 enhanced cell adhesion，motility and prolifera?tion of human CEC spheroids.Y-27632 treatment also allowed prostate colony cells to re-plate efficiently，in?creased the cloning efficiency of prostate stem cells in a prostate sphere assay，and suppressed cell dissociationinduced apoptosis.Our previous study also confirmed that Y-27632 decreased cell death inside spheroids，en?hanced cell proliferation in the spheroid periphery，and promoted the monolayer growth of injectable B-CEC spheroids.

There are internal and external approaches for sub?retinal RPE cell transplantation.Dorey et al reported that the internal technique was more precise while the external approach consistently resulted in a higher per?centage of transplanted cells on Bruch's membrane.Moreover，subretinal injection of hiPSC-RPE cells by the external approach was relatively easier and safer compared with the internal approach.Therefore，we chose subretinal injection of hiPSC-RPE cells through an external approachin vivostudy.

Schwartz et al［1］provided the first description of hESC-RPE transplanted into 2 patients with macular de?generation，which showed that hESC-RPE had no signs of hyperproliferation，tumorigenicity，ectopic tissue for?mation，or apparent rejection after 4 months.Schwartz et al［24］further reported that hESC-RPE was trans?planted into 9 patients with Stargardt's macular dystro?phy and 9 AMD patients for 22 months，suggesting that hESC-RPE transplantation improved patients' vision and had no serious ocular or systemic safety issues.Sig?nificantly，in 2017，Mandai et al［25］firstly performed iPS-RPE-based autologous cell transplantation for treat?ment of neovascular AMD in a patient，and no serious adverse events were noted after 1 year of follow-up.In addition，Westenskow et al［26］reported that the iPSRPE grafts remained viable and did not induce any ob?vious pathological changes in rats.One month after transplantation of hESC-derived retinal progenitors into the subretinal space of NaIO3-induced RPE degenera?tion adult rabbits，transplanted retinal cells survived，migrated into retinal layers，and restored a small but significant B-wave of ERG testing.

In our study，1 month after injection of hiPSCRPE cell spheroids into the subretinal space of RPE de?generation Chinchilla rabbits with NaIO3treatment，a segmental sheet of RPE cells was observed in the cell transplantation eyeballs.Other studies showed that mono-and multilayer RPEs were formed after injection of RPE micro-aggregates into the subretinal space，and apoptotic cells were rarely seen.In contrast to trans?plantation of dissociated cells，micro-aggregate trans?plantation developed in small patches in the outer seg?ment of the neural retina，and these transplanted cells survived and maintained their outer segments over the long term.Since the subretinal space is an immuneprivileged site，the post-transplantation hiPS-RPE cell spheroids could survive at least 1 month without ap?parently systemic immunosuppression in our model.In this study，our results showed that hiPSC-RPE cell spheroids tagged with PKH26 staining survived and maintained segmental sheet growth in the subretinal space.However，there are still some limitations that no comparison of the differences between hiPSC-RPE cell spheroid and hiPSC-RPE cell suspension transplanta?tion was conducted.In addition，we didn't perform op?tical coherence tomography imaging or assess retinal function after hiPSC-RPE cell spheroid transplantation.

In conclusion，our study suggested that non-colo?ny dissociated hiPSCs were effectively differentiated in?to functional RPE cells through the sequential adding defined factors but not bringing in exogenous genes.hiPSC-RPE cell spheroids maintained adherent mono?layer growth on the DCM scaffoldin vitroand segmental sheet growth in the subretinal space of RPE degenera?tion Chinchilla rabbitsin vivo.The preliminary hiPSCRPE cell spheroid transplantation may provide an alter?native cell transplantation method for the treatment of RPE degenerative diseases in the future.