Protocols and Manufacturing for Cell-Based Therapies

Defined Culture of Human Embryonic Stem Cells andXeno-Free Derivation of Retinal Pigmented EpithelialCells on a Novel Synthetic Substrate

BRITNEY O PENNINGTONa DENNIS O CLEGGab ZARA K MELKOUMIANc SHERRY T HIKITAad

Key Words Human embryonic stem cells x Retinal pigmented epithelium xSynthemax II-SC substrate x Age-related macular degeneration x Parylene-C

ABSTRACT

Age-related macular degeneration (AMD) a leading cause of blindness is characterized by thedeath of the retinal pigmented epithelium (RPE) which is a monolayer posterior to the retina thatsupports the photoreceptors Human embryonic stem cells (hESCs) can generate an unlimitedsource of RPE for cellular therapies and clinical trials have been initiated However protocolsfor RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferredfor clinical translation This avoids exposing AMD patients to animal-derived products which couldincite an immune response In this study we investigated the maintenance of hESCs and their differ-entiation into RPE using Synthemax II-SC which is a novel synthetic animal-derived component-freeRGD peptide-containing copolymer compliant with goodmanufacturing practices designed for xeno-free stem cell culture Cells on Synthemax II-SC were compared with cultures grownwith xenogeneicand xeno-free control substrates This report demonstrates that Synthemax II-SC supports long-termculture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPEExpression of RPE-specific markers was assessed by flow cytometry quantitative polymerase chainreaction and immunocytochemistry and RPE function was determined by phagocytosis of rod outersegments and secretion of pigment epithelium-derived factor Both hESCs and hESC-RPEmaintained nor-mal karyotypes after long-term culture on Synthemax II-SC Furthermore RPE generated on SynthemaxII-SC are functional when seeded onto parylene-C scaffolds designed for clinical use These experimentssuggest that Synthemax II-SC is a suitable defined substrate for hESC culture and the xeno-free derivationof RPE for cellular therapies STEM CELLS TRANSLATIONAL MEDICINE 20154165ndash177

INTRODUCTION

Age-related macular degeneration (AMD) is theleading cause of blindness in people aged 65and older in industrialized countries accountingfor 87 of the worldrsquos blind population and itis projected that annual costs to the US federalgovernment will exceed $845 million by 2021[1ndash3] The early stages of this disease involvethe dysfunction of the retinal pigmented epithe-lium (RPE) The RPE maintains the health of thephotoreceptors through phagocytosis of shedouter segments transporting and secreting ionsand growth factors across the blood-retina bar-rier isomerizing all-trans- to 11-cis-retinal to per-petuate the visual cycle absorbing stray light andlimiting oxidation in the eye [4ndash6] AMD mani-fests in two types Exudative or ldquowetrdquo AMD ischaracterized by choroidal neovascularization in-to the retina and can bemitigated by palliative in-traocular injections of angiogenesis inhibitorssuch as Avastin Lucentis or EYLEA However90 of AMD patients suffer from the ldquodryrdquo formwhich can lead to the geographic atrophy of the

RPE and photoreceptors in the macular regionof the retina [7] As AMD progresses the RPE de-grade causing thephotoreceptors to deterioratewhich leads to an incapacitating loss of vision [8]

Currently clinical studies are investigatingthe efficacy of transplanting RPE generatedfrom human embryonic stem cells (hESC-RPE)and induced pluripotent stem cells (iPSC-RPE)as a means to preserve vision in AMD patients[9ndash11] These pluripotent stem cell-derived RPEhave alreadydemonstrated visual rescue in an an-imalmodel of retinal degeneration the Royal Col-lege of Surgeons (RCS) rat [12 13] One approachto deliver these cells is to inject a bolus suspen-sion Subjects receiving hESC-RPE suspensionsin phase 12 clinical trials for AMDand Stargardtrsquosmacular dystrophy have not experienced adversesafety issues from the transplanted cells whereascomplications caused by surgery and immuno-suppression have been observed At the median22-month follow-up half of the patients havedemonstrated visual improvement [9 14] How-ever diseased retinas among patients will displayvarying degrees of degeneration and because

aCenter for Stem Cell Biologyand EngineeringNeuroscience ResearchInstitute BiomolecularScience and EngineeringProgram and bDepartment ofMolecular Cellular andDevelopmental BiologyUniversity of California SantaBarbara California USAcCorning Life SciencesDevelopment Corning IncCorning New York USAdAsterias BiotherapeuticsInc Menlo Park CaliforniaUSA

Correspondence Dennis OClegg PhD NeuroscienceResearch Institute University ofCalifornia Santa BarbaraCalifornia 93106 USATelephone 805-893-8490E-Mail clegglifesciucsbedu

Received August 28 2014accepted for publicationDecember 3 2014 first publishedonline in SCTM EXPRESS January15 2015

copyAlphaMed Press1066-50992015$20000

httpdxdoiorg105966sctm2014-0179

STEM CELLS TRANSLATIONAL MEDICINE 20154165ndash177 wwwStemCellsTMcom copyAlphaMed Press 2015

PROTOCOLS AND MANUFACTURING FOR CELL-BASEDTHERAPIES

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a suspension of RPE cells is extremely dissimilar to the endoge-nous organization it is difficult to predict the efficacy of bolusinjections as a therapy for the entire AMD population Anotherapproachundergoing clinical trials is to transplant a sheet consist-ing of autologous iPSC-RPE [11] Sheets of iPSC-RPE express RPEmarkers and demonstrate function in vitro and in RCS rats Fur-thermore transplantation of autologous nonhuman primateiPSC-RPE sheets does not invoke an immune response [11] Thestructural integrity of the sheet and its proper orientation aftersurgery in human patients is currently under investigation Alter-natively other groups are progressing to clinical trials using trans-plantable scaffolds which provide a solid surface to promotea polarized RPE monolayer enable site-specific delivery of thetherapeutic cells and confer support in a diseased environment[15 16] To this end ultrathin parylene-C scaffolds have beenengineered to provide a transplantable semipermeable surfaceto support hESC-RPE therapeutic cells in the subretinal space[17 18]

Parylene-C is a biostable polymer and its deposition results ina conformal coat (ie a surface without pinholes) [19 20] It hasa range of biomedical applications such as coating stents andpacemakers [20] and the circuitry of retinal prostheses [21 22]Established lithography techniques allow patterning ofparylene-C into an array of ultrathin regions (sim03 mm) thatpermit diffusion of biomolecules These submicron regions aresupported by a thicker meshwork of parylene-C (sim60 mm) thatprovidesmechanical support [23] Parylene-Cmaybe coatedwithfull-length human vitronectin to allow attachment polarizationand pigmentation of hESC-RPE [17]

Methods to culture and differentiate pluripotent stem cellsinto RPE often use xenogeneic mouse embryonic fibroblasts orMatrigel a substrate derived from the Engelbreth-Holm-Swarmmurine sarcoma [9 11ndash13 24] These conditions present a possi-bility of exposing therapeutic cells to animal-derived immuno-gens viruses and other undefined components such as growthfactors collagenases and plasminogen activators [25ndash27] Re-cent efforts to derive hESC-RPE without using nonhuman animalproducts have cocultured hESCs with human foreskin fibroblastsand substrates comprised of full-length human proteins [28ndash30]However because of the natural variability among fibroblast celllines or batches of purified proteins synthetic substrates may bemore desirable for consistent generation of clinical-grade thera-pies Synthemax-R is a synthetic animal-derived component-free(ACF) substrate designed for cell culture and is composed of a bi-ologically active peptide derived from the human extracellularmatrix (ECM) protein vitronectin that is covalently conjugatedto the tissue culture vessel by an acrylate moiety [31] ACF con-ditions donot use any animal-derived products including humanand this distinguishes it from xeno-free conditions Synthemax-Rhas been shown to support hESC and iPSC growth [31ndash37] andtheir differentiation into ocular cell types [32 37 38] HoweverSynthemax-R is only available in limited styles of tissue cultureplates thus restricting its range of applications and its ability toscale up the manufacture of therapeutic cells Synthemax-II SCSubstrate is a novel ACF good manufacturing practice (GMP)-compliant peptide-copolymer that self-adsorbs onto tissue cul-ture plastic or glass surfaces This feature imparts versatilitywhenscaling up production of therapeutic cells and facilitates a widerarray of characterization assays The Synthemax II-SC peptideincludes the RGD-containing sequence from the human ECMprotein vitronectin KGGPQVTRGDVFTMP which promotes

adhesion in a variety of cells [31 39 40] Synthemax II-SC differsfrom Synthemax-R in its coating chemistry and peptide densitySynthemax II-SC is a lyophilized powder that can be resus-pended to a desired concentration and its peptide noncova-lently adsorbs to a surface The covalently bound peptide onthe Synthemax-R surface ismore dense (8ndash12 pmolmm2) thanthe recommended coating density for Synthemax II-SC (5ndash10 pmolmm2) [31] Synthemax II-SC is also less expensivea six-well plate at the recommended coating density is less thanhalf the cost of a Synthemax-R plate

To date Synthemax II-SC has been shown to support humaniPSCs and humanmesenchymal stem cells [41 42] Here we val-idate Synthemax II-SC as a viable substrate for the defined cul-ture of hESCs and hESC-RPE We have found that pluripotent H9and H14 hESC lines can be maintained on Synthemax II-SC andretain a normal karyotype for more than 16 passages Theselines differentiate into hESC-RPEwith a significantly higher yieldof pigmented area than hESCs maintained on Synthemax-R Sur-face Also hESC-RPE cultured on Synthemax II-SC are similar tohESC-RPE derived onMatrigel and Synthemax-R with respect togene expression and function Furthermore hESC-RPE derivedon Synthemax II-SC retain RPE identity and function whenseeded onto transplantable parylene-C scaffolds

MATERIALS AND METHODS

hESC Culture

Cultures of hESC lines H9 andH14 (WiCell Research InstituteMad-ison WI httpwwwwicellorg) were maintained on MatrigelhESC-qualified matrix (catalog no 354277 Corning EnterprisesCorning NY httpwwwcorningcom) in mTeSR1 (catalog no05850 StemCell Technologies Vancouver BC Canada httpwwwstemcellcom) Stem cell colonies were manually passagedby hand every 4ndash7 days differentiated regionswere removed firstfollowed by a manual dissection of pluripotent colonies To com-pare hESCs grown on Synthemax II-SC Substrate with other com-mercially available substrates hESCs were passaged onto tissueculture-treated plates coated with Corning Matrigel CorningSynthemax-R Surface (catalog nos 3979 3984 3977XX1) and Syn-themax II-SC Substrate (catalog no 3536-XX1 lot no DEV45-10Corning Inc Corning NY httpwwwcorningcom) andwere seri-ally passaged bymanual dissection Similar number and size of col-onies were chosen for propagation on the three surfaces hESCsbetween passages 40 and 49 were used for characterization Fordifferentiation intogerm layershESCsonSynthemax II-SCweredif-ferentiated for 10 days in Dulbeccorsquos Modified Eaglersquos MediumF-12 + GlutaMAX I 20 knockout serum replacement 13 nones-sential amino acids and 01 mM b-mercaptoethanol (all reagentsfrom Life Technologies Carlsbad CA httpwwwlifetechcom)

Differentiation Enrichment and Culture of hESC-RPE

A methods schematic is provided in supplemental onlineFigure 1 Pluripotent hESCs were spontaneously differentiatedon the substrates of interest for 115 days in XVIVO10 medium(catalog no 04-743Q Lonza Walkersville MD httpwwwlonzacom) with Normocin (catalog no ant-nr-1 InvivogenSan Diego CA httpwwwinvivogencom) Pigmented cellswere enriched by treatment with TrypLE Select (catalog no12563-011 Gibco Grand Island NY httpwwwlifetechnologiescom) followed by manual removal of nonpigmented cells The

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remaining RPE were dissociated with TrypLE Select for 5minutes at 37degC passed through a 40-mm nylon cell strainerand seeded on the substrates of interest at 15 3 105 cells percm2 RPE culturesweremaintained in XVIVO10mediumandpas-saged with TrypLE Select every 28 days for 3months and seededat 10 3 105 cells per cm2 onto Matrigel Synthemax-R andSynthemax II-SC Substrate (catalog no 3536-XX1 lot nos28512016 14413006 22413018) Cells at passage 2 day 28wereused for characterization The ldquoMatrigelrdquo ldquoSynthemax-Rrdquo andldquoSynthemax-IIrdquo designations describe the condition of hESCspropagated differentiated enriched and passaged as RPEsolely on the Matrigel Synthemax-R and Synthemax II-SC sub-strates respectively ldquoMg-SRrdquodenotes the condition of undiffer-entiated hESCs grown and differentiated on Matrigel and thenenriched and grown as RPE on Synthemax-R as described previ-ously [18 43]

Human Fetal RPE Culture

Human fetal RPE (fRPE) were a kind gift of Dean Bok (University ofCalifornia LosAngeles) andLincoln Johnson (Center for theStudyofMacular Degeneration University of California Santa Barbara)fRPEweremaintained inMillermedium [44]a-modificationmini-mum essential medium (M4526 Sigma-Aldrich St Louis MOhttpwwwsigmaaldrichcom) supplemented with nonessentialamino acids (catalog no 11140-050 Gibco) GlutaMAX I (35050Life Technologies) N1 supplement (N6530 Sigma-Aldrich) 1 mlof THT per 500 ml of medium (00065 mgml triiodothyronineT5516 Sigma-Aldrich 10 mgml hydrocortisone Sigma-AldrichH0396 Sigma-Aldrich 125 mgml taurine T0625 Sigma-Aldrich)and heat-inactivated fetal bovine serum (15 for seeding fRPE5 for fRPE maintenance) (F-0500A Atlas Biologicals Fort CollinsCO httpwwwatlasbiocom)

Synthemax II-SC Substrate Vessel Coating

Tissue culture vessels were coated with Synthemax II-SC accord-ing to the manufacturerrsquos recommendations Briefly SynthemaxII-SC powderwas resuspended to 1mgml inHyClone HyPure CellCulture Grade Water (AWK21536) and either stored at 4degC for 1monthor further diluted 140 (0025mgml) to coat tissue culturevessels (CellBIND Surface catalog no 3335 Corning cell cultureplates and flasks catalog nos 3516 3603 430641 431082 Corn-ing Costar Acton MA httpwwwcorningcomlifesciences)Millicell-HA inserts (PIHA01250 Millipore Billerica MA httpwwwemdmilliporecom) and Lab-Tek PermanoxChamber Slides(catalog no 177445 Thermo Fisher Scientific Waltham MAhttpwwwthermofishercom) at 5 mgcm2 for 2 hours at roomtemperature The remaining Synthemax II-SC solution was aspi-rated and the air-dried vessels were used immediately or storedat 4degC for up to 3 months

Immunocytochemistry

Cells were rinsed with warm Dulbeccorsquos phosphate-bufferedsaline (DPBS) (catalog no 14190-144 Gibco) and fixed with anaqueous solution of 40 paraformaldehyde (catalog no15710 Electron Microscopy Sciences Hatfield PA httpwwwemsdiasumcommicroscopy) and 01 M sodium cacody-late buffer (catalog no 11652 Electron Microscopy Sciences)for 5 minutes at 4degC Fixed cells were blocked with 1 bovineserum albumin (BSA) (catalog no 15260 Gibco) 1 goat serum(catalog no G9023 Sigma-Aldrich) and 01 Triton X-100

(11332481001 Roche Indianapolis IN httpwwwrochecom) in phosphate-buffered saline (PBS) for 1 hour at 4degC Pri-mary antibodies diluted in block solutionwere applied overnightat 4degC (supplemental online Table 1) Cells were rinsed threetimes with PBS and secondary antibodies were diluted 1300 inblock solution and applied for 30 minutes at 4degC (supplementalonline Table 1) The cells were incubated with Hoechst nuclearstain (8mgml 33258 Sigma-Aldrich) for 5minutes at room tem-perature in the dark and rinsed three times with PBS Slides weremounted with Pro-Long Gold Antifade Reagent (catalog noP36930 Life Technologies) and imaged with QCapture Pro soft-ware on Olympus IX71 or BX51 fluorescence microscopes (Olym-pus Center Valley PA httpwwwolympusamericacom) Best1localization was assessed with Fluoview software on an OlympusFV1000 confocal scanning microscope

Quantitative Polymerase Chain Reaction

For RNA collection whole wells of hESCs ready for passage wereharvested from each substrate differentiated regions were notremoved prior to harvest RNA was harvested using the RNeasyPlus kit (catalog no 74136 Qiagen Valencia CA httpwwwqiagencom) and converted to cDNA (iScript cDNA Synthesis kitcatalog no 170-8891 Bio-Rad Hercules CA httpwwwbio-radcom) For hESC-RPE 153 105 cells were collected at passage2 day 28 and processed with the Cells-to-Ct kit (catalog no4399002 Ambion Austin TX httpwwwambioncom) Real-time quantitative polymerase chain reaction (qPCR) was com-pleted with TaqMan Gene Expression Assays (catalog no4351372 Life Technologies supplemental online Table 2) inTaqMan Gene Expression Master Mix (catalog no 4369016 LifeTechnologies) using a Bio-Rad CFX96 real-time system The datawere analyzedwith Bio-Rad CFXManager software Gene expres-sion was normalized using the geometric mean of expression ofthe housekeeping genes SERF2 UBE2R2 and EIF2B2

Flow Cytometry

Whole wells of hESCs ready for passage were harvested fromeach substrate differentiated regions were not removed priorto harvest hESCs were dissociated by 5- to 13-minute incuba-tions in TrypLE Select at 37degC and diluted 15 in fresh mediumFor hESC-RPE 1 3 106 cells were collected at passage 2 day28 For fixation cells were centrifuged at 1500 rpm for 3minutes washed in 05 BSA Fraction V (catalog no 15260-037 Gibco) in PBS and fixed in 4 paraformaldehyde in PBSfor 20 minutes at room temperature in the dark The cells werepelleted at 2300 rpm for 3minutes andpermeabilizedwith 02Triton X-100 (11332481001 Roche) 01 BSA Fraction V in PBSfor 3 minutes at room temperature Fixed cells were stored at4degC in 05 BSA until stained

Cells were stained with primary antibodies (supplementalonline Table 1) diluted in 3 BSA in PBS for 30 minutes at 4degC(hESCs) or room temperature (RPE) Samples were stained witha secondary antibody (Alexa Fluor 488 A21202 Life Technolo-gies) for 30 minutes at room temperature Cells were rinsedwith 05 BSA and data were collected on a BD AccuriC6 FlowCytometer with 10000 gated events per sample Gates wereset to exclude 990 of the population stained with the isotypecontrol of the primary antibody The data were analyzed withFCS Express (De Novo Software Glendale CA httpwwwdenovosoftwarecom)

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Karyotype

Cells were seeded in a T-25 flask (catalog no 430168 Corning)that was coated with Matrigel or Synthemax II-SC Substrateand shipped in logphaseovernight toCell LineGenetics Inc (Mad-ison WI httpwwwclgeneticscom) for G-band karyotyping

Pigmented Area Quantification

ImageJ was used to quantify pigmented area in the differentiatedhESC cultures Briefly photographs of the differentiated hESC-RPEplates were converted to 8-bit images Scale was set to 500 pixels173 mm for a six-well plate The well area was selected contrast-enhanced background-subtracted converted to binary and ana-lyzed for particle size (size 002ndash10 mm2)

PEDF Enzyme-Linked Immunosorbent Assay

To compare secretion of pigment epithelium-derived factor(PEDF) hESC-RPE derived on different substrates were seededonto TranswellMillicell-HA inserts (PIHA01250Millipore) coatedwith 263 mgcm2 human vitronectin (354238 BD BioSciencesSan Jose CA httpwwwbdbiosciencescom) or SynthemaxII-SC The cells were maintained in 400 ml of XVIVO10 withinthe insert and 600 ml in the well Apical and basal supernatantswere collected on passage 2 day 30 72 hours post-media changesnap-frozen in liquid nitrogen and storedat280degC SupernatantsfromhESC-RPE on parylene-Cmembraneswere collected 30 daysafter thawseed 24 hours postmedia change Supernatantswerediluted 15000 concentration of PEDF was determined with thePEDF enzyme-linked immunosorbent assay kit (catalog noPED613-Human Bioproducts MD Middletown MD httpwwwbioproductsmdcom) according to the manufacturerrsquosinstructions Optical density was measured at 450 nm

ROS Phagocytosis

The RodOuter Segment (ROS) phagocytosis assaywas performedas previously described [45] Briefly hESC-RPE human fRPE andARPE19 cells were seeded in quadruplicate onto gelatin Synthe-max II-SC or human vitronectin-coated 96-well plates at 13 105

cells per cm2 and maintained in Miller medium Rod outer seg-ments were isolated from fresh bovine retinas labeled with fluo-rescein isothiocyanate (FluoReporter fluorescein isothiocyanate[FITC] protein labeling kit catalog no F6434 Life Technologies)and resuspended in 25 sucrose in Miller medium Four weeksafter plating confluent cells were incubated with 13 106 labeledROS with or without 625mgml of the anti-avb5 function block-ing antibody (catalog no ab24694 Abcam Cambridge UKhttpwwwabcamcom) or the isotype control (catalog noab18447 Abcam) for 5 hours at 37degC in 5 CO2 Unbound ROSwere removed with six rinses of warm 25 sucrose in PBS andextracellular fluorescence was quenched with 04 trypan blue(catalog no 25-900Cl Corning) for 20 minutes at 37degC Trypanblue was replaced with 40 ml of PBS-sucrose and internalizedfluorescence was imaged with an Olympus IX71 ImageJ softwarewas used to calculate the internalized fluorescence intensity bypixel densitometry Fluorescent signals from each conditionwerenormalized to the ARPE19 signal

hESC-RPE on Parylene-C

Mesh-supported submicron parylene-C scaffolds were manufac-tured as previously described resulting in an array of ultrathin

regions (sim003 mm) supported by a thicker meshwork (sim6 mm)[17] These scaffolds were coated with human vitronectin ina 48-well plate Briefly scaffoldswereplaced in a 48well platewithTeflon forceps soaked in DPBS for 15 minutes air-dried for 48hours at room temperature secured to the bottom of the platewith a cloning cylinder (CLS316610 Sigma-Aldrich) and coatedwith 250 ml of human vitronectin (10 mgml) for 2 hours at roomtemperature Enriched hESC-RPE were cryopreserved and storedin liquid nitrogen (C Presbey M Tsie S Hikita et al manuscriptin preparation) The cells were thawed and seeded ontovitronectin-coated parylene-C scaffolds at 153 105 cells per welland grown for 30 days in XVIVO10 medium without antibiotics

Statistical Analysis

The two-tailed t test was used to determine the statistical signif-icance of the differences between the amount of secreted PEDFby hESC-RPE on parylene-C membranes The one-tailed t testwas used to determine the statistical significance betweenthe differences of internalized ROS fluorescence by hESC-RPEtreated with avb5 function blocking antibody versus isotypecontrol The one-tailed t testwas also used to compare secretionof apical versus basal PEDF of hESC-RPE cultured on inserts Thelevel ofp value thatwas considered significant has beendenotedas pp for p 01 and p for p 05

RESULTS

Synthemax II-SC Supports Long-Term Culture of H9 andH14 hESCs

Matrigel is one of the most widely used feeder-free substratesfor hESC culture [24 46] Therefore morphology and gene ex-pression of hESCs cultured on Synthemax II-SC were comparedwith hESCs on Matrigel Comparisons were also made to hESCsmaintained on Synthemax-R to determine whether SynthemaxII-SC is an acceptable alternative ACF substrate

Overall hESC colonies cultured on Synthemax II-SC ex-hibited morphology similar to hESCs grown on Matrigel char-acterized by a homogenous layer of small tightly packed cellsand a distinct colony border Colonies on Synthemax II-SC qual-itatively appeared larger than those grown on Synthemax-R butappeared to have thicker centers than the colonies grown onMatrigel Colonies on Synthemax-R generally appeared smallerand thicker than those on the other two substrates and seemedto manifest more regions with differentiated morphologythroughout the nine passages (Fig 1A supplemental onlineFig 2 arrowheads) Qualitatively hESC colonies were easierto passage on Synthemax II-SC than Synthemax-R because col-onies on the latter substrate remained small and developedcompact differentiated centers rendering the efficient passag-ing of undifferentiated cells difficult

Introducing hESCs to new culture conditions has been shownto elicit various fluctuations inmorphology and an increase in dif-ferentiation while the cells adapt to the new environment [4748] Previous studies have reported that hESCs undergo an adap-tion periodonce transferred to various feeder free substrates [4950] H9 and H14 hESCs transitioning to Synthemax II-SC at pas-sages2and4 respectivelymanifesteddensedifferentiated areasharboring small regions devoid of cells within several colonies(supplemental online Fig 2A 2C arrows) This extent of differen-tiation was not observed in other passages However it is known

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Figure 1 Characterization of human embryonic stem cells (hESCs) cultured on Synthemax II-SC Synthemax-R and Matrigel (A) Represen-tative phase-contrast micrographs of H9 and H14 hESC colonymorphology after nine passages on the indicated substrate Scale bars = 200mm(B) Representative flow cytometry histograms for pluripotency markers Oct4 and SSEA4 in H9 cultures grown on the indicated substrate atpassages 1 3 5 and 9 (C) Relative expression of pluripotency genes in H9 cultures grown on Synthemax II-SC Synthemax-R and Matrigelat passages 1 3 5 and 9 as detected by quantitative polymerase chain reaction (D)Normal karyotype of H9 hESCs maintained on SynthemaxII-SC for 23 passages (E)Relative gene expression of germ layermarkers is significantly higher in H9hESCs differentiated on Synthemax II-SC for10 days compared with hESCs at earlier passages pp p 001 t test (F) H9 hESCs on Synthemax II-SC stained for nuclear pluripotency tran-scription factors Oct4 and Sall4 and surface markers Tra-1-81 and Tra-1-60 Scale bars = 80 mm Abbreviation Diff differentiation

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that healthy hESC cultures will experience a slight degree of dif-ferentiation in every passage and some percentage of differen-tiation is expected [51] In this regard hESCs on Matrigelconsistently demonstrated the least amount of differentiatedmorphology throughout nine passages Because the original cul-tures for these experiments were previously adapted toMatrigelthe minimal differentiation observed in these cells is expectedcompared with the hESCs transitioning to a new substrate Cul-tures of hESCs on Synthemax II-SC and Synthemax-R generallyappeared slightly more differentiated than cells on Matrigel(supplemental online Fig 2 arrowheads)

To investigate the percentage of cells expressing pluripotencymarkers Oct4 and SSEA4 flow cytometry was carried out on cul-tures at passages 1 3 5 and 9 (Fig 1B supplemental online Fig3A) Although hESCs onMatrigel consistently expressed both plu-ripotency markers a decrease in Oct4 expression was observedon both xeno-free surfaces at some passages Expression ofSSEA4 however was consistent on all three substrates through-out these passages Additional markers of undifferentiated cellswere examined by qPCR and immunocytochemistry By passage7 hESCs on Synthemax II-SC andMatrigel expressed similar levelsof pluripotency-associated mRNAs (Fig 1C supplemental onlineFig 3B) and had similar colonymorphologies Throughout all pas-sages hESCs on Synthemax II-SC and Matrigel expressed the plu-ripotency surface antigens SSEA4 Tra-1-60 and Tra-1-81 alongwith nuclear hESC transcription factors Oct4 and Sall4 (Fig 1B1F supplemental online Fig 3A 3B) The H9 and H14 hESClines continued to be subcultured for 23 and 16 passages respec-tively and each line maintained a normal karyotype (Fig 1Dsupplemental online Fig 3D)

The differentiation potential of hESCs grown on SynthemaxII-SC was investigated by culturing the cells in a differentiationmedium for 10days These differentiated cells expressedmarkersfrom the three germ layers at significantly higher levels than theundifferentiated cultures (p 01) (Fig 1E supplemental onlineFig 3C) Markers of ectoderm (MAP2 [microtubule-associatedprotein 2]) [34] mesoderm (Tbx6 [T-box protein 6]) [35] and en-doderm (AFP [a-fetoprotein]) [52 53] were detected Taken to-gether thesedatademonstrate that Synthemax II-SC is a suitableACF substrate for the maintenance of hESCs that express canon-ical pluripotencymarkers and can differentiate into cells express-ing markers of all three germ layers

Synthemax II-SC Permits RPE DifferentiationFrom hESCs

Because Synthemax II-SC was found to support the defined cul-ture of hESCs we next investigated whether this substrate alsopermitted efficient xeno-free differentiation of hESCs into RPEH9 and H14 hESC lines adapted to Matrigel Synthemax-R andSynthemax II-SC for seven passageswere spontaneously differen-tiated into RPE by changing the growth medium to XVIVO10a xeno-free formulation lacking basic fibroblast growth factor(Fig 2) During this process pigmented foci appeared after 4weeks and continued to enlarge over the next few months At115 days postdifferentiation the area of pigmentation on eachsubstrate was quantified (Fig 2) The lowest yield of pigmentedarea occurred on Synthemax-R Surprisingly Synthemax II-SCyielded significantly more pigmented area than Synthemax-R(p 01) However there was no difference in the amount ofpigmented area produced onMatrigel and Synthemax II-SC This

suggests that Synthemax II-SC is an acceptable replacementfor Matrigel in the xeno-free production of clinical-grade RPEfrom hESCs

RPE Cultured on Synthemax II-SC Maintain RPE Identity

Pigmented regions produced by spontaneous differentiation(Fig 2) were manually isolated from nonpigmented cells in a pro-cess termed ldquoenrichmentrdquo (supplemental online Fig 1) Protocolshave been developedwhereby hESCs are differentiated to RPE onMatrigel and then enriched onto Synthemax-R (Mg-SR method)[17 18 52ndash54] To investigate whether Synthemax II-SC can en-tirely replaceMatrigel for the growth of hESCs and their differen-tiation into RPE we compared cultures maintained solely onSynthemax II-SC with those produced using the Mg-SR methodSynthemax II-SC hESC-RPE were also compared with cells pro-duced solely on Synthemax-R or Matrigel

After 30 days in culture the hESC-RPE produced on Synthe-max II-SC and Synthemax-R qualitatively appeared darker thanthose produced on Matrigel or by the Mg-SR method (Fig 3A

Figure 2 Spontaneous differentiation of retinal pigmented epithe-lia generated from human embryonic stem cells (hESC-RPE) onMatrigel and Synthemax II-SC yields significantly more pigmentedarea than hESC-RPE on Synthemax-R Top Pigmented area in indi-vidual wells was calculated with ImageJ software after 115 daysof differentiation Error bars denote standard deviation pp p 01t test Bottom Three representative wells from a six-well plateof differentiated hESC-RPE seeded on each substrate are shownScale bar = 1 cm

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Figure3 Characterization of RPE generated fromhumanembryonic stemcells (hESC-RPE) derivedonSynthemax II-SC (A)Phase-contrast andbright-field images show the typical pigmentation and cobblestonemorphology of H9 hESC-RPE cultured on different surfaces are shown Scalebar = 200 mm (B) Representative flow cytometry histograms are shown for the premelanosome pigmentation marker PMEL17 and the plu-ripotencymarker Oct4 (not detected) in H9-RPE at passage 2 day 28 on the indicated substrate (C) Expression of RPEmarker genes in H9 hESC-RPE on the indicated substrates fRPE served as a positive control (n = 3) p p 05 t test (D) Normal karyotype of H9 hESC-RPE afterspontaneous differentiation enrichment and three passages on Synthemax II-SC (E) Epifluorescent images are shown of H9 hESC-RPE derivedonSynthemax II-SC stained for the tight junctionmarker ZO-1 RPEmarkersOtx2 andPMEL17 and thepluripotencymarker Sall4 (not detected)Nucleiwere detected usingHoechst (bluemerged right panels) Scale bar = 50mmAbbreviations fRPE fetal retinal pigmentedepitheliumMg-SR condition of culturing and differentiating hESCs onMatrigel and then enriching and propagating the hESC-RPE on Synthemax-R RPE retinalpigmented epithelium

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supplemental online Fig 4A) Expression of three pigmentationmarkers was investigated in each condition Tyrosinase andtyrosinase-related protein-1 (Tyrp1) catalyze reactions in theme-lanogenesis pathway and PMEL17 is a structural protein in pre-melanosomes At passage 2 day 28 H9 hESC-RPE derived onSynthemax II-SC expressed Tyrp1 and PMEL17 similarly to thosederived on the other substrates but expressed significantly moretyrosinase (Tyr) mRNA than RPE on Synthemax-R or fRPE controls(Fig 3C) H14-derived RPE on Synthemax II-SC did not exhibit anydifference in pigmentation marker expression compared withthe other conditions (supplemental online Fig 5A)

All conditions of each line displayed the typical cobblestonemorphology of cultured RPE A few lacunae were observed in a mi-nority of hESC-RPE cultures on Synthemax II-SC at some passages(supplemental online Fig 6A 6B) These may be related to the effi-ciency of enrichment and the effects of paracrine signaling on tightjunctions by non-RPE cells [55] or could be caused by minisculebubbles that form during the Synthemax II-SC coating process thatresults in uneven peptide distribution However the hESC-RPEseemed to expand and fill the lacunaewhen cultured on SynthemaxII-SC for 3 months or longer (supplemental online Fig 6C)

The purity of the RPE population and efficiency of enrich-ment were measured at passage 2 day 28 by assessing the per-centage of cells that express PMEL17 an essential structuralprotein for melanogenesis [56 57] All cultures were more than95 positive for the protein PMEL17 and localization was con-firmed by immunocytochemistry (Fig 3B 3E supplementalonline Fig 4B 4C) There was no significant difference in the ex-pression of RPE65 and RLBP1 transcripts which are involved inthe visual cycle [6 58] nor theRPE chloride channel bestrophin-1 (BEST1) (Fig 3C supplemental online Fig 5A) FurthermoreRPE produced on Synthemax II-SC expressed RPE marker pro-teins with proper localization The RPEmaster transcription fac-tor Otx2 [59 60] was visualized in the nucleus and zonulaoccludens-1 (ZO-1) [6] immunoreactivity was observed at celljunctions consistent with localization to tight junctions (Fig3E) BEST1 localized to the basal surface which demonstratesthat hESC-RPE on Synthemax II-SC achieve a polarized morphol-ogy (supplemental online Fig 7)

One of the primary concerns regarding stem cell-derivedtherapies involves the potential of introducing tumorogenic un-differentiated stem cells into patients [61] Therefore the ex-pression of the pluripotency markers Oct4 Rex1 and Sall4was assessed for all conditions of H9 and H14 hESC-RPE All linestested negative for these embryonic stem cell markers (Fig 3Bsupplemental online Figs 4B 4C 5B 5C) Also the proliferativemarker mKi67 could not be detected in any of the hESC-RPE derived under different conditions which suggested thatthe cells are quiescent and have exited the cell cycle(supplemental online Fig 5B 5C) Furthermore H9 and H14hESCs that were grown differentiated enriched and main-tained as RPE for three passages on Synthemax II-SC retain a nor-mal human karyotype (Fig 3D supplemental online Fig 4D)These data demonstrate that hESC-RPE maintained in xeno-free conditions on Synthemax II-SC retain an RPE identity andare similar to RPE derived using the Mg-SR method

RPE Derived on Synthemax II-SC Are Functional

One of the primary functions of RPE in the retina is to internalizethe shed photoreceptor outer segments by phagocytosis [6 62

63] The phagocytic function of hESC-RPE derived on SynthemaxII-SCwas comparedwith RPE generated on the other substrateshESC-RPE on Synthemax II-SC internalized FITC-labeled ROS atlevels similar to the other hESC-RPE (Fig 4A supplementalonline Fig 4E) In the first steps of ROS phagocytosis integrinavb5 physically binds the ROS which activates focal adhesionkinase to stimulate receptor tyrosine kinase Mer to initiateROS engulfment [62] To test whether phagocytosis occurredvia this pathway function blocking antibodies to integrinavb5 or an isotype control antibody were added with theROS Inhibiting avb5 significantly reduced the amount of inter-nalized ROS compared with the isotype control indicating thathESC-RPE derived on Synthemax II-SC perform phagocytosissimilarly to hESC-RPE derived on Matrigel Synthemax-R orMg-SR (p 01) (Fig 4A supplemental online Fig 4E) Becausevitronectin-coated scaffolds are being developed as cellulartherapies for AMD [17] phagocytic function of hESC-RPE de-rived on Synthemax II-SC was analyzed on human vitronectin-coated plastic hESC-RPE derived under defined xeno-free cul-ture conditions using Synthemax II-SC were capable of ROSphagocytic function when seeded on either vitronectin-coatedsurfaces or on Synthemax II-SC Substrate (p 01) (Fig 4Bsupplemental online Fig 3F)

In addition to phagocytosis RPE are known to secretegrowth factors that support the health of the neural retinaand its structural integrity [6] Among these is the apically se-creted PEDF which protects photoreceptors from ischemiaand light damage [64 65] hESC-RPE derived on Synthemax II-SC secrete significantly more apical PEDF than basal PEDF whenseeded on either xeno-free substrate of human vitronectin orSynthemax II-SC (p 01) However the amount of apical PEDFsecreted by these cells is less than the apical PEDF secreted byhESC-RPE derived on Synthemax-R or by Mg-SR and fRPE (Fig4C) Taken together these data demonstrate that hESC-RPE de-rived under xeno-free conditions on the synthetic Synthemax II-SC surface perform phagocytosis and apically secrete PEDFwhen seeded on defined substrates

H9 hESC-RPE Derived on Synthemax II-SC Maintain RPEIdentity and Function on Parylene-C Scaffolds

Vitronectin-coatedmesh-supported submicronparylene-C scaf-folds have been developed as transplantable surfaces to deliverhESC-RPE produced by the Mg-SR method into AMD patients[17 18 54] To determine whether Synthemax II-SC can be usedas a xeno-free alternative to the Mg-SR method to producehESC-RPE cells from these two conditions were cultured for30 days on vitronectin-coated parylene-C scaffolds No differ-ence in initial attachment was observed and hESC-RPE derivedunder both conditions grew to confluent monolayers with cob-blestonemorphology (Fig 5A) Cells derived on Synthemax II-SCdid not form lacunae when seeded onto vitronectin-coatedparylene-C (n = 8 scafolds) Once again darker pigmentationwas observed in the hESC-RPE cultures generated on SynthemaxII-SC by day 30 (Fig 5A 5B) However there was no significantdifference in the expression levels of genes associated with pig-mentation as measured by qPCR (Fig 5C) The mRNA encodingthe melanocyte-specific Mitf-4 isoform (Mitf-M) was notdetected whereas variant 2 (Mitf-H) which is expressed duringRPE development along with isoforms 1 and 7 (Mitf-A and Mitf-D) was observed (Fig 5C) [66] Although hESC-RPE derived on

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Synthemax II-SC expressed higher levels of RPE marker genesRPE65 Best1 and RLBP1 than the Mg-SR cells this differencewas not significant The Synthemax II-SC hESC-RPE secretedsim18 less PEDF than hESC-RPE derived using theMg-SRmethod(16 vs 196 ngmm2) (Fig 5D) However both amounts are withinthe expected range of PEDF secretion by H9-derived RPE onparylene-C scaffolds (C Hinman D Zhu S Hikita et al manu-script in preparation) Taken together these data suggest thatSynthemax II-SC is a viable alternative to Matrigel for the xeno-free derivation and culture of hESC-RPE on parylene-C scaffolds

DISCUSSION

The advent of using pluripotent stem cell-derived therapies totreat human diseases necessitates good manufacturing practi-ces and preferably defined culture conditions that do notuse xenogeneic products [25] However some of the currentmethods used to produce hESC-RPE use Matrigel a mixture ofECM proteins harvested from the Engelbreth-Holm-Swarmmouse sarcoma [17 18 54] Coculturing therapeutic cellswith xenogeneic derivatives may inadvertently introduce non-human viruses that could potentially inoculate the patientpost-transplantation Synthemax II-SC is a novel ACF GMP-compliant copolymer containing an RGD peptide To date ithas been shown to support the growth of pluripotent humaniPSCs as well as the large scale production of human mesenchy-mal stem cells [41 42] Here we present the first characteriza-tion of hESC cultures and the xeno-free derivation of RPE usingSynthemax II-SC Furthermore we have shown that hESC-RPEmaintained on Synthemax II-SC express RPE markers and se-crete PEDF when seeded onto transplantable parylene-C scaf-folds These data show that Matrigel can be entirelyeliminated from the production of hESC-RPE and replaced withSynthemax II-SC

Qualitatively hESCs on Synthemax II-SC appeared largerthan those grown on Synthemax-R The observed restrictionin colony size on the latter substrate may be due to the slightlyhigher density of peptide on the Synthemax-R Surface (8ndash12pmolmm2) compared with the recommended coating densityfor Synthemax II-SC (5ndash10 pmolmm2) [31] Consistent with thisobservation a previous study has shown that human iPSC colo-nies on Synthemax-Rweremore compact than theMatrigel con-trols [36] It is not uncommon to observe varying sizes of hESCcolonies on various feeder-free substrates as demonstratedby a comparison of Matrigel CELLstart and vitronectin con-ducted by Yoon et al [46]

The compact hESC colonies on Synthemax-R may also ex-plain the significant decrease in the yield of pigmented areaafter spontaneous differentiation For these experiments pig-mentation potential seemed to be augmented by a high conflu-ence of hESCs and a lowpercentage of differentiation at the timeof applying the XVIVO10 differentiation medium Varyingdegrees of culture confluence have previously been implicatedin determining the ultimate fate of differentiated stem cells [6768] This may explain why the apparently smaller colonies onSynthemax-R gave a lower yield of pigmented area after spon-taneous differentiation Perhaps the lower peptide density ofthe Synthemax II-SC substrate permitted hESC colonies to ex-pand more freely and achieve confluence faster than the cellson Synthemax-R This could have augmented the yield of

Figure 4 Functional characterization of H9 RPE generated from hu-man embryonic stem cells (hESC-RPE) derived on Synthemax II-SC(A) Phagocytosis of ROS by H9 hESC-RPE derived on MatrigelSynthemax-R Synthemax II-SC or Mg-SR A function-blocking anti-body against avb5 integrin which is necessary for phagocytosis byRPE significantly decreased internalization of ROS in all hESC-RPEwhen compared with the IgG isotype control pp p 01 t test fRPEserved as a positive control (B) Phagocytosis of ROS by H9 hESC-RPEderived on Synthemax II-SC after seeding on the defined substratesSynthemax II-SC and human vitronectin Phagocytic activity was sig-nificantly decreased with the function-blocking avb5 antibody whencomparedwith the IgG isotype control pp p 01 t test (C)All con-ditions secrete PEDF significantly more on the apical surface com-pared with the basal side when seeded on surfaces coated withhuman vitronectin or Synthemax II-SC pp p 01 t test hESC-RPE derived on Synthemax II-SC secrete less apical PEDF comparedwith fRPE and cells derived on Synthemax-R Abbreviations fRPE fe-tal retinal pigmented epithelium HuVn human vitronectin ROS rodouter segment S2 hESC-RPE derived on Synthemax II-SC Mg-SRhESCs grown and differentiated on Matrigel and then enriched ashESC-RPEon Synthemax-R PEDF pigment epithelium-derived factorSR hESC-RPE derived on Synthemax-R

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pigmented area after 115 days of spontaneous differentiationon Synthemax II-SC

Flow cytometric analysis of hESCs at passages 1 3 5 and 9revealed stable expressionof thepluripotency-associated surface

antigen SSEA4 [69] on all three substrates and a small subpopu-lation of cells negative for Oct4 on the synthetic surfaces at somepassages Aprevious studydemonstrated that hESCs beginning todifferentiate into anearly neural lineagemaintainedhigh levels of

Figure5 CharacterizationofH9RPEgenerated fromhumanembryonic stemcells (hESC-RPE)derivedonSynthemax II-SC versusMg-SR seededonto parylene-C scaffolds (A)Bright-field and phase-contrast images of H9 hESC-RPE cells grown for 30 days on parylene-C scaffolds hESC-RPEwere derived on Synthemax II-SC or by theMg-SRmethod Ultrathin regions of the membrane appear as an array of circles in the micrographsScale bar = 200 mm (B) Bright-field images of three representative parylene-C scaffolds seeded with hESC-RPE in a 24-well plate (n = 8) Scalebar = 1 cm Note darker pigmentation in the RPE derived on Synthemax II-SC (C) Expression of RPE and pigmentation markers after 30 days onparylene-C scaffolds as detected by quantitative polymerase chain reaction (n = 3) (D) Quantification of PEDF protein secreted by hESC-RPEderived on Synthemax II-SC or by theMg-SRmethod after 30 days on parylene-C scaffolds (n = 6 error is SEM) p p 05 t test AbbreviationsMg-SR hESCs grown and differentiated onMatrigel and then enriched as hESC-RPE on Synthemax-R PEDF pigment epithelium-derived factorRPE retinal pigmented epithelium

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SSEA4 expression whereas Oct4 was progressively down-regulated [70] The differentiated morphology seen in the cul-tures on the Synthemax surfaces could likely account for the smallpopulation of cells negative for Oct4 by flow cytometry Despitethe slightly smaller populationpositive forOct4 at somepassageswe conclude that Synthemax II-SC is a suitable defined substratefor the maintenance of hESCs

It is interesting that the H9-derived RPE on Synthemax II-SCexpress significantly higher TyrmRNA than those on Synthemax-R despite their similarities of darker pigmentation comparedwith theMatrigel cultures RPE pigmentation performs amyriadof essential roles for vision such as protecting against harmfulshort-wavelength light participating as an antioxidant to reduceoxidative damage and even contributing to proper retinal struc-ture during development [6 71] The macula is also reported tocontain a higher concentration of pigment granules than the pe-ripheral retina [72] The discrepancy between theH9 Synthemaxcultures could be due to differential rates of RPE maturation onthe various substrates During embryonic development of theoptic cup activation of the tyrosinase promoter initiates RPEmaturation and melanogenesis [59 71] RPE then begin toproduce melanosomes which are specialized organelles formelanin synthesis Tyrosinase a heavily post-translationallymodified enzyme catalyzes the rate limiting step of melanin syn-thesis in developing melanosomes [57 71 73] Pigment produc-tion in the RPE occurs during embryonic development howeverand no melanogenesis nor melanosome assembly is thought tooccur after gestation [73] RPE manifesting darker pigmentationmay be more mature and could thus be starting to reduce theexpressionofmelanogenesis geneswhile theproteinspersistDif-ferential expression of gene transcripts and proteins for tyrosi-nase have been previously reported [74] RPE pigmentation canvary considerably in vivo however and does not necessarily re-flect on other functional capacities [12] H14-derived RPE grownon the given substrates however did not have significant differ-ences in thegeneexpressionofpigmentationmarkers so itwill beinteresting to determine if this trend in H9-derived RPE persistswith further experimental repetitions

hESC-RPE derived under xeno-free conditions with Synthe-max II-SC demonstrated polarized secretory function and theability to internalize ROS by phagocytosis Regarding the secre-tory function of hESC-RPE polarized secretion of PEDF testifiesto the integrity of the tight junctions that separate the apicaland basal environments as well as to the maturity of the cellsH9 hESC-RPE on parylene-C scaffolds have been observed tosecrete a range of apical PEDF concentrations between 11and 20 ngmm2ofprotein (CHinmanD Zhu SHikitaetalmanu-script in preparation)When seeded on parylene-C scaffolds andculture inserts coated with human vitronectin hESC-RPE de-rived on Synthemax II-SC secreted less PEDF than hESC-RPE de-rived by the Mg-SR method However the secreted amount ofPEDF by the Synthemax II-SC cells on the scaffolds was stillwithin the expected range Whether these cells will increasethe amount secreted PEDF as they mature in culture remainsto be determined It will also be interesting to investigate cellmorphology and function when seeded on parylene-C scaffoldscoatedwith Synthemax II-SC because the synthetic peptide con-tains the RGD binding site from vitronectin

In summary we have found that Matrigel can be replaced bythe GMP-compliant substrate Synthemax II-SC for the culture ofhESCs and the production of hESC-RPE hESCs cultured on

Synthemax II-SC express pluripotency markers and are capableof differentiation into cells expressing markers from the threegerm layers The yield of pigmented area on Synthemax II-SC afterspontaneous differentiation of hESCs is significantly more thanthe yield on an alternative ACF substrate Synthemax-R hESC-RPE derived on Synthemax II-SC express RPEmarkers and internal-ize ROS by phagocytosis similarly to hESC-RPE derived onMatrigeland are also capable of PEDF secretion Taken together this workvalidates theuseofanovel ACF substrate for thedefinedcultureofhESCs and the xeno-free production of hESC-RPE that can translateto stem cell-based therapies for human maladies such as AMD

CONCLUSION

hESC-derived therapies are expected to be a powerful tool in re-generativemedicine that requires defined xeno-free culture con-ditions as the field moves forward Here we demonstrate thata novel synthetic substrate Synthemax II-SC supports pluripo-tent hESC cultures and promotes their efficient differentiationinto functional RPE

ACKNOWLEDGMENTS

We thank the staff of the Center for Stem Cell Biology and Engi-neering as well as the Laboratory for Stem Cell Biology and En-gineering This work was supported by the Richard amp KatherineGee Breaux Fellowship in Vision Research the Garland Initiativefor Vision a grant-in-aid for research from the Scientific Re-search Society Sigma Xi theWynn-Gund Translational ResearchAcceleration Program the University of California SantaBarbara Institute forCollaborativeBiotechnologies throughGrantW911NF-09-0001 from the US Army Research Office andCalifornia Institute for RegenerativeMedicineGrants DR1-01444CL1-00521 TG2-01151 and FA1-00616 (to DOC) Financialsupport from Fight for Sight is gratefully acknowledged BOPis a fellow of the California Institute for Regenerative MedicineThe content of the information does not necessarily reflectthe position or the policy of the US government and no officialendorsement should be inferred

AUTHOR CONTRIBUTIONS

BOP conception and design collection andor assembly of datadata analysis and interpretation financial support manuscriptwritingDOC conceptionanddesign financial supportdataanal-ysis and interpretation final approval ofmanuscript ZKM provi-sion of study material final approval of manuscript STHconception and design data analysis and interpretation finalapproval of the manuscript

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

DOC has uncompensated employment uncompensated intel-lectual property rights and uncompensated ownership interestas co-owner of Regenerative Patch Technologies ZKM is a com-pensated employee of Corning Inc whichmanufactures and sellsSynthemax Surface product a compensated inventor of CorningInc which is an assignee of the patent rights for Synthemax andhas been compensated with Corning stock options STH has un-compensated intellectual property rights The other author indi-cated no potential conflicts of interest

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supported submicron parylene-C membranesfor culturing retinal pigment epithelial cellsBiomed Microdevices 201214659ndash667

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et al Implantation and explantation of an activeepiretinal visual prosthesis 2-year follow-updata from the EPIRET3 prospective clinical trialEye (Lond) 201226501ndash50923 Lu B Liu Z Tai Y-C Ultrathin parylene-C

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Feeder-free growth of undifferentiated humanembryonic stem cells Nat Biotechnol 200119971ndash97425 OrsquoConnor MD The 3R principle Advanc-

ing clinical application of human pluripotentstem cells Stem Cell Res Ther 201342126 Vukicevic S KleinmanHK Luyten FP et al

Identification of multiple active growth factorsin basement membrane Matrigel suggests cau-tion in interpretation of cellular activity relatedto extracellular matrix components Exp CellRes 19922021ndash827 McGuire PG SeedsNW The interaction

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K et al Toward thedefined and xeno-freediffer-entiation of functional human pluripotent stemcell-derived retinal pigment epithelial cellsMolVis 201117558ndash57530 Sorkio A Hongisto H Kaarniranta K et al

Structure and barrier properties of human em-bryonic stem cell-derived retinal pigment epi-thelial cells are affected by extracellularmatrix protein coating Tissue Eng Part A201420622ndash63431 Melkoumian Z Weber JL Weber DM

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relevant patient specific induced pluripotentstem (iPS) cells PLoS ONE 20138e8162234 Lin P-Y Hung S-H Yang Y-C et al A syn-

thetic peptide-acrylate surface for productionof insulin-producing cells from human embry-onic stem cells Stem Cells Dev 201423372ndash37935 Li Y Gautam A Yang J et al Differentia-

tion of oligodendrocyte progenitor cells fromhuman embryonic stem cells on vitronectin-derived synthetic peptide acrylate surfaceStem Cells Dev 2013221497ndash150536 Jin S Yao H Weber JL et al Synthetic

xeno-free peptide surface for expansion and di-rected differentiation of human induced plurip-otent stem cells PLoS ONE 20127e5088037 Maruotti J Wahlin K Gorrell D et al A

simple and scalable process for the differentia-tion of retinal pigment epithelium from humanpluripotent stem cells STEM CELLS TRANSLATIONALMEDICINE 20132341ndash35438 Baranov P Regatieri C Melo G et al Syn-

thetic peptide-acrylate surface for self-renewalof human retinal progenitor cells Tissue EngPart C Methods 201319265ndash27039 HaymanEGPierschbacherMDOhgrenY

et al Serum spreading factor (vitronectin) ispresent at the cell surface and in tissues ProcNatl Acad Sci USA 1983804003ndash400740 Ruoslahti E RGD and other recognition

sequences for integrins Annu Rev Cell Dev Biol199612697ndash71541 HervyMWeber JL PecheulMet al Long

term expansion of bone marrow-derivedhMSCs on novel synthetic microcarriers inxeno-free defined conditions PLoS ONE20149e9212042 Kelly J Zhou J Henry D et al Feeder-Free

Expansion of Human Induced Pluripotent StemCells (hiPSC)onCorningSynthemax-II SCSubstrateCoated Surface in Defined Medium SnAPPShots2012 Available at httpwwwcorningcomlifesciencessurfacesensynthemaxtechnical_resourcesaspxlinkidentifier=idampitemid=47257ampmenutitle=posters Accessed July 15 201443 Buchholz DE Hikita ST Rowland TJ et al

Derivation of functional retinal pigmented epi-thelium from induced pluripotent stem cellsSTEM CELLS 2009272427ndash243444 Maminishkis A Miller SS Experimental

models for study of retinal pigment epithelialphysiology and pathophysiology J Vis Exp201045203245 Buchholz DE Pennington BO Croze RH

et al Rapid and efficient directed differentia-tionof humanpluripotent stemcells into retinalpigmented epithelium STEM CELLS TRANSLATIONALMEDICINE 20132384ndash39346 Yoon T-M Chang B KimH-T et al Human

embryonic stem cells (hESCs) cultured underdistinctive feeder-free culture conditions dis-play global gene expression patterns similarto hESCs from feeder-dependent culture condi-tions Stem Cell Rev 20106425ndash43747 Bigdeli N Andersson M Strehl R et al Ad-

aptationofhumanembryonicstemcells tofeeder-free andmatrix-free culture conditions directly onplastic surfaces J Biotechnol 2008133146ndash15348 Stover AE Schwartz PH Adaptation of

human pluripotent stem cells to feeder-freeconditions in chemically defined medium withenzymatic single-cell passaging Methods MolBiol 2011767137ndash146

176 Synthetic Substrate for Stem Cell and RPE Culture

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49 Tompkins JD Hall C Chen VC-Y et al Epi-genetic stability adaptability and reversibilityin human embryonic stem cells Proc Natl AcadSci USA 201210912544ndash1254950 Ramos-Mejia V Bueno C Roldan M

et al The adaptation of human embryonicstem cells to different feeder-free cultureconditions is accompanied by amitochondrialresponse Stem Cells Dev 2012211145ndash115551 Perkel J Tricks for Human Embryonic

Stem Cells The Scientistcom 2005 Availableat httpwwwthe-scientistcomarticlesviewarticleNo16566titleTricks-For-Human-Embryonic-Stem-Cells Accessed July 16 201452 Kwon GS Fraser ST Eakin GS et al Tg

(Afp-GFP) expression marks primitive anddefinitive endoderm lineages during mousedevelopment Dev Dyn 20062352549ndash255853 Dziadek M Adamson E Localization

and synthesis of alphafoetoprotein in post-implantation mouse embryos J Embryol ExpMorphol 197843289ndash31354 Diniz B Thomas P Thomas B et al Subre-

tinal implantation of retinal pigment epithelialcells derived fromhumanembryonic stemcellsImproved survival when implanted as a mono-layer Invest Ophthalmol Vis Sci 2013545087ndash509655 Lagunoff D Rickard A Mast cell granule

heparinproteoglycan induces lacunae in conflu-ent endothelial cell monolayers Am J Pathol19991541591ndash160056 Kobayashi T Urabe K Orlow SJ et al The

Pmel 17silver locus protein Characterizationand investigation of its melanogenic functionJ Biol Chem 199426929198ndash29205

57 Wasmeier C Hume AN Bolasco G et alMelanosomes at a glance J Cell Sci 20081213995ndash399958 Saari JC NawrotM Kennedy BN et al Vi-

sual cycle impairment in cellular retinaldehydebinding protein (CRALBP) knockoutmice resultsin delayed dark adaptation Neuron 200129739ndash74859 Clegg DO Buchholz D Hikita S et al Ret-

inalpigmentepithelial cellsDevelopment invivoand derivation from human embryonic stemcells invitro for treatmentofage-relatedmaculardegeneration Stem Cell Research and Thera-peutics New York NY Springer Science + Busi-ness Media 20081ndash2460 Meyer JS Shearer RL Capowski EE et al

Modeling early retinal development with hu-man embryonic and induced pluripotent stemcells Proc Natl Acad Sci USA 200910616698ndash1670361 Tung P-Y Knoepfler PS Epigeneticmech-

anisms of tumorigenicity manifesting in stemcells Oncogene 2014 [Epub ahead of print]62 Finnemann SC Focal adhesion kinase

signaling promotes phagocytosis of integrin-bound photoreceptors EMBO J 2003224143ndash415463 DrsquoCruz PMP Yasumura D Weir J et al

Mutation of the receptor tyrosine kinase geneMertk in the retinal dystrophic RCS rat HumMol Genet 20009645ndash65164 Cao W Tombran-Tink J Elias R et al In

vivo protection of photoreceptors from lightdamage by pigment epithelium-derived fac-tor Invest Ophthalmol Vis Sci 2001421646ndash165265 Ogata N Wang L Jo N et al Pigment ep-

ithelium derived factor as a neuroprotective

agent against ischemic retinal injury Curr EyeRes 200122245ndash25266 Bharti K Liu W Csermely T et al Alterna-

tive promoter use in eye development The com-plex roleand regulationof the transcription factorMITF Development 20081351169ndash117867 Chambers SMFasanoCA PapapetrouEP

et al Highly efficient neural conversion of hu-man ES and iPS cells by dual inhibition of SMADsignaling Nat Biotechnol 200927275ndash28068 Lane A Philip LR Ruban L et al Engineer-

ing efficient retinal pigment epithelium differ-entiation from human pluripotent stem cellsSTEM CELLS TRANSLATIONAL MEDICINE 201431295ndash130469 Adewumi O Aflatoonian B Ahrlund-

Richter L et al Characterization of human em-bryonic stemcell lines by the International StemCell InitiativeNatBiotechnol200725803ndash81670 Noisa P Ramasamy TS Lamont FR et al

Identification and characterisation of the earlydifferentiating cells in neural differentiation ofhuman embryonic stem cells PLoS ONE 20127e3712971 JefferyG The retinal pigment epithelium

as a developmental regulator of the neural ret-ina Eye (Lond) 199812499ndash50372 BoultonM Dayhaw-Barker P The role of

the retinal pigment epithelium Topographicalvariation and ageing changes Eye (Lond)200115384ndash38973 Sarna T Properties and function of the

ocularmelanin A photobiophysical view J Pho-tochem Photobiol B 199212215ndash25874 Hashimoto Y Ito Y Kato T et al Expres-

sion profiles of melanogenesis-related genesand proteins in acquired melanocytic nevus JCutan Pathol 200633207ndash215

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Britney O Pennington Dennis O Clegg Zara K Melkoumian and Sherry T HikitaRetinal Pigmented Epithelial Cells on a Novel Synthetic Substrate

Defined Culture of Human Embryonic Stem Cells and Xeno-Free Derivation of

doi 105966sctm2014-0179 originally published online January 15 20152015 4165-177Stem Cells Trans Med

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