STEM CELLSVolume 32, Issue 4 p. 1032-1042 Regenerative Medicine Free Access Defined Human Pluripotent Stem Cell Culture Enables Highly Efficient Neuroepithelium Derivation Without Small Molecule Inhibitors Ethan Scott Lippmann, Wisconsin Institute for Discovery and University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USASearch for more papers by this authorMaria Carolina Estevez-Silva, Wisconsin Institute for Discovery and University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USASearch for more papers by this authorRandolph Scott Ashton, Corresponding Author Wisconsin Institute for Discovery and University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USACorrespondence: Randolph S. Ashton, Ph.D., Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53715, USA. Telephone: 608-316-4312; Fax: 608-316-4606; e-mail: rashton2@wisc.eduSearch for more papers by this author Ethan Scott Lippmann, Wisconsin Institute for Discovery and University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USASearch for more papers by this authorMaria Carolina Estevez-Silva, Wisconsin Institute for Discovery and University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USASearch for more papers by this authorRandolph Scott Ashton, Corresponding Author Wisconsin Institute for Discovery and University of Wisconsin-Madison, Madison, Wisconsin, USA Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USACorrespondence: Randolph S. Ashton, Ph.D., Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53715, USA. Telephone: 608-316-4312; Fax: 608-316-4606; e-mail: rashton2@wisc.eduSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The embryonic neuroepithelium gives rise to the entire central nervous system in vivo, making it an important tissue for developmental studies and a prospective cell source for regenerative applications. Current protocols for deriving homogenous neuroepithelial cultures from human pluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followed by a manual isolation step or adherent differentiation using small molecule inhibitors. Here, we report that hPSCs maintained under chemically defined, feeder-independent, and xeno-free conditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax6+/N-cadherin+ with widespread rosette formation) within 6 days under adherent conditions, without small molecule inhibitors, and using only minimalistic medium consisting of Dulbecco\'s modified Eagle\'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 this high 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 neural compliant state upon transfer to these defined conditions while still maintaining their ability to generate all three germ layers. Overall, this fully defined and scalable protocol should be broadly useful for generating therapeutic neural cells for regenerative applications. Stem Cells 2014;32:1032–1042 Introduction Over the past decade, significant advancements have been made in differentiating human pluripotent stem cells (hPSCs) toward diverse neural lineages of the central nervous system (CNS) and peripheral nervous system including GABA neurons 1, floor plate precursors 2, dopaminergic neurons 3-5, spinal motor neurons 6, 7, nociceptors 8, astrocytes 9, and oligodendrocytes 10-13. These achievements have culminated in recent demonstrations that engraftment of hPSC-derived oligodendrocytes, GABA, and dopaminergic neurons can alleviate symptoms in rodent models of spinal cord injury 13, 14, Huntington\'s 1, and Parkinson\'s 3, 5 disease, respectively, thereby highlighting the prospective use of hPSC-derived neural cells for translational medicine. However, these prior examples continue to use undefined or xenogeneic culture components for hPSC maintenance and differentiation (e.g., mouse embryonic fibroblast [MEF] feeder layers supplemented with knockout serum replacer [KSR], Matrigel, or medium containing serum albumin), which could ultimately limit the scalability and clinical utility of these protocols 3, 5, 15. Additionally, many neuroepithelium derivation protocols still require manual enrichment 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 differentiation. Therefore, an important advancement in this field would be the adaptation and optimization of current hPSC neural differentiation protocols to xeno-free and defined systems to facilitate clinical implementation of therapeutic cell products and assist in standardizing differentiation procedures from lab-to-lab. In the developing human embryo, the entire CNS arises from neuroepithelial cells that constitute the primordial neural tube. Thus, protocols for generating CNS cells from hPSCs typically proceed from the Oct-4 (POU5F1)+/Nanog+ pluripotent state through an initial Pax6+/N-cadherin+ neuroepithelial state 1, 7, 9, 17, which can be identified in culture by columnar cell morphologies and the formation of polarized rosette structures 25, although polarization is not required for neural differentiation 3. Robust generation of hPSC-derived neuroepithelial cells has been achieved through either embryoid body (EB) formation followed by plating 25 or adherent differentiation using small molecule inhibition of SMAD signaling followed by passaging 26. However, these protocols were developed and standardized using hPSCs cultured on feeder layers and differentiated with xenogenic or undefined reagents 1, 3, 5, 8, 17, 25-27. With the recent establishment of novel defined protocols for hPSC derivation and culture 23, it remains to be investigated whether traditional neuroepithelial cell derivation methods are still optimal for hPSCs maintained under such defined conditions. Indeed, the variability in source 28, 29 and derivation protocols between different hPSC lines can also affect neural differentiation efficiency due to variations in epigenetic regulation and basal gene expression 30. As such, the overall goal for this study was to merge advancements in defined culture systems with existing protocols to neuralize hPSCs under chemically defined, minimalistic, xeno-free, and scalable conditions. Using hPSCs maintained in chemically defined E8 medium 23 (E8) on either Matrigel-coated or recombinant human vitronectin peptide (VTN-NC)-coated substrates 23, we demonstrate a procedure where hPSCs can be differentiated into neuroepithelium with high purity after only 6 days and without the presence of small molecule SMAD inhibitors. Differentiation is initiated by high density hPSC seeding and culture in defined medium (i.e., Dulbecco\'s modified Eagle\'s medium [DMEM]/F-12, ascorbic acid, sodium bicarbonate, selenium, human transferrin, and human insulin, termed E6 medium) under adherent conditions. No difference in differentiation efficiency was observed using culture surfaces coated with either Matrigel or VTN-NC (98% ± 2% and 99% ± 1% Pax6+/N-cadherin+, respectively), and further analysis of E6 medium revealed that DMEM/F-12, sodium bicarbonate, selenium, and insulin were the minimum components necessary for neuroepithelial differentiation and survival. The E6-derived neuroepithelium could be differentiated to motor neurons, indicating the neural progenitors are responsive to lineage patterning cues, and astrocytes, indicating the neuroepithelium is multipotent. Interestingly, hPSCs maintained on MEFs prior to differentiation in E6 medium conditions do not efficiently generate neuroepithelium, but if transferred from MEFs to E8/feeder-independent (FI) maintenance conditions, they acquire the capacity for efficient neuroepithelial differentiation. This change in differentiation capacity is accompanied by increased expression of genes associated with epiblast and neuroectoderm fates and decreased expression of genes associated with mesoderm and endoderm differentiation, indicating a shift in differentiation bias. However, while hPSCs maintained in E8/FI conditions become biased toward a neuroectodermal fate, they maintain their pluripotent capabilities and can still be effectively differentiated toward mesodermal and endodermal fates. hPSCs were obtained as frozen vials and banked under FI conditions in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada (www.stemcell.com)). hPSCs were then thawed and cultured 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 µg/l sodium selenite (Sigma), 19.4 mg/l insulin (Sigma), 10.7 mg/l transferrin (Sigma), 100 µg/l FGF2 (Waisman Clinical Biomanufacturing Facility, University of Wisconsin-Madison), and 2 µg/l TGFβ1 (Peprotech, Rocky Hill, NJ (www.peprotech.com)). pH of E8 medium was adjusted to 7.4 and osmolarity was adjusted to 340 mOsm with NaCl. hPSCs were maintained on Matrigel (BD Biosciences, San Jose, CA (www.bdbiosciences.com)) or VTN-NC (provided by Dr. James Thomson) 23. Cell lines used in this study were H9 human embryonic stem cells (hESCs) (passages 25–50), H1 hESCs (passages 28–36), IMR90-4 iPSCs (passages 26–62), and 004A iPSCs (passages 3–7). For some comparative experiments, hPSCs were maintained on irradiated MEFs in standard unconditioned medium: DMEM/F-12 containing 20% KSR (Life Technologies), 1× Minimum Essential Medium (MEM) nonessential amino acids (Life Technologies), 1 mM l-glutamine (Sigma), 0.1 mM β-mercaptoethanol (Sigma), and 4 ng/ml FGF2. Cells were routinely passaged with Versene (Life Technologies) as previously described 23. ROCK inhibitor (1 µM) (Y-27632; R&D Systems, Minneapolis, MN (www.rndsystems.com)) was included when passaging H1 hESCs onto VTN-NC to facilitate attachment and removed 24 hours after attachment. hPSCs were washed once with phosphate-buffered saline (PBS; Life Technologies), incubated with accutase (Life Technologies) for 3 minutes, and collected by centrifugation. hPSCs were then plated onto Matrigel or VTN-NC at a density of 2 × 105 cells per square centimeter in E8 medium containing 10 µM ROCK inhibitor and cultured overnight. The following morning, cells were changed to E6 medium, E6 containing 10 µM SB431542 (Cellagentech, San Diego, CA (www.cellagentech.com)), or E6 containing 10 µM SB431542 and 200 ng/ml recombinant human noggin (R&D Systems) to initiate differentiation. E6 medium is the same formulation as E8 medium but without FGF2 and TGFβ1. Medium was changed every day until cells were used for analysis. After these initial experiments, additional seeding densities of 1 × 105 cells per square centimeter, 5 × 104 cells per square centimeter, and 1 × 104 cells per square centimeter were tested with E6 medium. Further experiments removed individual components from E6 medium while qualitatively analyzing cell viability. H9 hESCs were singularized and plated overnight in E8 medium at a density of 1.5 × 105 cells per square centimeter on Matrigel as described above. Mesoderm and endoderm differentiation schemes were adapted from previous protocols 31, 32. To generate mesoderm, cells were differentiated for 4 days in E6 medium containing 6 µM CHIR99021. To generate endoderm, cells were differentiated for 1 day in E6 medium containing 6 µM CHIR99021 and 100 ng/ml activin A (R&D Systems), followed by 3 days of activin A alone. To form EBs, H9 hESCs were incubated with 2 mg/ml dispase (Life Technologies) for 10–15 minutes to facilitate colony detachment, washed twice with DMEM/F-12, and transferred to low-attachment six-well plates (Corning, Corning, NY (www.corning.com)) in E6 medium. Medium was changed every other day. At day 4 of differentiation, whole EBs were transferred to standard tissue culture polystyrene dishes or glass chamber slides coated with Matrigel. Resultant cells were maintained in E6 medium for the duration of each experiment. H9 hESCs were singularized and plated overnight in E8 medium at a density of 1.5 × 105 cells per square centimeter on Matrigel as described above. Cells were differentiated in E6 medium for 3 days, at which point single rosettes were manually isolated and replated in Matrigel-coated chamber slides. Cells were then patterned with 1 µM retinoic acid (RA; Sigma) and 0.1 µM purmorphamine (PM; EMD Millipore, Billerica, MA (www.millipore.com)) as described in Results. Cyclic AMP (1 µM) (cAMP; Sigma), 10 ng/ml brain-derived neurotrophic factor (BDNF; Peprotech), and 10 ng/ml glial-derived neurotrophic factor (GDNF; Peprotech) were added as indicated to support neuronal survival. H9 hESCs were singularized and plated overnight in E8 medium at a density of 1.5 × 105 cells per square centimeter on Matrigel as described above. Cells were differentiated in E6 medium for 6 days, at which point single rosettes were manually isolated and replated in Matrigel-coated chamber slides. Cells were then differentiated for 4 weeks as described in Results. cAMP (1 µM), BDNF (10 ng/ml), and GDNF (10 ng/ml) were added as indicated. Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Picric acid (0.1%) (Fisher, Pittsburgh, PA (www.fishersci.com)) was included when labeling for choline acetyltransferase (ChAT). After additional washes in PBS, cells were blocked and permeabilized in tris-buffered saline (TBS)-DT (containing 5% donkey serum [Sigma] and 0.3% Triton X-100 [TX-100; Fisher]) for at least 1 hour at room temperature. Primary antibodies were diluted in TBS-DT and cells were incubated in these antibodies overnight at 4°C. Antibodies are listed in Supporting Information Table S1. The following day, chambers were rinsed once with TBS containing 0.3% Triton X-100 (TBST) and then washed five times, 15 minutes apiece, with TBST. Secondary antibodies (Supporting Information Table S1) were diluted in TBS-DT and incubated on the cells for 1 hour at room temperature and nuclei were subsequently counterstained with 300 nM 4′,6-diamidino-2-pheny-lindoldihydrochloride (DAPI) for 10 minutes. Afterward, cells were washed once for 25 minutes with TBS and three additional times for 10 minutes apiece. Cells cultured in chamber slides were then mounted with Prolong Gold Antifade Reagent (Life Technologies) and visualized using a Nikon A1R confocal microscope. Cells cultured in 6- or 12-well plates were visualized using a Nikon Ti-E microscope. Nikon NIS-Elements software was used for image analysis. Bright-field images were acquired using a Nikon TS100 microscope. Cells were harvested from 6- or 12-well plates by washing once with PBS and incubating with accutase for 3–5 minutes. Cells were then recovered by centrifugation and fixed in 4% paraformaldehyde for 10 minutes at room temperature. After blocking with PBS containing 10% normal serum (goat or donkey serum depending on the species of primary antibody; Sigma) and 0.1% Triton X-100 for at least 30 minutes at room temperature, cells were incubated with primary antibodies for 1 hour at room temperature or overnight at 4°C. Antibodies are listed in Supporting Information Table S1. IgG controls were included for each species of antibody (Life Technologies). After washing twice with PBS containing 0.75% bovine serum albumin (BSA; Life Technologies), cells were incubated for 30–60 minutes at room temperature in PBS containing 10% normal serum and secondary antibodies (Supporting Information Table S1). After washing twice with PBS containing 0.75% BSA, cells were analyzed on a FACSCanto (BD Biosciences), and data were analyzed using Cyflogic software. Positive events were determined by gating above the top 1% of the IgG control histograms. Total RNA was extracted from cells using Trizol reagent (Life Technologies) according to the manufacturer\'s instructions. Five micrograms of total RNA was then subjected to reverse-transcription using a Thermoscript RT-PCR kit (Life Technologies) in a 20 µl mixture according to the manufacturer\'s instructions. Resultant cDNA (0.5 µl) was then amplified in a 25 µl mixture containing 10× PCR buffer, 0.2 mM dNTP, 1.5 mM MgCl2, 0.5 µM of each primer, and 1 U Taq DNA polymerase (Life Technologies). Amplified products were resolved on 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 Supporting Information Table S2. To confirm the fidelity of primers designed for genes not detected in our samples, genomic DNA or positive control cDNA was used. Quantitative PCR analysis was conducted between H9 hESCs maintained on MEFs and hESCs transferred to E8/FI conditions for two passages. cDNA was mixed with Taqman Gene Expression Master Mix (Life Technologies) and loaded onto Human Stem Cell Taqman Array Plates (Life Technologies). Amplification was carried out on a BioRad CFX96 thermocycler according to the manufacturer\'s instructions. Relative gene expression was quantified using the comparative cycle threshold (CT) method using GAPDH as the housekeeping gene. SOX1 and OTX2 expression were quantified using individual Taqman primers with RSP18 as the housekeeping gene. H9 hESCs cultured in chamber slides were fixed in 2% paraformaldehyde for 10 minutes and permeabilized in 70% ethanol overnight at 4°C. Cells were then incubated in wash buffer consisting of 10% formamide (Life Technologies) and 2× 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 hybridization probes were purchased from Biosearch Technologies, Petaluma, CA (www.biosearchtech.com) and diluted to 1.25 µM in hybridization buffer consisting of 100 mg/ml dextran sulfate (Sigma) and 10% formamide in 2× SSC. Cells were incubated with probes overnight in a 37°C humidified incubator. The following day, after three washes and a DAPI counterstain, the slides were mounted with Prolong Gold Antifade Reagent and immediately visualized on a Nikon Ti-E microscope. hPSCs Cultured and Differentiated in E8/FI Conditions Efficiently Generate Neuroepithelium Without the Use of Small Molecule Inhibitors A recent report by Chambers et al. demonstrated rapid and efficient hPSC neuralization using small molecule inhibitors and recombinant proteins, yielding [mt]80% neuroepithelium after 11 days 26. The original protocol neuralized hESCs maintained on MEFs in KSR-containing media (MEFs/KSR) using SB431542 (an inhibitor of TGFβ signaling) and noggin (an inhibitor of bone morphogenetic protein [BMP] signaling), and follow-up protocols have replaced noggin with the small molecules dorsomorphin or LDN-193189 8, 33. Such factors were hypothesized in these previous studies to promote ectoderm neuralization and suppress endoderm and mesoderm formation by inhibiting endogenous BMP and activin/TGFβ signaling. Here, we hypothesized that hPSCs maintained in defined medium under FI conditions 23 might have different basal signaling activity compared to hPSCs maintained on MEFs/KSR. Therefore, we tested whether suppression of activin/TGFβ and BMP signaling is still necessary to efficiently derive neuroepithelium from hPSCs cultured under defined conditions. We cultured H9 hESCs in E8/FI conditions 23 on Matrigel-coated substrates and verified their expression of pluripotency markers Sox2, Oct4, and Nanog by immunocytochemistry (ICC) and flow cytometry (FC) (Supporting Information Fig. S1A-S1C). Upon positive confirmation, these cells were subcultured onto Matrigel-coated plates at 2 × 105 cells per square centimeter in E8 medium containing ROCK inhibitor (Fig. 1A). The following day, differentiation was initiated by replacing E8 with E6 medium (i.e., E8 without FGF2 and TGFβ1), E6 medium supplemented with SB431542, or E6 medium supplemented with both SB431542 and noggin for an additional 6 days to probe inhibitor requirements for efficient neuroepithelial derivation. Differentiation within the cultures was monitored every other day and time course reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed a gradual decrease in the expression of pluripotency genes POU5F1 (Oct4) and NANOG under all differentiation conditions (Fig. 1B and Supporting Information Fig. S2). After 2–4 days of differentiation, all cultures acquired a neuroectodermal gene expression profile as indicated by the absence of Sox17 (a definitive endoderm marker 34), diminishing expression of T (brachyury) which is expressed in primitive streak mesoderm 35, and activation of the neuroectoderm fate determinant 36 PAX6. As previously observed 17, SOX2 was expressed in both undifferentiated H9 hPSCs and neuroectodermal cells, but interestingly, we also detected increasing levels of SOX1 (a neuroectoderm marker 36), OTX2 (a midbrain and forebrain marker 17), and FOXG1 (a forebrain marker 26) expression throughout differentiation. By day 6, the cultures under all conditions acquired a rostral/dorsal neuroectodermal fate as indicated by OTX2 and FOXG1 expression in the absence of HOXB4 (a hindbrain/spinal cord marker 17) and OLIG2 (a ventral transcription factor 7) expression. Figure 1Open in figure viewerPowerPoint 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 expression in differentiating H9 human embryonic stem cells (hESCs). \"SB” indicates addition of SB431542 and \"N” indicates addition of noggin. (C): Flow cytometry analysis of Pax6. Data are presented as mean ± SD calculated from at least two biological replicates. Differentiation was conducted on Matrigel-coated substrates unless otherwise specified. (D): Images of neural rosette formation on day 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 coated with recombinant vitronectin peptide. Scale bars in Pax6/N-cadherin-stained images are 250 µm; scale bars in Otx2/Sox2-stained images are 50 µm. (E): Representative flow cytometry histograms of N-cadherin, Otx2, and Sox2 expression at day 6 of differentiating H9 hESCs in E6 medium. Data are representative of two biological replicates and mean ± SD are listed in Results. Gray histogram, IgG control; red histogram, label of interest. (F): Progression of Sox1 expression in E6/FI conditions with and without retinoic acid. Scale bars = 100 µm. To further assess neural conversion, we quantitatively analyzed the percentage of Pax6+ neuroectodermal cells by FC and qualitatively affirmed acquisition of a neuroepithelial state by ICC (Fig. 1C, 1D). By day 2, Pax6 expression was not detected in differentiating H9 hESCs under any experimental conditions (E6, E6+SB432542, or E6+SB431542+noggin), but by day 3 of differentiation in E6 medium alone, 72% ± 4% of cells expressed Pax6. By days 4–6 of differentiation, this percentage increased to [mt]90% Pax6+ neuroectodermal cells under all differentiation conditions, and at day 6, H9 hESCs differentiated in just E6 medium were uniform in their expression of Pax6 (98% ± 2%), N-cadherin (100% ± 0%), Otx2 (95% ± 0%), and Sox2 (98% ± 1%) (Fig. 1C-1E). As an additional indicator of neural conversion, N-cadherin expression was observed to increase throughout the 6 days of differentiation while E-cadherin, an hPSC marker 37, concurrently decreased (Supporting Information Fig. S3). This pattern of cadherin expression aligns with recent in vivo results indicating that E-cadherin is coexpressed with N-cadherin at the midbrain portion of the recently polarized Hamburger and Hamilton (HH) stage 9 neural tube before eventually disappearing by HH stage 11 38. As a final qualitative assessment of H9 hESC neuroepithelial conversion, ICC at day 6 of differentiation under all experimental conditions confirmed nuclear Pax6 expression and widespread polarization of N-cadherin cell membrane proteins toward apical lumens within neural rosette structures (Fig. 1D), a definitive hallmark of neuroepithelium 16, 17, 25. Thus, H9 hESCs maintained in E8/FI conditions can be efficiently differentiated into highly pure neuroepithelium on Matrigel-coated substrates using E6 medium without SMAD inhibitors, and this is not a cell line-dependent phenomena as IMR90-4 iPSCs cultured and differentiated in the same manner also undergo efficient neuroepithelial conversion (87% ± 9% Pax6+; Fig. 1C). All experiments thus far used Matrigel as the culture substrate during maintenance and differentiation. Therefore, to construct a completely defined system, we maintained H9 hESCs in E8/FI conditions on VTN-NC-coated substrates 23 and then differentiated the cells in E6 medium also on similar substrates. This yielded 99% ± 1% Pax6+ cells from H9 hESCs after 6 days (Fig. 1C) again with high levels of N-cadherin (100% ± 0%), Otx2 (91% ± 6%), and Sox2 (98% ± 1%) expression (Fig. 1E) and uniform neural rosette formation (Fig. 1D). Additionally, H1 hESCs were tested under these conditions and yielded 90% ± 1% Pax6+ cells again with widespread neural rosette formation (Fig. 1C and Supporting Information Fig. S4). Therefore, the effectiveness of the differentiation procedure does not rely on Matrigel as a substrate. To demonstrate the clinical applicability of this protocol, we also differentiated the iPSC line 004A, which was derived under E8/FI conditions 23 and never exposed to MEF coculture, for 6 days in E6 medium on VTN-NC-coated substrates. Similar to prior results, this also yielded cultures that were 90% ± 1% Pax6+, 90% ± 1% Otx2+, and 99% ± 0% Sox2+ with widespread formation of Pax6+/N-cadherin+ neural rosettes (Supporting Information Fig. S5). Thus, the fully defined E6/VTN-NC culture system alone is sufficient to efficiently generate nearly pure neuroepithelium from hPSC lines maintained in E8/FI conditions. Previous reports have demonstrated that Pax6 precedes Sox1 expression in human neuroectodermal tissues generated both in vitro 17 and in vivo 36. However, SOX1 mRNA was detectable at basal levels in our undifferentiated hPSC cultures and appeared to increase in relative abundance throughout the differentiation process (Fig. 1B). Therefore, we investigated the progression of Sox1 protein expression within H9 hESCs differentiated using E6/FI culture. By ICC analysis, Pax6 expression was uniform by day 6 of differentiation in E6/Matrigel or VTN-NC culture, while in contrast Sox1 was only sparsely expressed by day 6 and not even uniformly expressed by day 9 of differentiation (Fig. 1F). If RA was added to facilitate neural conversion at day 3 of differentiation, increased Sox1 expression was observed at days 6 and 9 on both Matrigel- and VTN-NC-coated substrates, but this was still preceded by uniform Pax6 expression. Therefore, Pax6 precedes Sox1 expression in neuroepithelium derived under E6/FI conditions, which is in good agreement with human developmental principles and previously established differentiation protocols 17, 36. To determine whether prior hPSC maintenance in E8/FI culture conditions is critical to the E6/FI protocol, we evaluated the efficiency of neuroepithelium derivation from H9 hESCs maintained in the undifferentiated state using MEFs/KSR culture. Whereas H9 hESCs from E8/FI conditions formed neural rosettes with 98%–99% Pax6+ cells after 6 days of differentiation in E6 medium, hESCs from MEFs/KSR conditions generate few polarized rosettes and only 39% ± 0% of the cells were Pax6+ (Fig. 2A). Thus, hESCs maintained on MEFs/KSR do not efficiently form neuroepithelium when differentiated in E6 medium alone. Furthermore, to determine whether adherent conditions were critical for neuroepithelial differentiation, we formed EBs from H9 hESCs maintained in E8/FI conditions, differentiated these EBs for 4 days in E6 medium, and plated the EBs onto Matrigel-coated dishes for an additional 2 days of differentiation. Some EBs produced regions with polarized rosette morphology but only 51% ± 2% of the cells was Pax6+ (Fig. 2A), suggesting adherent conditions are also required for the E6 medium protocol\'s high differentiation efficiency. Figure 2Open in figure viewerPowerPoint Neuroepithelium derivation from H9 human embryonic stem cells (hESCs) maintained and differentiated under various conditions. (A): H9 hESCs maintained in E8/Matrigel or VTN-NC conditions were differentiated in E6 medium under adherent conditions or free-floating EBs as described in Materials and Methods. Alternatively, hESCs were maintained in MEF/KSR conditions and differentiated in E6/Matrigel culture under adherent conditions. After 6 days of differentiation, cultures were examined for rosette formation by immunofluorescence. Red, Pax6; green, N-cadherin. Scale bars = 50 µm. Cultures were also probed for Pax6 expression by flow cytometry at this time point. Data are presented as mean ± SD calculated from two biological replicates. (B): Human pluripotent stem cells transferred from MEFs to defined conditions were analyzed for neuroectoderm differentiation efficiency. H9 hESCs and IMR90-4 induced pluripotent stem cells transferred from MEFs/KSR to E8/Matrigel or VTN-NC conditions for two passages. After each passage, cells were differentiated in E6/FI culture for 6 days and Pax6 expression was measured by flow cytometry. Data are presented as mean ± SD calculated from two biological replicates. (C): Quantitative polymerase chain reaction was used to compare gene expression between H9 hESCs maintained under E8/FI or MEF/KSR conditions. Mean ± SD were calculated from duplicate reactions. Statistical significance was calculated using the Student\'s unpaired t test (*, p   .05; **, p   .01; ***, p   .005). Results for all analyzed genes are presented in Supporting Information Table S3. (D): H9 hESCs were seeded onto Matrigel or VTN-NC-coated substrates at 1 × 104, 5 × 104, 1 × 105, or 2 × 105 cells per square centimeter, differentiated for 6 days in E6/FI culture, and analyzed for neural rosette formation by immunofluorescence. Red, Pax6; green, N-cadherin. Scale bars = 100 µm. Abbreviations: EB, embryoid body; MEF, mouse embryonic fibroblast. Since hPSC maintenance in E8/FI conditions appeared to be required for efficient, adherent, and small molecule-independent neuroepithelium derivation, we explored whether E8/FI conditions could convert hPSCs previously maintained in MEF/KSR culture to a more compliant state. Thus, we transferred H9 hESCs and IMR90-4 iPSCs maintained in MEF/KSR conditions to E8/Matrigel or VTN-NC culture, and conducted differentiation in E6/FI conditions after one and two rounds of passaging. When differentiated directly from MEF/KSR coculture, these lines exhibited low neuroectoderm differentiation efficiency as assessed by FC for Pax6 expression (27% ± 4% for H9 hESCs and 2% ± 1% for IMR90-4 iPSCs; Fig. 2B). However, after one passage under E8/FI conditions, their differentiation efficiency increased dramatically (82%–90% and 43%–53% Pax6+ cells derived from H9 hESCs and IMR90-4 iPSCs, respectively). After a second passage, H9 hESCs could form 95%–97% Pax6+ cultures, and IMR90-4 iPSCs differentiated on VTN-NC-coated substrates were able to reach 80% ± 13% Pax6+ cultures. Thus, hPSC maintenance in E8/FI culture conditions appears to be responsible for efficient neuroepithelium derivation using the small molecule inhibitor-independent, E6/FI protocol. Interestingly, the transition of hPSCs from MEF/KSR to E8/FI culture conditions and their subsequent enhanced neuroepithelial conversion using the E6/FI protocol were accompanied by a shift in gene expression within the undifferentiated cultures. Quantitative comparison of gene expression between H9 hESCs maintained on MEFs/KSR and after their transfer to E8/FI conditions for two passages revealed significant differences in basal expression levels of genes associated with primitive germ layers. For instance, both sets of hESCs expressed similar levels of pluripotency-associated genes LIN28 and POU5F1, while hESCs maintained under E8/FI conditions expressed significantly lower amounts of NANOG, SOX2, and UTF1 (a transcriptional coactivator that promotes pluripotency 39). Meanwhile, hESCs maintained on MEFs/KSR expressed significantly higher levels of genes associated with primitive mesoderm (T, EOMES, and CDH5) 40, 41 and endoderm (GATA4, GATA6, and SOX17) 42, whereas hESCs maintained in E8/FI conditions expressed higher levels of genes associated with epiblast (FGF5 and OTX2) and neuroectoderm (SOX1, which was confirmed by fluorescence in situ hybridization in Supporting Information Fig. S1E). Interestingly, hESCs maintained on MEFs/KSR also expressed significantly higher levels of genes associated with TGFβ signaling, such as NODAL, LEFTY1, LEFTY2, and NOG. To confirm hPSCs maintained under E8/FI conditions were still competent to form all primitive germ layers, we differentiated H9 hESCs to mesoderm and endoderm fates using modifications of previously published protocols 31, 32. Whereas differentiation in E6/FI conditions yields neuroectoderm that is uniformly Sox2+/Pax6+/Brachyury−/Sox17− (Fig. 1B-1D and Supporting Information Fig. S6), 4 days of differentiation in E6 medium containing CHIR99021 (CHIR; a small molecule antagonist of GSK3 that promotes Wnt/β-catenin signaling 32) led to Brachyury+ mesoderm (85% ± 1%) that lacked Sox2, Pax6, and Sox17 expression (Supporting Information Fig. S6). Furthermore, differentiation for 1 day in E6 medium containing CHIR and activin A followed by an additional 3 days in E6 medium containing activin A alone yielded primitive endoderm as indicated by widespread Sox17 (75% ± 4%) and Brachyury (58% ± 21%) expression without Sox2 and Pax6 (Supporting Information Fig. S6). Thus, while differentiation in E6/FI conditions alone favors neuroectoderm, mesoderm and endoderm fates can be obtained by modulating Wnt/β-catenin and TGFβ signaling pathways. Based on quantitative differences in basal gene expression, we propose that culturing hPSCs in E8/FI conditions primes them for highly efficient neural induction in E6 medium alone possibly due to decreased endogenous TGFβ signaling that would otherwise be inhibited by the addition of exogenous small molecule inhibitors. We also investigated whether cell seeding density was an important variable for efficient neuroepithelial differentiation under E6/FI adherent conditions. We varied the seeding density of H9 hESCs in E8 medium containing ROCK inhibitor from 1 × 104 to 2 × 105 cells per square centimeter on either Matrigel or VTN-NC-coated substrates, and then differentiated the cultures under E6/FI conditions. As shown in Figure 2D, cell seeding density could be reduced to 1 × 105 per square centimeter on either substrate and the cells still readily formed neuroepithelium with [mt]98% Pax6+ expression (Table 1). Seeding densities of 1 × 104 or 5 × 104 cells per square centimeter led to decreased cell outgrowth and a qualitative decrease in the amount of rosette formation, although the majority of cells seeded at these densities still became Pax6+. Therefore, higher densities are optimal for transitioning the neuroectoderm cells to definitive neuroepithelium. Pax6 was measured after 6 days of differentiating H9 human embryonic stem cells in E6 medium. Data are presented as mean ± SD calculated from two biological replicates. Cells were not analyzed by flow cytometry due to limited outgrowth on VTN-NC-coated substrates. We demonstrated that a minimal medium consisting of DMEM/F-12 containing five extra factors (i.e., E6 medium) is sufficient to generate high purity neuroepithelium from hPSCs maintained in E8/FI conditions. Of these five factors, we asked which were absolutely essential to the differentiation process by removing individual components and probing for Pax6 expression and neural rosette formation at day 6 of differentiation. Removal of insulin resulted in substantial cell death after 48 hours of differentiation (Fig. 3A, panel i), while removal of transferrin from E6 medium yielded no change in neural rosette formation or purity of Pax6+ cells (Fig. 3A, panel ii). Removal of selenium permitted neural rosette formation (Fig. 3A, panel iii) but also led to decreased cell viability by day 6 (Fig. 3A, panel iv), whereas removal of ascorbic acid had 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 with added buffer (sodium bicarbonate), insulin, and selenium is sufficient to support derivation and survival of neuroepithelium, establishing the minimum conditions necessary for neural differentiation of hPSCs maintained under E8/FI conditions. Figure 3Open in figure viewerPowerPoint 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 differentiation. Cells in panel i are shown at day 2 due to widespread cell death. Circle in panel iv denotes a region of cell detachment. Differentiation was conducted on VTN-NC-coated substrates in all cases. For immunocytochemistry: red, Pax6; green, N-cadherin. Scale bars on bright-field images = 250 µm; scale bars on fluorescent images = 100 µm. (B): Analysis of neural markers by flow cytometry at day 6 of differentiation for cells cultured in DMEM/F-12, sodium bicarbonate, insulin, and selenium. Gray histograms, IgG control; red histograms, label of interest. Mean ± SD was calculated from two biological replicates. Abbreviation: DMEM, Dulbecco\'s modified Eagle\'s medium. To demonstrate the responsiveness of neuroepithelium derived using E6/FI conditions to lineage patterning cues, we modified previously published protocols 6, 7, 26, 43 to differentiate the neuroepithelium into a culture containing motor neuron progenitors. H9 hESCs were differentiated for 3 days in E6/FI conditions on Matrigel, which corresponds to the onset of Pax6 expression and neural rosette formation, and then subjected to RA and PM (a small molecule agonist of the hedgehog pathway 43) treatment for 2 weeks to caudalize and ventralize the neural progenitors (Fig. 4A). Immunocytochemical analysis at day 10 demonstrated induction of the hindbrain/spinal cord marker HoxB4 due to the RA/PM treatment 43, whereas control cultures do not express any HoxB4 (Fig. 4A). At day 16, ventral transcription factors Nkx6.1 and Olig2 were widespread and Hb9+ motor neuron precursors were intermingled with other βIII-tubulin+ neurons (Fig. 4A). After extending differentiation for an additional 3 weeks, ChAT was colocalized with Hb9+ motor neurons, indicating their maturation (Fig. 4A). Synapsin was also detected at this time point, further indicating neuronal maturation (Fig. 4A). Thus, neuroepithelium derived using E6/FI culture conditions has the capacity to undergo morphogenetic patterning to generate mature neuronal subtypes. Figure 4Open in figure viewerPowerPoint Differentiation of E6-derived neuroepithelium. (A): Differentiation to motor neurons. Immunofluorescence at day 10 demonstrated that only cells treated with RA and PM express HoxB4 (scale bars = 250 µm). Immunofluorescence at day 16 revealed positive labeling for Olig2, Nkx6.1, Hb9, and βIII-tubulin (scale bars = 50 µm). Immunofluorescence at day 37 revealed Hb9+/ChAT+ motor neurons and positive labeling for synapsin (scale bars = 20 µm). (B): Differentiation to GFAP+ astrocytes as detected by immunofluorescence at day 39 (scale bar = 100 µm). Reverse transcriptase polymerase chain reaction demonstrated that the neuronal marker MAP2 was detected prior to GFAP during the differentiation process. Abbreviations: DAPI, 4′,6-diamidino-2-pheny-lindoldihydrochloride; GFAP, glial fibrillary acidic protein; PM, purmorphamine; RA, retinoic acid. To determine the ability of E6-derived neuroepithelium to form other neural lineages, differentiation in E6 medium was carried out for 4 weeks in the absence of patterning factors (Fig. 4B). While MAP2 (a neuronal marker) was detected at day 26 of differentiation by RT-PCR, glial fibrillary acidic protein (GFAP) (an astrocyte marker) was not detected until day 39 (Fig. 4B). At this time point, GFAP expression was also observed by immunofluorescence (Fig. 4B). These findings agree with previous hPSC studies demonstrating neuronal differentiation occurs prior to astrocyte differentiation 9 and demonstrate that neuroepithelium derived under E8/FI conditions is indeed multipotent. This work represents a simple, efficient, and completely defined method for differentiating hPSCs to definitive neuroepithelium with high purity and without the use of exogenous small molecules or growth factors. Methods originally presented by Chambers et al. 26 and used or modified by others 5, 44, 45 have suggested that direct suppression of mesoderm and endoderm differentiation by small molecules or proteins is required during hPSC differentiation to reach highly pure Pax6+ neuroepithelium. In contrast, the methods presented in this manuscript do not require any pathway inhibitors, instead demonstrating that E6 medium, consisting only of DMEM/F-12, ascorbic acid, sodium bicarbonate, selenium, insulin, and transferrin, can yield [mt]90% Pax6+ neuroepithelium from multiple hPSC lines maintained in E8/FI conditions after only 6 days of differentiation, with H9 hESCs routinely achieving 99%–100% purity. Furthermore, transferrin and ascorbic acid can be removed from E6 medium without any negative impact on neuroepithelial differentiation and survival. The most critical component of the differentiation protocol is the prior maintenance of hPSCs in fully defined E8/FI culture conditions. When E6 medium was used to differentiate hPSCs maintained in MEF/KSR culture, only a few polarized rosettes were observed and Pax6 expression was substantially diminished. However, once transferred to E8/FI maintenance conditions, hPSCs gained the ability to reach high purity Pax6+ cultures when differentiated using E6/FI conditions. Interestingly, this transition in differentiation capacity was accompanied by changes in basal gene expression within the pluripotent cultures, including decreased expression of mesoderm- and endoderm-associated genes and increased expression of epiblast- and neuroectoderm-associated genes. Moreover, hPSCs maintained under E8/FI conditions exhibit diminished expression of several TGFβ pathway modulators, and it is possible that this decrease in endogenous signaling favors spontaneous differentiation to neuroectoderm in E6 medium alone. Despite this bias toward neuroectoderm differentiation, mesoderm and endoderm can be efficiently generated from E8/FI hPSCs using Wnt/β-catenin and TGFβ pathway agonists. Thus, hPSCs maintained under E8/FI conditions can be efficiently directed to all three primitive germ layers under completely defined conditions. As outlined in this manuscript, the completely defined, xeno-free properties of this differentiation system make it attractive for clinical applications. Overall, the high purity of neuroepithelium achieved without an enrichment step and rapidity of neural specification are attractive for scale-up procedures. In addition, the neuroepithelium has the capacity to form multiple neural lineages (e.g., neurons and astrocytes) as well as specialized cell types (e.g., motor neurons). Therefore, it may be of great interest to adapt existing protocols for derivation of neurons, astrocytes, and oligodendrocytes to this system, which could improve the translation of therapeutically relevant neural cells from the bench top to the clinic. Furthermore, the use of minimal media compositions and the elimination of MEFs should make the techniques outlined here more broadly accessible to laboratories that do not routinely culture hPSCs. We would like to thank Dr. James Thomson, Nick Propson, and Mitch Probasco for providing reagents, providing the iPS 004A cell line, assisting with flow cytometry, and helpful discussions on E8 medium and hPSC culture. We would also like to thank Dr. Su-Chun Zhang, Cindy Huang, and Jeffrey Jones for providing H9 hESCs and IMR90-4 iPSCs cultured on MEFs. The Pax6, Nkx6.1, HoxB4, and Hb9 antibodies used in this study were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. This work was supported by funding from the Wisconsin Institutes for Discovery and the Wisconsin Alumni Research Foundation. 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