defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation...

Defined Human Pluripotent Stem Cell CultureEnables Highly Efficient NeuroepitheliumDerivation Without Small Molecule Inhibitors

ETHAN SCOTT LIPPMANN,a,b MARIA CAROLINA ESTEVEZ–SILVA,a,b RANDOLPH SCOTT ASHTONa,b*

Key Words. Neural stem cells • Neuroectoderm • Neuroepithelial cells • Defined tissue culture

ABSTRACT

The embryonic neuroepithelium gives rise to the entire central nervous system in vivo, makingit an important tissue for developmental studies and a prospective cell source for regenerativeapplications. Current protocols for deriving homogenous neuroepithelial cultures from humanpluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followedby a manual isolation step or adherent differentiation using small molecule inhibitors. Here, wereport that hPSCs maintained under chemically defined, feeder-independent, and xeno-free con-ditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax61/N-cadherin1 with widespread rosette formation) within 6 days under adherent conditions, withoutsmall molecule inhibitors, and using only minimalistic medium consisting of Dulbecco’s modifiedEagle’s medium/F-12, sodium bicarbonate, selenium, ascorbic acid, transferrin, and insulin (i.e.,E6 medium). Furthermore, we provide evidence that the defined culture conditions enable thishigh level of neural conversion in contrast to hPSCs maintained on mouse embryonic fibroblasts(MEFs). In addition, hPSCs previously maintained on MEFs could be rapidly converted to a neu-ral compliant state upon transfer to these defined conditions while still maintaining their abilityto generate all three germ layers. Overall, this fully defined and scalable protocol should bebroadly useful for generating therapeutic neural cells for regenerative applications. STEM CELLS

2014;32:1032–1042

INTRODUCTION

Over the past decade, significant advancementshave been made in differentiating human pluri-potent stem cells (hPSCs) toward diverse neurallineages of the central nervous system (CNS) andperipheral nervous system including GABA neu-rons [1], floor plate precursors [2], dopaminergicneurons [3–5], spinal motor neurons [6, 7], noci-ceptors [8], astrocytes [9], and oligodendrocytes[10–13]. These achievements have culminated inrecent demonstrations that engraftment of hPSC-derived oligodendrocytes, GABA, and dopaminer-gic neurons can alleviate symptoms in rodentmodels of spinal cord injury [13, 14], Hunting-ton’s [1], and Parkinson’s [3, 5] disease, respec-tively, thereby highlighting the prospective use ofhPSC-derived neural cells for translational medi-cine. However, these prior examples continue touse undefined or xenogeneic culture compo-nents for hPSC maintenance and differentiation(e.g., mouse embryonic fibroblast [MEF] feederlayers supplemented with knockout serumreplacer [KSR], Matrigel, or medium containingserum albumin), which could ultimately limit thescalability and clinical utility of these protocols[3, 5, 15]. Additionally, many neuroepithelium

derivation protocols still require manual enrich-

ment steps that are also undesirable for scale-up

[16, 17]. In efforts to facilitate widespread use of

hPSCs for diverse applications, researchers have

recently developed defined surfaces [18–22] and

media [23, 24] for hPSC culture and differentia-

tion. Therefore, an important advancement in

this field would be the adaptation and optimiza-

tion of current hPSC neural differentiation proto-

cols to xeno-free and defined systems to

facilitate clinical implementation of therapeutic

cell products and assist in standardizing differen-

tiation procedures from lab-to-lab.In the developing human embryo, the

entire CNS arises from neuroepithelial cellsthat constitute the primordial neural tube.Thus, protocols for generating CNS cells fromhPSCs typically proceed from the Oct-4(POU5F1)1/Nanog1 pluripotent state throughan initial Pax61/N-cadherin1 neuroepithelialstate [1, 7, 9, 17], which can be identified inculture by columnar cell morphologies and theformation of polarized rosette structures [25],although polarization is not required for neuraldifferentiation [3]. Robust generation of hPSC-derived neuroepithelial cells has been achieved

aWisconsin Institute forDiscovery and University ofWisconsin-Madison, Madison,Wisconsin, USA, bDepartmentof Biomedical Engineering,University of Wisconsin-Madison, Madison,Wisconsin, USA

Correspondence: Randolph S.Ashton, Ph.D., Department ofBiomedical Engineering,University of Wisconsin-Madison, Madison, Wisconsin53715, USA. Telephone: 608-316-4312; Fax: 608-316-4606;e-mail: [email protected]

Received August 2, 2013;accepted for publicationNovember 21, 2013; firstpublished online in STEM CELLS

EXPRESS December 19, 2013.

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http://dx.doi.org/10.1002/stem.1622

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REGENERATIVE MEDICINE

through either embryoid body (EB) formation followed byplating [25] or adherent differentiation using small moleculeinhibition of SMAD signaling followed by passaging [26]. How-ever, these protocols were developed and standardized usinghPSCs cultured on feeder layers and differentiated with xeno-genic or undefined reagents [1, 3, 5, 8, 17, 25–27]. With therecent establishment of novel defined protocols for hPSC deri-vation and culture [23], it remains to be investigated whethertraditional neuroepithelial cell derivation methods are stilloptimal for hPSCs maintained under such defined conditions.Indeed, the variability in source [28, 29] and derivation proto-cols between different hPSC lines can also affect neural differ-entiation efficiency due to variations in epigenetic regulationand basal gene expression [30]. As such, the overall goal forthis study was to merge advancements in defined culture sys-tems with existing protocols to neuralize hPSCs under chemi-cally defined, minimalistic, xeno-free, and scalable conditions.

Using hPSCs maintained in chemically defined E8 medium[23] (E8) on either Matrigel-coated or recombinant humanvitronectin peptide (VTN-NC)-coated substrates [23], we dem-onstrate a procedure where hPSCs can be differentiated intoneuroepithelium with high purity after only 6 days and withoutthe presence of small molecule SMAD inhibitors. Differentiationis initiated by high density hPSC seeding and culture in definedmedium (i.e., Dulbecco’s modified Eagle’s medium [DMEM]/F-12, ascorbic acid, sodium bicarbonate, selenium, human trans-ferrin, and human insulin, termed E6 medium) under adherentconditions. No difference in differentiation efficiency wasobserved using culture surfaces coated with either Matrigel orVTN-NC (98%6 2% and 99%6 1% Pax61/N-cadherin1, respec-tively), and further analysis of E6 medium revealed thatDMEM/F-12, sodium bicarbonate, selenium, and insulin werethe minimum components necessary for neuroepithelial differ-entiation and survival. The E6-derived neuroepithelium couldbe differentiated to motor neurons, indicating the neural pro-genitors are responsive to lineage patterning cues, and astro-cytes, indicating the neuroepithelium is multipotent.Interestingly, hPSCs maintained on MEFs prior to differentiationin E6 medium conditions do not efficiently generate neuroepi-thelium, but if transferred from MEFs to E8/feeder-independent(FI) maintenance conditions, they acquire the capacity for effi-cient neuroepithelial differentiation. This change in differentia-tion capacity is accompanied by increased expression of genesassociated with epiblast and neuroectoderm fates anddecreased expression of genes associated with mesoderm andendoderm differentiation, indicating a shift in differentiationbias. However, while hPSCs maintained in E8/FI conditionsbecome biased toward a neuroectodermal fate, they maintaintheir pluripotent capabilities and can still be effectively differen-tiated toward mesodermal and endodermal fates.

MATERIALS AND METHODS

Maintenance of hPSCs

hPSCs were obtained as frozen vials and banked under FI condi-tions in mTeSR1 medium (STEMCELL Technologies, Vancouver,Canada (www.stemcell.com)). hPSCs were then thawed and cul-tured directly into E8 medium [23] consisting of DMEM/F-12(Life Technologies, Carlsbad, CA (www.lifetechnologies.com)),64 mg/l ascorbic acid (Sigma), 543 mg/l sodium bicarbonate

(Sigma, St. Louis, MO (www.sigmaaldrich.com)), 14 mg/l sodiumselenite (Sigma), 19.4 mg/l insulin (Sigma), 10.7 mg/l transferrin(Sigma), 100 mg/l FGF2 (Waisman Clinical BiomanufacturingFacility, University of Wisconsin-Madison), and 2 mg/l TGFb1(Peprotech, Rocky Hill, NJ (www.peprotech.com)). pH of E8medium was adjusted to 7.4 and osmolarity was adjusted to340 mOsm with NaCl. hPSCs were maintained on Matrigel (BDBiosciences, San Jose, CA (www.bdbiosciences.com)) or VTN-NC(provided by Dr. James Thomson) [23]. Cell lines used in thisstudy were H9 human embryonic stem cells (hESCs) (passages25–50), H1 hESCs (passages 28–36), IMR90-4 iPSCs (passages26–62), and 004A iPSCs (passages 3–7). For some comparativeexperiments, hPSCs were maintained on irradiated MEFs instandard unconditioned medium: DMEM/F-12 containing 20%KSR (Life Technologies), 13 Minimum Essential Medium (MEM)nonessential amino acids (Life Technologies), 1 mM L-glutamine(Sigma), 0.1 mM b-mercaptoethanol (Sigma), and 4 ng/mlFGF2. Cells were routinely passaged with Versene (Life Technol-ogies) as previously described [23]. ROCK inhibitor (1 mM) (Y-27632; R&D Systems, Minneapolis, MN (www.rndsystems.com))was included when passaging H1 hESCs onto VTN-NC to facili-tate attachment and removed 24 hours after attachment.

Differentiation to Neuroepithelium Under AdherentConditions

hPSCs were washed once with phosphate-buffered saline(PBS; Life Technologies), incubated with accutase (Life Tech-nologies) for 3 minutes, and collected by centrifugation. hPSCswere then plated onto Matrigel or VTN-NC at a density of2 3 105 cells per square centimeter in E8 medium containing10 mM ROCK inhibitor and cultured overnight. The followingmorning, cells were changed to E6 medium, E6 containing10 mM SB431542 (Cellagentech, San Diego, CA (www.cellagentech.com)), or E6 containing 10 mM SB431542 and 200 ng/mlrecombinant human noggin (R&D Systems) to initiate differen-tiation. E6 medium is the same formulation as E8 mediumbut without FGF2 and TGFb1. Medium was changed everyday until cells were used for analysis. After these initialexperiments, additional seeding densities of 1 3 105 cells persquare centimeter, 5 3 104 cells per square centimeter, and 13 104 cells per square centimeter were tested with E6medium. Further experiments removed individual componentsfrom E6 medium while qualitatively analyzing cell viability.

Differentiation to Mesoderm and Endoderm Fates

H9 hESCs were singularized and plated overnight in E8medium at a density of 1.5 3 105 cells per square centimeteron Matrigel as described above. Mesoderm and endodermdifferentiation schemes were adapted from previous protocols[31, 32]. To generate mesoderm, cells were differentiated for4 days in E6 medium containing 6 mM CHIR99021. To gener-ate endoderm, cells were differentiated for 1 day in E6medium containing 6 mM CHIR99021 and 100 ng/ml activin A(R&D Systems), followed by 3 days of activin A alone.

EB Formation

To form EBs, H9 hESCs were incubated with 2 mg/ml dispase(Life Technologies) for 10–15 minutes to facilitate colonydetachment, washed twice with DMEM/F-12, and transferredto low-attachment six-well plates (Corning, Corning, NY(www.corning.com)) in E6 medium. Medium was changed every

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other day. At day 4 of differentiation, whole EBs were trans-ferred to standard tissue culture polystyrene dishes or glasschamber slides coated with Matrigel. Resultant cells were main-tained in E6 medium for the duration of each experiment.

Differentiation to Motor Neurons

H9 hESCs were singularized and plated overnight in E8medium at a density of 1.5 3 105 cells per square centimeteron Matrigel as described above. Cells were differentiated inE6 medium for 3 days, at which point single rosettes weremanually isolated and replated in Matrigel-coated chamberslides. Cells were then patterned with 1 mM retinoic acid (RA;Sigma) and 0.1 mM purmorphamine (PM; EMD Millipore, Bill-erica, MA (www.millipore.com)) as described in Results. CyclicAMP (1 mM) (cAMP; Sigma), 10 ng/ml brain-derived neurotro-phic factor (BDNF; Peprotech), and 10 ng/ml glial-derivedneurotrophic factor (GDNF; Peprotech) were added as indi-cated to support neuronal survival.

Differentiation to Astrocytes

H9 hESCs were singularized and plated overnight in E8medium at a density of 1.5 3 105 cells per square centimeteron Matrigel as described above. Cells were differentiated inE6 medium for 6 days, at which point single rosettes weremanually isolated and replated in Matrigel-coated chamberslides. Cells were then differentiated for 4 weeks as describedin Results. cAMP (1 mM), BDNF (10 ng/ml), and GDNF (10 ng/ml) were added as indicated.

Immunocytochemistry and Microscopy

Cells were washed twice with PBS and fixed with 4% parafor-maldehyde for 10 minutes at room temperature. Picric acid(0.1%) (Fisher, Pittsburgh, PA (www.fishersci.com)) was includedwhen labeling for choline acetyltransferase (ChAT). After addi-tional washes in PBS, cells were blocked and permeabilized intris-buffered saline (TBS)-DT (containing 5% donkey serum[Sigma] and 0.3% Triton X-100 [TX-100; Fisher]) for at least 1hour at room temperature. Primary antibodies were diluted inTBS-DT and cells were incubated in these antibodies overnightat 4�C. Antibodies are listed in Supporting Information Table S1.The following day, chambers were rinsed once with TBS con-taining 0.3% Triton X-100 (TBST) and then washed five times, 15minutes apiece, with TBST. Secondary antibodies (SupportingInformation Table S1) were diluted in TBS-DT and incubated onthe cells for 1 hour at room temperature and nuclei were sub-sequently counterstained with 300 nM 40,6-diamidino-2-pheny-lindoldihydrochloride (DAPI) for 10 minutes. Afterward, cellswere washed once for 25 minutes with TBS and three additionaltimes for 10 minutes apiece. Cells cultured in chamber slideswere then mounted with Prolong Gold Antifade Reagent (LifeTechnologies) and visualized using a Nikon A1R confocal micro-scope. Cells cultured in 6- or 12-well plates were visualizedusing a Nikon Ti-E microscope. Nikon NIS-Elements softwarewas used for image analysis. Bright-field images were acquiredusing a Nikon TS100 microscope.

Flow Cytometry

Cells were harvested from 6- or 12-well plates by washing oncewith PBS and incubating with accutase for 3–5 minutes. Cellswere then recovered by centrifugation and fixed in 4% parafor-maldehyde for 10 minutes at room temperature. After blocking

with PBS containing 10% normal serum (goat or donkey serumdepending on the species of primary antibody; Sigma) and 0.1%Triton X-100 for at least 30 minutes at room temperature, cellswere incubated with primary antibodies for 1 hour at roomtemperature or overnight at 4�C. Antibodies are listed in Sup-porting Information Table S1. IgG controls were included foreach species of antibody (Life Technologies). After washingtwice with PBS containing 0.75% bovine serum albumin (BSA;Life Technologies), cells were incubated for 30–60 minutes atroom temperature in PBS containing 10% normal serum andsecondary antibodies (Supporting Information Table S1). Afterwashing twice with PBS containing 0.75% BSA, cells were ana-lyzed on a FACSCanto (BD Biosciences), and data were analyzedusing Cyflogic software. Positive events were determined bygating above the top 1% of the IgG control histograms.

Reverse Transcriptase Polymerase Chain Reaction andQuantitative PCR

Total RNA was extracted from cells using Trizol reagent (LifeTechnologies) according to the manufacturer’s instructions.Five micrograms of total RNA was then subjected to reverse-transcription using a Thermoscript RT-PCR kit (Life Technolo-gies) in a 20 ml mixture according to the manufacturer’sinstructions. Resultant cDNA (0.5 ml) was then amplified in a25 ml mixture containing 103 PCR buffer, 0.2 mM dNTP, 1.5mM MgCl2, 0.5 mM of each primer, and 1 U Taq DNA poly-merase (Life Technologies). Amplified products were resolvedon 2% agarose gels containing SYBR Safe (Life Technologies)and visualized with a VersaDoc (BioRad, Hercules, CA(www.bio-rad.com)). Primer sequences can be found in Sup-porting Information Table S2. To confirm the fidelity of pri-mers designed for genes not detected in our samples,genomic DNA or positive control cDNA was used.

Quantitative PCR analysis was conducted between H9hESCs maintained on MEFs and hESCs transferred to E8/FIconditions for two passages. cDNA was mixed with TaqmanGene Expression Master Mix (Life Technologies) and loadedonto Human Stem Cell Taqman Array Plates (Life Technolo-gies). Amplification was carried out on a BioRad CFX96 ther-mocycler according to the manufacturer’s instructions.Relative gene expression was quantified using the compara-tive cycle threshold (CT) method using GAPDH as the house-keeping gene. SOX1 and OTX2 expression were quantifiedusing individual Taqman primers with RSP18 as the house-keeping gene.

Fluorescence In Situ Hybridization

H9 hESCs cultured in chamber slides were fixed in 2% parafor-maldehyde for 10 minutes and permeabilized in 70% ethanolovernight at 4�C. Cells were then incubated in wash bufferconsisting of 10% formamide (Life Technologies) and 23

saline-sodium citrate (SSC; VWR, Radnor, PA (www.vwr.com))for 5 minutes. Fluorescein-labeled SOX1 and Quasar 570-labeled GAPDH Stellaris fluorescence in situ hybridizationprobes were purchased from Biosearch Technologies, Petal-uma, CA (www.biosearchtech.com) and diluted to 1.25 mM inhybridization buffer consisting of 100 mg/ml dextran sulfate(Sigma) and 10% formamide in 23 SSC. Cells were incubatedwith probes overnight in a 37�C humidified incubator. The fol-lowing day, after three washes and a DAPI counterstain, the

1034 Defined Culture Enables Neuroepithelium Derivation

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slides were mounted with Prolong Gold Antifade Reagent andimmediately visualized on a Nikon Ti-E microscope.

RESULTS

hPSCs Cultured and Differentiated in E8/FI ConditionsEfficiently Generate Neuroepithelium Without the Useof Small Molecule Inhibitors

A recent report by Chambers et al. demonstrated rapid andefficient hPSC neuralization using small molecule inhibitors andrecombinant proteins, yielding [mt]80% neuroepithelium after11 days [26]. The original protocol neuralized hESCs maintainedon MEFs in KSR-containing media (MEFs/KSR) using SB431542(an inhibitor of TGFb signaling) and noggin (an inhibitor ofbone morphogenetic protein [BMP] signaling), and follow-upprotocols have replaced noggin with the small molecules dorso-morphin or LDN-193189 [8, 33]. Such factors were hypothe-sized in these previous studies to promote ectodermneuralization and suppress endoderm and mesoderm formationby inhibiting endogenous BMP and activin/TGFb signaling.Here, we hypothesized that hPSCs maintained in definedmedium under FI conditions [23] might have different basal sig-naling activity compared to hPSCs maintained on MEFs/KSR.Therefore, we tested whether suppression of activin/TGFb andBMP signaling is still necessary to efficiently derive neuroepi-thelium from hPSCs cultured under defined conditions.

We cultured H9 hESCs in E8/FI conditions [23] on Matrigel-coated substrates and verified their expression of pluripotencymarkers Sox2, Oct4, and Nanog by immunocytochemistry (ICC)and flow cytometry (FC) (Supporting Information Fig. S1A-S1C).Upon positive confirmation, these cells were subcultured ontoMatrigel-coated plates at 2 3 105 cells per square centimeter inE8 medium containing ROCK inhibitor (Fig. 1A). The followingday, differentiation was initiated by replacing E8 with E6medium (i.e., E8 without FGF2 and TGFb1), E6 medium supple-mented with SB431542, or E6 medium supplemented with bothSB431542 and noggin for an additional 6 days to probe inhibitorrequirements for efficient neuroepithelial derivation. Differentia-tion within the cultures was monitored every other day andtime course reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed a gradual decrease in the expression ofpluripotency genes POU5F1 (Oct4) and NANOG under all differ-entiation conditions (Fig. 1B and Supporting Information Fig.S2). After 2–4 days of differentiation, all cultures acquired aneuroectodermal gene expression profile as indicated by theabsence of Sox17 (a definitive endoderm marker [34]), diminish-ing expression of T (brachyury) which is expressed in primitivestreak mesoderm [35], and activation of the neuroectodermfate determinant [36] PAX6. As previously observed [17], SOX2was expressed in both undifferentiated H9 hPSCs and neuroec-todermal cells, but interestingly, we also detected increasing lev-els of SOX1 (a neuroectoderm marker [36]), OTX2 (a midbrainand forebrain marker [17]), and FOXG1 (a forebrain marker [26])expression throughout differentiation. By day 6, the culturesunder all conditions acquired a rostral/dorsal neuroectodermalfate as indicated by OTX2 and FOXG1 expression in the absenceof HOXB4 (a hindbrain/spinal cord marker [17]) and OLIG2 (aventral transcription factor [7]) expression.

To further assess neural conversion, we quantitatively ana-lyzed the percentage of Pax61 neuroectodermal cells by FC

and qualitatively affirmed acquisition of a neuroepithelialstate by ICC (Fig. 1C, 1D). By day 2, Pax6 expression was notdetected in differentiating H9 hESCs under any experimentalconditions (E6, E61SB432542, or E61SB4315421noggin), butby day 3 of differentiation in E6 medium alone, 72%6 4% ofcells expressed Pax6. By days 4–6 of differentiation, this per-centage increased to [mt]90% Pax61 neuroectodermal cellsunder all differentiation conditions, and at day 6, H9 hESCsdifferentiated in just E6 medium were uniform in their expres-sion of Pax6 (98%6 2%), N-cadherin (100%6 0%), Otx2(95%6 0%), and Sox2 (98%6 1%) (Fig. 1C-1E). As an addi-tional indicator of neural conversion, N-cadherin expressionwas observed to increase throughout the 6 days of differen-tiation while E-cadherin, an hPSC marker [37], concurrentlydecreased (Supporting Information Fig. S3). This pattern ofcadherin expression aligns with recent in vivo results indicat-ing that E-cadherin is coexpressed with N-cadherin at themidbrain portion of the recently polarized Hamburger andHamilton (HH) stage 9 neural tube before eventually disap-pearing by HH stage 11 [38]. As a final qualitative assessmentof H9 hESC neuroepithelial conversion, ICC at day 6 of differ-entiation under all experimental conditions confirmed nuclearPax6 expression and widespread polarization of N-cadherincell membrane proteins toward apical lumens within neuralrosette structures (Fig. 1D), a definitive hallmark of neuroepi-thelium [16, 17, 25]. Thus, H9 hESCs maintained in E8/FI con-ditions can be efficiently differentiated into highly pureneuroepithelium on Matrigel-coated substrates using E6medium without SMAD inhibitors, and this is not a cell line-dependent phenomena as IMR90-4 iPSCs cultured and differ-entiated in the same manner also undergo efficient neuroepi-thelial conversion (87%6 9% Pax61; Fig. 1C).

All experiments thus far used Matrigel as the culture sub-strate during maintenance and differentiation. Therefore, toconstruct a completely defined system, we maintained H9hESCs in E8/FI conditions on VTN-NC-coated substrates [23]and then differentiated the cells in E6 medium also on similarsubstrates. This yielded 99%6 1% Pax61 cells from H9 hESCsafter 6 days (Fig. 1C) again with high levels of N-cadherin(100%6 0%), Otx2 (91%6 6%), and Sox2 (98%6 1%) expres-sion (Fig. 1E) and uniform neural rosette formation (Fig. 1D).Additionally, H1 hESCs were tested under these conditions andyielded 90%6 1% Pax61 cells again with widespread neuralrosette formation (Fig. 1C and Supporting Information Fig. S4).Therefore, the effectiveness of the differentiation proceduredoes not rely on Matrigel as a substrate. To demonstrate theclinical applicability of this protocol, we also differentiated theiPSC line 004A, which was derived under E8/FI conditions [23]and never exposed to MEF coculture, for 6 days in E6 mediumon VTN-NC-coated substrates. Similar to prior results, this alsoyielded cultures that were 90%6 1% Pax61, 90%6 1% Otx21,and 99%6 0% Sox21 with widespread formation of Pax61/N-cadherin1 neural rosettes (Supporting Information Fig. S5).Thus, the fully defined E6/VTN-NC culture system alone is suffi-cient to efficiently generate nearly pure neuroepithelium fromhPSC lines maintained in E8/FI conditions.

Sox1 Expression in Neuroepithelium Derived UsingE6/FI Culture

Previous reports have demonstrated that Pax6 precedes Sox1expression in human neuroectodermal tissues generated both



in vitro [17] and in vivo [36]. However, SOX1 mRNA wasdetectable at basal levels in our undifferentiated hPSC cul-tures and appeared to increase in relative abundancethroughout the differentiation process (Fig. 1B). Therefore, weinvestigated the progression of Sox1 protein expression withinH9 hESCs differentiated using E6/FI culture. By ICC analysis,Pax6 expression was uniform by day 6 of differentiation inE6/Matrigel or VTN-NC culture, while in contrast Sox1 wasonly sparsely expressed by day 6 and not even uniformly

expressed by day 9 of differentiation (Fig. 1F). If RA wasadded to facilitate neural conversion at day 3 of differentia-tion, increased Sox1 expression was observed at days 6 and 9on both Matrigel- and VTN-NC-coated substrates, but this wasstill preceded by uniform Pax6 expression. Therefore, Pax6precedes Sox1 expression in neuroepithelium derived underE6/FI conditions, which is in good agreement with humandevelopmental principles and previously established differen-tiation protocols [17, 36].

Figure 1. Differentiation of human pluripotent stem cells to neuroepithelium under defined conditions. (A): Experimental timeline.(B): Reverse transcriptase polymerase chain reaction analysis of pluripotency, mesoderm, endoderm, and neuroectoderm gene expres-sion in differentiating H9 human embryonic stem cells (hESCs). “SB” indicates addition of SB431542 and “N” indicates addition of nog-gin. (C): Flow cytometry analysis of Pax6. Data are presented as mean6 SD calculated from at least two biological replicates.Differentiation was conducted on Matrigel-coated substrates unless otherwise specified. (D): Images of neural rosette formation onday 6 of H9 hESC differentiation. The inset shows the magnified rosette structure. All images are of cells differentiated on Matrigel-coated substrates except for the one labeled “E6 (VTN-NC),” which indicates cells differentiated in E6 medium on substrates coatedwith recombinant vitronectin peptide. Scale bars in Pax6/N-cadherin-stained images are 250 mm; scale bars in Otx2/Sox2-stainedimages are 50 mm. (E): Representative flow cytometry histograms of N-cadherin, Otx2, and Sox2 expression at day 6 of differentiatingH9 hESCs in E6 medium. Data are representative of two biological replicates and mean6 SD are listed in Results. Gray histogram, IgGcontrol; red histogram, label of interest. (F): Progression of Sox1 expression in E6/FI conditions with and without retinoic acid. Scalebars5 100 mm.



Efficient Neuroepithelium Derivation Depends on E8/FIMaintenance Conditions

To determine whether prior hPSC maintenance in E8/FI cul-ture conditions is critical to the E6/FI protocol, we evaluatedthe efficiency of neuroepithelium derivation from H9 hESCsmaintained in the undifferentiated state using MEFs/KSR cul-ture. Whereas H9 hESCs from E8/FI conditions formed neuralrosettes with 98%–99% Pax61 cells after 6 days of differentia-tion in E6 medium, hESCs from MEFs/KSR conditions generatefew polarized rosettes and only 39%6 0% of the cells werePax61 (Fig. 2A). Thus, hESCs maintained on MEFs/KSR do notefficiently form neuroepithelium when differentiated in E6medium alone. Furthermore, to determine whether adherentconditions were critical for neuroepithelial differentiation, weformed EBs from H9 hESCs maintained in E8/FI conditions,differentiated these EBs for 4 days in E6 medium, and platedthe EBs onto Matrigel-coated dishes for an additional 2 daysof differentiation. Some EBs produced regions with polarizedrosette morphology but only 51%6 2% of the cells wasPax61 (Fig. 2A), suggesting adherent conditions are also

required for the E6 medium protocol’s high differentiationefficiency.

Since hPSC maintenance in E8/FI conditions appeared to berequired for efficient, adherent, and small molecule-independent neuroepithelium derivation, we explored whetherE8/FI conditions could convert hPSCs previously maintained inMEF/KSR culture to a more compliant state. Thus, we transferredH9 hESCs and IMR90-4 iPSCs maintained in MEF/KSR conditionsto E8/Matrigel or VTN-NC culture, and conducted differentiationin E6/FI conditions after one and two rounds of passaging.When differentiated directly from MEF/KSR coculture, theselines exhibited low neuroectoderm differentiation efficiency asassessed by FC for Pax6 expression (27%6 4% for H9 hESCs and2%6 1% for IMR90-4 iPSCs; Fig. 2B). However, after one passageunder E8/FI conditions, their differentiation efficiency increaseddramatically (82%–90% and 43%–53% Pax61 cells derivedfrom H9 hESCs and IMR90-4 iPSCs, respectively). After a secondpassage, H9 hESCs could form 95%–97% Pax61 cultures, andIMR90-4 iPSCs differentiated on VTN-NC-coated substrateswere able to reach 80%6 13% Pax61 cultures. Thus, hPSC

Figure 2. Neuroepithelium derivation from H9 human embryonic stem cells (hESCs) maintained and differentiated under various condi-tions. (A): H9 hESCs maintained in E8/Matrigel or VTN-NC conditions were differentiated in E6 medium under adherent conditions orfree-floating EBs as described in Materials and Methods. Alternatively, hESCs were maintained in MEF/KSR conditions and differentiatedin E6/Matrigel culture under adherent conditions. After 6 days of differentiation, cultures were examined for rosette formation byimmunofluorescence. Red, Pax6; green, N-cadherin. Scale bars5 50 mm. Cultures were also probed for Pax6 expression by flow cytome-try at this time point. Data are presented as mean6 SD calculated from two biological replicates. (B): Human pluripotent stem cellstransferred from MEFs to defined conditions were analyzed for neuroectoderm differentiation efficiency. H9 hESCs and IMR90-4 inducedpluripotent stem cells transferred from MEFs/KSR to E8/Matrigel or VTN-NC conditions for two passages. After each passage, cells weredifferentiated in E6/FI culture for 6 days and Pax6 expression was measured by flow cytometry. Data are presented as mean6 SD calcu-lated from two biological replicates. (C): Quantitative polymerase chain reaction was used to compare gene expression between H9hESCs maintained under E8/FI or MEF/KSR conditions. Mean6 SD were calculated from duplicate reactions. Statistical significance wascalculated using the Student’s unpaired t test (*, p< .05; **, p< .01; ***, p< .005). Results for all analyzed genes are presented in Sup-porting Information Table S3. (D): H9 hESCs were seeded onto Matrigel or VTN-NC-coated substrates at 1 3 104, 5 3 104, 1 3 105, or2 3 105 cells per square centimeter, differentiated for 6 days in E6/FI culture, and analyzed for neural rosette formation by immunofluo-rescence. Red, Pax6; green, N-cadherin. Scale bars5 100 mm. Abbreviations: EB, embryoid body; MEF, mouse embryonic fibroblast.



maintenance in E8/FI culture conditions appears to be responsi-ble for efficient neuroepithelium derivation using the small mol-ecule inhibitor-independent, E6/FI protocol.

Interestingly, the transition of hPSCs from MEF/KSR to E8/FI culture conditions and their subsequent enhanced neuroe-pithelial conversion using the E6/FI protocol were accompa-nied by a shift in gene expression within the undifferentiatedcultures. Quantitative comparison of gene expression betweenH9 hESCs maintained on MEFs/KSR and after their transfer toE8/FI conditions for two passages revealed significant differen-ces in basal expression levels of genes associated with primi-tive germ layers. For instance, both sets of hESCs expressedsimilar levels of pluripotency-associated genes LIN28 andPOU5F1, while hESCs maintained under E8/FI conditionsexpressed significantly lower amounts of NANOG, SOX2, andUTF1 (a transcriptional coactivator that promotes pluripotency[39]). Meanwhile, hESCs maintained on MEFs/KSR expressedsignificantly higher levels of genes associated with primitivemesoderm (T, EOMES, and CDH5) [40, 41] and endoderm(GATA4, GATA6, and SOX17) [42], whereas hESCs maintainedin E8/FI conditions expressed higher levels of genes associatedwith epiblast (FGF5 and OTX2) and neuroectoderm (SOX1,which was confirmed by fluorescence in situ hybridization inSupporting Information Fig. S1E). Interestingly, hESCs main-tained on MEFs/KSR also expressed significantly higher levelsof genes associated with TGFb signaling, such as NODAL,LEFTY1, LEFTY2, and NOG. To confirm hPSCs maintained underE8/FI conditions were still competent to form all primitivegerm layers, we differentiated H9 hESCs to mesoderm andendoderm fates using modifications of previously publishedprotocols [31, 32]. Whereas differentiation in E6/FI conditionsyields neuroectoderm that is uniformly Sox21/Pax61/Brachyury2/Sox172 (Fig. 1B-1D and Supporting InformationFig. S6), 4 days of differentiation in E6 medium containingCHIR99021 (CHIR; a small molecule antagonist of GSK3 thatpromotes Wnt/b-catenin signaling [32]) led to Brachyury1

mesoderm (85%6 1%) that lacked Sox2, Pax6, and Sox17expression (Supporting Information Fig. S6). Furthermore, dif-ferentiation for 1 day in E6 medium containing CHIR and acti-vin A followed by an additional 3 days in E6 mediumcontaining activin A alone yielded primitive endoderm as indi-cated by widespread Sox17 (75%6 4%) and Brachyury(58%6 21%) expression without Sox2 and Pax6 (SupportingInformation Fig. S6). Thus, while differentiation in E6/FI condi-tions alone favors neuroectoderm, mesoderm and endodermfates can be obtained by modulating Wnt/b-catenin and TGFbsignaling pathways. Based on quantitative differences in basalgene expression, we propose that culturing hPSCs in E8/FIconditions primes them for highly efficient neural induction inE6 medium alone possibly due to decreased endogenousTGFb signaling that would otherwise be inhibited by the addi-tion of exogenous small molecule inhibitors.

Efficient Neuroepithelium Formation Depends on CellSeeding Density

We also investigated whether cell seeding density was animportant variable for efficient neuroepithelial differentiationunder E6/FI adherent conditions. We varied the seeding densityof H9 hESCs in E8 medium containing ROCK inhibitor from 1 3

104 to 2 3 105 cells per square centimeter on either Matrigel

or VTN-NC-coated substrates, and then differentiated the cul-tures under E6/FI conditions. As shown in Figure 2D, cell seed-ing density could be reduced to 1 3 105 per squarecentimeter on either substrate and the cells still readily formedneuroepithelium with [mt]98% Pax61 expression (Table 1).Seeding densities of 1 3 104 or 5 3 104 cells per square centi-meter led to decreased cell outgrowth and a qualitativedecrease in the amount of rosette formation, although themajority of cells seeded at these densities still became Pax61.Therefore, higher densities are optimal for transitioning theneuroectoderm cells to definitive neuroepithelium.

Minimum Medium Requirements for NeuroepithelialDifferentiation and Survival

We demonstrated that a minimal medium consisting ofDMEM/F-12 containing five extra factors (i.e., E6 medium) issufficient to generate high purity neuroepithelium from hPSCsmaintained in E8/FI conditions. Of these five factors, we askedwhich were absolutely essential to the differentiation processby removing individual components and probing for Pax6expression and neural rosette formation at day 6 of differen-tiation. Removal of insulin resulted in substantial cell deathafter 48 hours of differentiation (Fig. 3A, panel i), whileremoval of transferrin from E6 medium yielded no change inneural rosette formation or purity of Pax61 cells (Fig. 3A,panel ii). Removal of selenium permitted neural rosette for-mation (Fig. 3A, panel iii) but also led to decreased cell viabilityby day 6 (Fig. 3A, panel iv), whereas removal of ascorbic acidhad no effect on neural rosette formation (Fig. 3A, panel v)and the resultant cells were uniformly pure for Pax6, N-cadherin, and Sox2 (Fig. 3B). Therefore, DMEM/F-12 withadded buffer (sodium bicarbonate), insulin, and selenium is suf-ficient to support derivation and survival of neuroepithelium,establishing the minimum conditions necessary for neural dif-ferentiation of hPSCs maintained under E8/FI conditions.

Differentiation of Neuroepithelium Derived Under E6/FI Conditions

To demonstrate the responsiveness of neuroepithelium derivedusing E6/FI conditions to lineage patterning cues, we modifiedpreviously published protocols [6, 7, 26, 43] to differentiatethe neuroepithelium into a culture containing motor neuronprogenitors. H9 hESCs were differentiated for 3 days in E6/FIconditions on Matrigel, which corresponds to the onset of Pax6expression and neural rosette formation, and then subjected to

Table 1. Effect of Seeding Density on Neural Conversion

Seeding density (cells persquare centimeter) Substrate % Pax61

1 3 104 Matrigel 986 0VTN-NC N.T.a

5 3 104 Matrigel 946 2VTN-NC 996 0

1 3 105 Matrigel 996 0VTN-NC 996 0

2 3 105 Matrigel 996 0VTN-NC 996 0

Pax6 was measured after 6 days of differentiating H9 human embry-onic stem cells in E6 medium. Data are presented as mean6 SD cal-culated from two biological replicates.aCells were not analyzed by flow cytometry due to limited outgrowthon VTN-NC-coated substrates.



RA and PM (a small molecule agonist of the hedgehog pathway[43]) treatment for 2 weeks to caudalize and ventralize theneural progenitors (Fig. 4A). Immunocytochemical analysis atday 10 demonstrated induction of the hindbrain/spinal cordmarker HoxB4 due to the RA/PM treatment [43], whereas con-trol cultures do not express any HoxB4 (Fig. 4A). At day 16,ventral transcription factors Nkx6.1 and Olig2 were widespreadand Hb91 motor neuron precursors were intermingled withother bIII-tubulin1 neurons (Fig. 4A). After extending differen-tiation for an additional 3 weeks, ChAT was colocalized withHb91 motor neurons, indicating their maturation (Fig. 4A). Syn-apsin was also detected at this time point, further indicatingneuronal maturation (Fig. 4A). Thus, neuroepithelium derivedusing E6/FI culture conditions has the capacity to undergo mor-phogenetic patterning to generate mature neuronal subtypes.

To determine the ability of E6-derived neuroepithelium toform other neural lineages, differentiation in E6 medium wascarried out for 4 weeks in the absence of patterning factors(Fig. 4B). While MAP2 (a neuronal marker) was detected atday 26 of differentiation by RT-PCR, glial fibrillary acidic pro-tein (GFAP) (an astrocyte marker) was not detected until day39 (Fig. 4B). At this time point, GFAP expression was alsoobserved by immunofluorescence (Fig. 4B). These findingsagree with previous hPSC studies demonstrating neuronal dif-ferentiation occurs prior to astrocyte differentiation [9] anddemonstrate that neuroepithelium derived under E8/FI condi-tions is indeed multipotent.

DISCUSSION

This work represents a simple, efficient, and completelydefined method for differentiating hPSCs to definitive neuroe-

pithelium with high purity and without the use of exogenoussmall molecules or growth factors. Methods originally pre-sented by Chambers et al. [26] and used or modified byothers [5, 44, 45] have suggested that direct suppression ofmesoderm and endoderm differentiation by small moleculesor proteins is required during hPSC differentiation to reachhighly pure Pax61 neuroepithelium. In contrast, the methodspresented in this manuscript do not require any pathwayinhibitors, instead demonstrating that E6 medium, consistingonly of DMEM/F-12, ascorbic acid, sodium bicarbonate, sele-nium, insulin, and transferrin, can yield [mt]90% Pax61 neuro-epithelium from multiple hPSC lines maintained in E8/FIconditions after only 6 days of differentiation, with H9 hESCsroutinely achieving 99%–100% purity. Furthermore, transferrinand ascorbic acid can be removed from E6 medium withoutany negative impact on neuroepithelial differentiation andsurvival.

The most critical component of the differentiation proto-col is the prior maintenance of hPSCs in fully defined E8/FIculture conditions. When E6 medium was used to differenti-ate hPSCs maintained in MEF/KSR culture, only a few polar-ized rosettes were observed and Pax6 expression wassubstantially diminished. However, once transferred to E8/FImaintenance conditions, hPSCs gained the ability to reachhigh purity Pax61 cultures when differentiated using E6/FIconditions. Interestingly, this transition in differentiationcapacity was accompanied by changes in basal gene expres-sion within the pluripotent cultures, including decreasedexpression of mesoderm- and endoderm-associated genes andincreased expression of epiblast- and neuroectoderm-associated genes. Moreover, hPSCs maintained under E8/FIconditions exhibit diminished expression of several TGFb path-way modulators, and it is possible that this decrease in

Figure 3. Minimum conditions for neuroepithelial differentiation and survival. (A): Individual components were removed from E6 medium,followed by analysis of neural rosette formation by immunocytochemistry and cell viability by bright-field microscopy after 6 days of differ-entiation. Cells in panel i are shown at day 2 due to widespread cell death. Circle in panel iv denotes a region of cell detachment. Differen-tiation was conducted on VTN-NC-coated substrates in all cases. For immunocytochemistry: red, Pax6; green, N-cadherin. Scale bars onbright-field images5 250 mm; scale bars on fluorescent images5 100 mm. (B): Analysis of neural markers by flow cytometry at day 6 of dif-ferentiation for cells cultured in DMEM/F-12, sodium bicarbonate, insulin, and selenium. Gray histograms, IgG control; red histograms, labelof interest. Mean6 SD was calculated from two biological replicates. Abbreviation: DMEM, Dulbecco’s modified Eagle’s medium.



endogenous signaling favors spontaneous differentiation toneuroectoderm in E6 medium alone. Despite this bias towardneuroectoderm differentiation, mesoderm and endoderm canbe efficiently generated from E8/FI hPSCs using Wnt/b-cateninand TGFb pathway agonists. Thus, hPSCs maintained underE8/FI conditions can be efficiently directed to all three primi-tive germ layers under completely defined conditions.

CONCLUSION

As outlined in this manuscript, the completely defined, xeno-free properties of this differentiation system make it attrac-tive for clinical applications. Overall, the high purity of neu-

roepithelium achieved without an enrichment step andrapidity of neural specification are attractive for scale-upprocedures. In addition, the neuroepithelium has thecapacity to form multiple neural lineages (e.g., neurons andastrocytes) as well as specialized cell types (e.g., motor neu-rons). Therefore, it may be of great interest to adapt existingprotocols for derivation of neurons, astrocytes, and oligoden-drocytes to this system, which could improve the translationof therapeutically relevant neural cells from the bench topto the clinic. Furthermore, the use of minimal media compo-sitions and the elimination of MEFs should make the techni-ques outlined here more broadly accessible to laboratoriesthat do not routinely culture hPSCs.

Figure 4. Differentiation of E6-derived neuroepithelium. (A): Differentiation to motor neurons. Immunofluorescence at day 10 demon-strated that only cells treated with RA and PM express HoxB4 (scale bars5 250 mm). Immunofluorescence at day 16 revealed positivelabeling for Olig2, Nkx6.1, Hb9, and bIII-tubulin (scale bars5 50 mm). Immunofluorescence at day 37 revealed Hb91/ChAT1 motor neu-rons and positive labeling for synapsin (scale bars5 20 mm). (B): Differentiation to GFAP1 astrocytes as detected by immunofluores-cence at day 39 (scale bar5 100 mm). Reverse transcriptase polymerase chain reaction demonstrated that the neuronal marker MAP2was detected prior to GFAP during the differentiation process. Abbreviations: DAPI, 40,6-diamidino-2-pheny-lindoldihydrochloride; GFAP,glial fibrillary acidic protein; PM, purmorphamine; RA, retinoic acid.



ACKNOWLEDGMENTS

We would like to thank Dr. James Thomson, Nick Propson,and Mitch Probasco for providing reagents, providing the iPS004A cell line, assisting with flow cytometry, and helpful dis-cussions on E8 medium and hPSC culture. We would also liketo thank Dr. Su-Chun Zhang, Cindy Huang, and Jeffrey Jonesfor providing H9 hESCs and IMR90-4 iPSCs cultured on MEFs.The Pax6, Nkx6.1, HoxB4, and Hb9 antibodies used in thisstudy were obtained from the Developmental Studies Hybrid-oma Bank developed under the auspices of the NICHD andmaintained by the University of Iowa. This work was sup-ported by funding from the Wisconsin Institutes for Discoveryand the Wisconsin Alumni Research Foundation.

AUTHOR CONTRIBUTION

E.S.L.: conception and design, data collection, data analysis,manuscript writing, and final approval of manuscript; M.C.E.-S.: data collection, data analysis, and final approval of manu-script; R.S.A.: conception and design, data analysis, manu-script writing, final approval of manuscript, and financialsupport.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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