Human embryonic stem cells (hESCs) can exit the self‐renewal programme, through the action of signalling molecules, at any given time and differentiate along the three germ layer lineages. We have systematically investigated the specific roles of three signalling pathways, TGFβ/SMAD2, BMP/SMAD1, and FGF/ERK, in promoting the transition of hESCs into the neuroectoderm lineage. In this context, inhibition of SMAD2 and ERK signalling served to cooperatively promote exit from hESC self‐renewal through the rapid downregulation of NANOG and OCT4. In contrast, inhibition of SMAD1 signalling acted to maintain SOX2 expression and prevent non‐neural differentiation via HAND1. Inhibition of FGF/ERK upregulated OTX2 that subsequently induced the neuroectodermal fate determinant PAX6, revealing a novel role for FGF2 in indirectly repressing PAX6 in hESCs. Combined inhibition of the three pathways hence resulted in highly efficient neuroectoderm formation within 4 days, and subsequently, FGF/ERK inhibition promoted rapid differentiation into peripheral neurons. Our study assigns a novel, biphasic role to FGF/ERK signalling in the neural induction of hESCs, which may also have utility for applications requiring the rapid and efficient generation of peripheral neurons.
The TGFβ and BMP pathways, two evolutionary conserved signalling cascades, activate the set of transcription factors SMAD2/3 and SMAD1/5/8, respectively, which in turn either activate or repress the expression of target genes (Schmierer and Hill, 2007; Figure 1A; ‘SMAD1’ and ‘SMAD2’ hereafter). The balance between these pathways has a major impact on cell fate specification in human embryonic stem cells (hESCs; Yao et al, 2006; Greber et al, 2008; Vallier et al, 2009b). hESCs resemble epiblast stem cells, which are derived from pregastrulation mouse embryos, in their responsiveness to extracellular signals (Brons et al, 2007; Tesar et al, 2007). Consistent with the absence of inductive signals in the anterior mouse epiblast, neuroectoderm formation from hESCs is facilitated by the inhibition of either TGFβ signalling (Smith et al, 2008), BMP signalling (Pera et al, 2004), or both (Chambers et al, 2009).
FGF2 is a major factor involved in the self‐renewal of hESCs and has been shown to activate the ERK cascade (Kang et al, 2005; Figure 1A). The effects of FGF2 signalling in hESCs appear to be complex and relatively subtle. In addition to positive effects on cell survival and/or proliferation, FGF2 signalling sustains expression of the pluripotency controlling gene NANOG (Greber et al, 2010; Yu et al, 2011), in collaboration with TGFβ/SMAD2 signalling (Greber et al, 2008; Xu et al, 2008; Vallier et al, 2009a). On the other hand, FGF2 signalling antagonizes BMP signalling, a major (non‐neural) differentiation‐inducing pathway (Xu et al, 2005, 2008; Greber et al, 2007; Peerani et al, 2007). FGF2 signalling has also been implicated in neuroectoderm formation from hESCs, and from the related mouse epiblast stem cells, but whether it bears a positive or negative effect remains controversial: on the one hand, some studies reported negative effects on neural commitment when inactivating FGF signalling using pharmacological inhibitors (LaVaute et al, 2009; Vallier et al, 2009b; Cohen et al, 2010; Na et al, 2010). On the other hand, data by others suggest that neuroectodermal gene expression in hESCs can be induced by withdrawing FGF2 from the culture medium (Greber et al, 2008; Chambers et al, 2009; Rosa and Brivanlou, 2011). Here, we seek to clarify these seeming discrepancies by systematically investigating the interplay of the three pathways in neural induction of hESCs, with an emphasis on the role played by FGF signalling in this context.
Combined TGFβ/SMAD2 and FGF/ERK inhibition promotes rapid exit from self‐renewal
We sought to systematically investigate the specific roles of the three key pathways by using recombinant proteins and pharmacological inhibitors to selectively activate or repress their activity (Figure 1A). Four types of treatment conditions were designed to monitor the effects of the individual cascades on global gene expression over a period of 6 days (Figure 1B). As a baseline, hESCs were treated with FGF2 and the BMP antagonist Noggin (FGF2 NOG) in chemically defined medium, a condition that has been reported to promote self‐renewal (Xu et al, 2005). A comparison between FGF2 NOG and FGF2 SB NOG‐treated cells is expected to reveal the transcriptional response of SMAD2 inactivation, whereas a comparison of FGF2 SB NOG with PD SB NOG treatment would reveal the effects of the FGF/ERK cascade. Finally, a comparison of PD SB NOG with PD SB treatment should help to clarify the role of BMP inactivation (Figure 1A and B).
A prerequisite for hESC differentiation is the shutdown of the self‐renewal machinery. Analysis of the array data confirmed that FGF2 NOG treatment largely sustained the expression of pluripotency markers. A decline in the expression of certain markers was observed towards the end of the time course, which could have been due to the near confluency of the cultures by day 6. In contrast, additional supplementation with SB led to a strong decline in the expression of self‐renewal markers over the course of 6 days, indicating loss of hESC identity (Figure 1C). NANOG downregulation occurred very rapidly, consistent with the finding that it is a direct target of SMAD2. It is therefore likely that the expression of other members of the hESC self‐renewal network, such as OCT4, were affected by the downregulation of NANOG. Interestingly, expression of SOX2, which is required not only by undifferentiated hESCs but also by neuroectoderm, was maintained with FGF2 SB NOG treatment (Figure 1C). This configuration—low NANOG and OCT4 but high SOX2 levels—is compatible with neuroectoderm formation. However, a time point‐by‐time point comparison of the two conditions revealed that the expression of only a few genes was induced by SB treatment (Supplementary Table SI). Based on this observation, we conclude that inactivation of TGFβ/SMAD2 signalling is effective in downregulating the expression of hESC‐specific genes, but that in the presence of FGF2 and NOG, inhibition of TGFβ/SMAD2 signalling is not sufficient for activating the expression of prominent differentiation‐associated markers.
Consistent with these negative effects of SB treatment on hESC self‐renewal, immunocytochemistry analysis revealed that most—albeit not all—cells stained negative for NANOG and OCT4 after 4 days in culture (Figure 1D, top). Like TGFβ/SMAD2 signalling, FGF/ERK signalling has been suggested to also sustain NANOG expression in hESCs (Greber et al, 2010; Yu et al, 2011). We therefore compared the downregulation kinetics of hESC markers in two treatment conditions: FGF2 SB NOG and PD SB NOG. More effective downregulation of prominent hESC markers was found with PD SB NOG compared with FGF2 SB NOG. An 80% downregulation in NANOG mRNA levels was seen within 6 h of treatment with PD SB NOG; NANOG and OCT4 mRNA levels were virtually undetectable by day 4 (Supplementary Figure S1; Figure 1D, bottom). The cooperativity between inactivation of SMAD2 and ERK signalling was confirmed by RT–qPCR in two hESC lines—HuES6 and NCL3—on day 4 (Figure 1E). We therefore conclude that inhibition of both TGFβ/SMAD2 and FGF/ERK signalling produces the most prominent downregulation of hESC‐specific gene expression (Supplementary Figure S1).
FGF/ERK inhibition specifically induces PAX6
Next, we investigated the induction of neuroectodermal markers with an emphasis on PAX6 expression (Zhang et al, 2010). A time‐course comparison revealed a pronounced upregulation of PAX6 in the PD SB and PD SB NOG‐treated samples, but not in the FGF2 NOG or FGF2 SB NOG‐treated samples, suggesting the presence of a strong association between FGF/ERK inhibition and PAX6 induction (Figure 2A). This result was confirmed by RT–qPCR of samples that had been exposed to different degrees of FGF/ERK signalling activity (as well as using alternative pharmacological inhibitors or PD in the presence of FGF2; Supplementary Figure S2A). These data also revealed that inhibition of the FGF/ERK cascade was superior in inducing PAX6 expression over simply withdrawing FGF2 from the culture medium (Figure 2B). Immunocytochemistry analysis confirmed the suppression of PAX6 induction by FGF2, whereas many—albeit, by far, not all—cells grown in the absence of FGF2 or with FGF/ERK inhibition showed pronounced PAX6 staining (Figure 2C). These data strongly suggest that FGF2/ERK signalling specifically represses PAX6 expression in hESCs. This effect of FGF2/ERK signalling must be independent of its NANOG‐maintaining function, as SB treatment alone was effective in reducing NANOG expression, but in the presence of FGF2, SMAD2 inhibition was not sufficient in causing strong PAX6 upregulation.
BMP/SMAD1 inhibition enhances the specificity of neuroectodermal induction
We next sought to assess the effect of BMP/SMAD1 suppression. Global time‐course analysis suggested a high overall similarity in gene expression changes between the PD SB and PD SB NOG‐treated samples. 3D principal component analysis, however, suggested differences in a subset of genes (Figure 2D). Strikingly, SOX2 expression was not well maintained with PD SB treatment, whereas HAND1, encoding a basic helix‐loop‐helix transcription factor involved in extraembryonic as well as mesodermal development, was strongly induced in the absence of NOG (Figure 2E). These effects are reminiscent of the activation of BMP signalling in hESCs (Greber et al, 2008). The rapid suppression of SOX2 expression concomitant with the induction of HAND1 expression by BMP4 stimulation was confirmed using an independent hESC line (Supplementary Figure S2B). We hypothesized that FGF/ERK suppression has ambivalent effects on neuroectodermal induction: on the one hand, it leads to NANOG downregulation as well PAX6 induction (productive), while on the other hand, it activates autocrine BMP/SMAD1 signalling (Greber et al, 2007; Peerani et al, 2007), which may induce non‐neural differentiation (Li et al, 2007) (counterproductive). Consistent with this, HAND1, a BMP/SMAD1 target, became induced within only 6 h of FGF/ERK inactivation; additional NOG supplementation was able to rescue this effect (Figure 2F). Hence, FGF/ERK inhibition may cause differentiation into different types of lineages—neuroectodermal or extraembryonic/mesodermal. Not surprisingly, hESCs treated with PD or PD SB stained heterogeneously positive for both PAX6 and HAND1. However, addition of NOG suppressed the induction of HAND1 in favour of PAX6 expression (Figure 2G). Based on these data, we conclude that the addition of NOG is required to antagonize the BMP/SMAD1 activating effect of FGF/ERK inhibition. Hence, in this context, NOG acts as a specificity enhancer for neuroectodermal commitment, consistent with its role in model organisms. This interpretation was confirmed by substituting NOG with a small molecule inhibitor of BMP/SMAD1 signalling, dorsomorphin (Yu et al, 2008), yielding pronounced PAX6 expression in 80–90% of all cells by day 4 (Figure 2H; Supplementary Figure S2C and D).
FGF/ERK inactivation upregulates OTX2 that acts as a positive regulator of PAX6
The above data suggest that FGF/ERK inactivation is the key trigger for PAX6 induction in hESCs, which could imply that concomitant inactivation of TGFβ/SMAD2 is dispensable. Indeed, by day 6, FGF plus BMP inactivation was almost as effective in inducing PAX6 as triple pathway inhibition (Supplementary Figure S3A and B). Notably, however, PAX6 induction started only once NANOG and OCT4 were almost completely downregulated. NANOG and OCT4 have been found to bind to the PAX6 promoter in hESCs, and we confirmed these data using ChIP–qPCR (Figure 3A; Boyer et al, 2005). Next, we seeked to functionally assess whether the two factors indeed repress PAX6 induction. Following stable overexpression and 4 days of triple pathway inhibition, immunocytochemical analysis revealed that NANOG‐overexpressing cells were largely—and OCT4‐overexpressing cells completely—non‐overlapping with transgene‐negative/PAX6‐positive cells (Figure 3B). These observations strongly suggest that NANOG and, especially, OCT4 do indeed act as repressors of neural fate in hESCs and of PAX6 in particular. Since PD treatment cooperates with SB in diminishing NANOG and OCT4 expression, this must constitute one mechanism by which FGF inactivation promotes PAX6 induction.
When treating hESCs with SB plus FGF2 for up to 8 days, NANOG and OCT4 got eventually diminished to essentially undetectable levels. Still, however, PAX6 was not strongly induced under these conditions (Figures 1C and 4A, below). We concluded that downregulation of NANOG/OCT4 is necessary for PAX6 induction—but not sufficient. We therefore filtered the array data set of Supplementary Table SI for potential positive inducers of PAX6, requiring that such a factor shall become immediately upregulated following FGF/ERK inactivation. One of the very few candidates passing our filtering criteria was the homeobox transcription factor OTX2. Although it is already moderately expressed in undifferentiated hESCs, RT–qPCR and western blot analyses of independent samples confirmed that OTX2 became rapidly and markedly upregulated by day 2—just before the main induction of PAX6 and other neuroectodermal genes like NR2F2 (Figure 3C and D; Rosa and Brivanlou, 2011). Interestingly, the overall OTX2 upregulation appeared to be driven by isoform ‘a’, which is only expressed at low levels in the undifferentiated state (Figure 3C; Supplementary Figure S3C and D). The upregulation of OTX2 was specific to inactivation of the FGF/ERK signalling cascade (Figure 3E, left; Supplementary Figure S3F) and detectable within just 2 h of PD treatment (Supplementary Figure S3E). In line with a putative role in neuroectoderm formation, OTX2 colocalized with PAX6 in neuroectoderm cells (Figure 3E, right).
Given the rapid and pronounced induction kinetics upon neuroectoderm formation, we next tested in functional assays whether OTX2—in collaboration with downregulation of NANOG/OCT4—may act as a positive inducer of PAX6. Indeed, counteracting the upregulation of OTX2 during neuroectoderm formation by RNA interference clearly compromised the induction of PAX6 and also of NR2F2 (Figure 3F and G). Conversely, overexpressing OTX2 ‘a’ under FGF2 culture conditions clearly induced PAX6 to some degree (Figure 3H, left panel). Not surprisingly, this effect on PAX6 expression was rather moderate given that lentiviral infection rates were imperfect and that NANOG/OCT4 were fully expressed under these conditions. However, addition of SB (to selectively diminish NANOG/OCT4) lead to a pronounced increase of PAX6 (and NR2F2) levels, as expected—despite the presence of FGF2 in the culture medium (Figure 3H and I). Similarly, we observed synergistic effects on PAX6 induction between OTX2 overexpression and NANOG RNA interference (Supplementary Figure S3G). Finally, we scanned conserved upstream regions of the PAX6 locus for the presence of canonical OTX motifs and tested these for OTX2 occupancy using ChIP–qPCR. Two out of three upstream regions tested displayed clear OTX2 binding in hESCs undergoing neuroectoderm induction (Figure 3J). Taken together, these data strongly suggest that besides negative effects on NANOG/OCT4, inactivation of FGF/ERK signalling upregulates OTX2, which then acts as a positive inducer of PAX6.
FGF/ERK inhibition promotes peripheral neuron differentiation following neuroectoderm formation
We next asked whether prolonged triple pathway inhibition of hESCs would give rise to neuron‐like cells, as was the case for mouse epiblast stem cells (Greber et al, 2010). Cells staining positive for β‐III Tubulin were readily observed within 8 days in adherent culture. However, these cultures still contained many PAX6+ cells, indicating asynchronous differentiation (Figure 4A). We therefore switched to a modified procedure involving neuroectoderm formation in embryoid bodies (EBs) for 4 days, followed by plating these EBs on matrigel‐coated dishes for another 4 days (Figure 4B). Strikingly, a large number of neuron‐like cells were seen to grow out of the plated EBs, in a radial manner, which stained strongly positive for β‐III Tubulin (Figure 4C). About two‐thirds (line HuES6) or up to 100% (line NCL3) of the plated EBs displayed this pattern. Matrigel appeared to be a preferred substrate for the outgrowth of these cells (Supplementary Figure S4B). Fluorescence‐activated cell sorting (FACS) of bulk cultures revealed that most cells were positive for β‐III Tubulin, with an apparent correlation between the size of the plated EBs and the efficiency of neuronal marker induction (Figure 4D; Supplementary Figure S4C).
Cells at the periphery of the outgrowths stained positive for doublecortin, a marker for immature neurons. MAP2 expression was rather confined to the intersection between the centre and the periphery of the outgrowths. Only rarely did cells stain positive for specific markers such as GABA or tyrosine hydroxylase (TH). However, many cells appeared positive for vesicular glutamate transporter 2, indicative of glutamatergic identity, which was also confirmed by pronounced VGLUT2 mRNA levels (Figure 4E; Supplementary Table SIIA). Electrophysiological cell characterization on days 8 and 12 using the whole‐cell patch clamp technique revealed the ability of the cells to fire one or more action potentials upon injecting depolarizing currents, with an average amplitude of 69±3 mV (n=9, resting membrane potential: −44±1.3 mV). Addition of tetrodotoxin (TTX) reversibly erased the action potentials, demonstrating the functionality of fast voltage‐gated sodium channels (Figure 4F). Finally, when induced pluripotent stem cells (iPSCs) were subjected to the differentiation procedure, results were similar to those obtained with hESCs (Figure 4G; Supplementary Figure S4D). Collectively, these data demonstrate that extended inhibition of the three pathways in human pluripotent cells rapidly gives rise to functional neurons at high efficiency.
To delve deeper into the dispensability of FGF/ERK signalling in this entire differentiation process, we took a closer look at marker gene expression in these neuronal cultures (Supplementary Table SIIA). Mapping panels of known markers of diverse neuronal fates to these data as well as Gene Ontology analysis revealed that the neurons generated upon continuous pathway inhibition were likely to be neural crest‐derived neurons of the peripheral nervous system (PNS), not neurons of the central nervous system (CNS) (green dots in Figure 5A and Supplementary Table SIIB). The strong upregulation of neural crest and peripheral neuron markers was confirmed in independent samples (Figure 5B). Confirming this interpretation, between 60 and 80% of all cells in the EB outgrowths stained positive for BRN3A (also known as POU4F1), a prominent marker for sensory neurons (Howard, 2005; Figure 5C).
To determine the signalling requirements for the formation of these neurons from neuroectoderm, we varied the exogenous factors added during the adherent phase of the procedure outlined in Figure 4B. The addition of FGF2 between days 4 and 8 almost completely suppressed terminal differentiation into peripheral neurons, whereas withdrawal of inhibitors of the other two pathways did not appear to have any effect (Figure 5D and E). The suppression of peripheral neuron fate by FGF2 may fit well with the common use of FGF2 to stabilize an accepted precursor stage in CNS development (Elkabetz et al, 2008). Consistent with this, we found that when PD SB NOG‐treated EBs were plated out on day 3 (but not later) into FGF2‐containing medium, we reproducibly observed formation of neural rosettes that displayed sustained PAX6 and Nestin expression (Figure 5F). Taken together, these data suggest that in addition to repressing the initial induction of PAX6 in hESCs, FGF2 later acts as a repressor of peripheral neuron fate to arrest differentiation and stabilize neural precursor stages.
Overall, our data set assigns a novel role to FGF2 signalling in hESCs, namely prevention of neuroectoderm induction by the specific repression of PAX6, the key fate determinant of that lineage in human (Zhang et al, 2010; Figure 5G). As FGF2 supplementation inhibited PAX6 induction throughout all our experiments and as sheer FGF2 withdrawal or FGF/ERK inhibition alone was sufficient in activating PAX6 expression, we conclude that inactivation of FGF2 signalling—by FGF2 withdrawal from the culture medium or by pharmacological inhibition—may be the key trigger underlying neuroectoderm conversion from hESCs. According to our time‐course data, inhibition of TGFβ/SMAD2 signalling mainly serves to promote downregulation of hESC‐specific genes (Figure 5G; Brown et al, 2011). FGF inactivation cooperates with TGFβ/SMAD2 inhibition to downregulate NANOG and OCT4 levels, and overexpression of these factors strongly interfered with PAX6 induction. Hence, as a necessary requirement, NANOG and OCT4 need to be turned off to enable derepression of PAX6—in principle.
In the presence of FGF2, however, SMAD2 inhibition was not sufficient for inducing neuroectoderm development—even after prolonged time periods of treatment when NANOG and OCT4 were essentially shut down. We therefore conclude that NANOG and OCT4 downregulation alone is not sufficient for promoting substantial induction of PAX6. Rather, we hypothesized that FGF inactivation additionally promotes the upregulation of a positively acting inducer of PAX6. Rosa and Brivanlou (2011) recently implicated the transcription factor NR2F2 in promoting neuroectoderm differentiation of hESCs, in part acting by repressing OCT4. Notably, in their study, differentiation was induced by switching to FGF2‐free culture media. Consistent with the data by Rosa and Brivanlou, NR2F2 was strictly coregulated with PAX6 throughout all our experiments. This was, however, also true for the initial time points in which PAX6 and NR2F2 were only very modestly upregulated. Here, we identified OTX2 to be one of the earliest upregulated genes following FGF inactivation. OTX2 silencing in differentiating cells interfered with the induction of both PAX6 and NR2F2, and OTX2 overexpression—in cooperation with NANOG/OCT4 downregulation—promoted induction of both these key genes. OTX2 therefore appears to be an early driver of neuroectoderm commitment, being immediately linked to inactivation of FGF signalling (Figure 5G). Our hESC data are consistent with the in‐vivo expression pattern of mouse Otx2 in the Fgf‐expressing pregastrulation epiblast and in anterior neuroectoderm (Kurokawa et al, 2004). In future studies, it will be interesting to investigate the link between FGF/ERK and OTX2 in more detail.
The model of Figure 5G initially appears to be at odds with published work, as FGF signalling has frequently been implicated in promoting neural induction. However, most of these data have been generated in model organisms, such as chick and Xenopus, and little is known about pregastrulation‐stage mammalian embryos or hESCs. Moreover, inactivation of FGF/ERK signalling has been reported to disable neural induction from mouse ESCs (Ying et al, 2003; Kunath et al, 2007; Stavridis et al, 2007), but these data may better be interpreted as a failure of mouse ESCs to enter an epiblast‐like intermediate stage under these conditions (Lanner and Rossant, 2010). Furthermore, models suggesting FGF2 as a driver of neuroectodermal induction in hESCs were based partly on the observation that inhibition of FGF signalling alone or in combination with TGFβ/SMAD2 inactivation may give rise to non‐neural fates (LaVaute et al, 2009; Vallier et al, 2009b; Na et al, 2010). We argue, however, that these observations rather highlight yet another role of FGF signalling in hESCs, namely to antagonize endogenous BMP signalling (Xu et al, 2005, 2008; Greber et al, 2007; Peerani et al, 2007). When FGF2 is withdrawn from the media or when the pathway is inhibited by pharmacologic agents, autocrine BMP signalling becomes activated enough to interfere with neuroectodermal commitment. Addition of NOG or DM was therefore necessary to prevent BMP‐mediated effects, such as activation of HAND1 and repression of SOX2, to enable the most pronounced induction of neuroectoderm (Chambers et al, 2009; Figure 5G).
After neuroectoderm formation, FGF signalling appears to play another critical role, as prolonged inhibition of the FGF/ERK cascade immediately gave rise to PNS derivatives—mostly sensory neurons—at high efficiency. The occasional emergence of TH‐positive cells is consistent with this finding, as TH is also a marker for sympathetic neurons (Howard, 2005; Figure 4E). The procedure may also present a valuable platform for modelling PNS‐associated diseases or for compound screens requiring the rapid generation of neurons from human pluripotent cells. FGF2 almost completely blocked peripheral neuron formation to redirect cell fate into neural rosettes—an accepted CNS precursor state—or putative Nestin+/PAX6− neural crest cells. Indeed, FGF2 is frequently used to promote the self‐renewal of neural stem and precursor cells, and protocols describing their derivation from ESCs incorporate the addition of FGF2 at some point (Zhang et al, 2001; Conti et al, 2005; Koch et al, 2009). However, this is not inconsistent with our finding that FGF2 represses the onset of PAX6 induction in hESCs (Figure 5G). Rather, it appears that FGF signalling plays a biphasic role in the neural induction of hESCs (Figure 5H).
Materials and methods
Fully characterized hESC lines HuES6 (Cowan et al, 2004) and NCL3 (Zhang et al, 2006) were received from the Harvard Office of Technology Development and the UK Stem Cell Bank, respectively, and used at a passage number below 50. Both lines were confirmed to give rise to derivatives of all three germ layers by spontaneous in vitro differentiation. hESCs tested negative for mycoplasma. All experimental conditions were carried out under adherence using matrigel‐coated 6‐ or 12‐well dishes. Proper matrigel coating was crucial for both maintenance of self‐renewal and efficient neuronal differentiation: matrigel HC (growth factor‐reduced, BD) was thawed on ice overnight and then diluted 1:3 with ice‐cold knockout DMEM (Invitrogen). In all, 1‐ml stocks were then kept frozen at −20°C. For coating of plates, each 1‐ml stock was resuspended in 24 ml of conventional hESC medium (Amit et al, 2004) (without growth factors) on ice (1:75 total dilution, final protein concentration: ∼0.25 mg/ml). Precooled (at 4°C) 6‐ or 12‐wells were covered with 1 ml or 0.4 ml of diluted matrigel, respectively. Plates were then wrapped in parafilm and kept at room temperature overnight. The next day, plates were transferred to 4°C for at least 1 day. Before use, coating quality was verified under a phase contrast cell culture microscope (a network of polymerized matrigel, such as e.g., in Figure 4C, must readily be visible).
hESCs were routinely maintained in MEF‐conditioned medium (Xu et al, 2001) and split using colony precutting with sterile injection needles, followed by treatment with dispase (2 mg/ml), repeated washing in PBS with Mg2+/Ca2+, lifting off the cell clumps using a sharp plastic scraper (PAA), and several seconds of centrifugation to collect the aggregates without single cells. Experiments were exclusively conducted in chemically defined medium. hESCs were always preadapted to defined medium for 1–2 days (see Supplementary Figure S4A). Defined medium—which was modified from Yao et al (2006)—consisted of DMEM/F12 (Hyclone), supplemented with 1 × N2 supplement, 1 × B27 supplement, 0.1 mM β‐mercaptoethanol (Invitrogen), 1 × non‐essential amino acids, 1 × l‐glutamine, 1 × penicillin/streptomycin (PAA), and 0.2% human serum albumin (Biological Industries). For maintenance of self‐renewal, N2NB27 medium was supplemented with 20 ng/ml FGF2 or FGF2 plus dorsomorphin/Noggin. Upon splitting, 10 μM of ROCK inhibitor Y‐27632 (Watanabe et al, 2007) (Ascent) was added to the medium as well. Growth factor or inhibitor treatments were typically initiated 1–2 days after splitting: FGF2 (PeproTech; 20 ng/ml), Noggin (‘NOG’; PeproTech; 250 ng/ml), BMP4 (PeproTech; 20 ng/ml), TGFβ1 (PeproTech; 1 ng/ml), SB431542 (‘SB’; Sigma; 15 μM), PD0325901 (‘PD'; Axon Medchem; 0.5–1 μM), dorsomorphin (‘DM’; Merck; 0.5 μM). For some of the time‐course experiments, for genetic manipulation, and for quantifying PAX6‐positive cells by immunocytochemistry, cells were treated with inhibitors 2 days after splitting with Accutase (PAA), which resulted in colonies of more homogeneous sizes. For EB formation, cell aggregates were collected as for routine splitting and then transferred to bacteriological 6‐cm dishes containing defined medium with FGF2 and ROCK inhibitor. For media changes, dishes were slowly rotated under a stereo microscope and EBs were collected from the centre and then transferred to new dishes with minimal liquid. ROCK inhibitor Y‐27632 was added to all EB cultures for the first 3 days of treatment to support cell survival. Medium was changed daily in all experiments.
RT–qPCR, RNA profiling, and computational analysis
Cells were lysed directly in culture dishes and RNA was isolated using RNeasy kits with on‐column DNA digestion (Qiagen). RT–qPCR and microarray analyses were carried out as described (Greber et al, 2010), using fully validated primers with normalization to two housekeeping genes (Supplementary Table SIII) and HumanRef‐8 v3 bead chips (Illumina), respectively. Processed array data are in Supplementary Tables SI and SII. Principal component analysis was performed using an in‐house developed algorithm in Matlab. Gene Ontology analysis was carried out using DAVID. RNA sequencing was performed on Illumina HiScan SQ instrumentation. hESC library preparation, cluster generation, and 2 × 50 bp paired‐end sequencing was carried out using kits by the manufacturer. Data were analysed using freely available software packages. For the identification of putative OTX2 binding sites in the PAX6 locus, we used an in‐house database of putative regulatory elements in upstream regions of known genes. As a general procedure, the binding energy m for the affinity of a given motif with a position weight matrix (PWM) σ to a sequence X=(x1,…,xw) of length w was calculated by:
where σi is the background frequency of the four nucleotides. P(xi∣σi) denotes the normalized frequency of a nucleotide xi in the PMW column σi. The multiple alignment format data set was obtained from the UCSC genome browser website. Binding energies were obtained for each sequence in 40 000 conserved upstream regions of known genes, followed by calculating the binding energy z‐score for each motif. Only hits with an energy z‐score ⩾2.0 were accepted. OTX binding matrix information was obtained from Transfac.
Immunocytochemistry, western blotting, ChIP–qPCR, and FACS
For immunocytochemistry, cells in 12‐well dishes were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2–1% Triton X‐100 for 10 min, and blocked with a solution comprising 2% BSA, 5% FCS, and 0.1% Tween 20 in PBS for 45 min. Primary and secondary antibodies were applied overnight at 4°C and for 1 h at room temperature, respectively, in 0.5% BSA in PBS‐T. Hoechst stain was applied in the second‐to‐last washing step at 1 μg/ml. Samples were mounted with Aqua‐Poly/Mount (Polysciences) and round glass coverslips. SDS–PAGE and western blotting was performed according to standard procedures with loading 40 μg of protein per gel lane. ChIP–qPCR was performed as described, using about 5 × 106 formaldehyde‐fixed cells as starting material (Greber et al, 2010). FACS analysis was performed using the Fix and Perm intracellular staining kit (Invitrogen GAS‐003) and BD FACSAria instrumentation. Primary antibodies were α AFP (Sigma A8452, IF 1:500); α Doublecortin (Santa Cruz sc‐8067, IF 1:100); α BRN3A (Santa Cruz sc‐8429, IF 1:500); α GABA (Sigma A2052, IF 1:1000); α GAPDH (Ambion 4300, WB 1:5000); α HAND1 (Santa Cruz sc‐9413, IF 1:25); α MAP2 (Millipore MAB3418, IF 1:200); α MAPK (Cell Signaling Technology 9102, WB 1:1000); α NANOG (R&D AF1997, IF 1:50, WB 1:250, ChIP 2 μg); α Nestin (Chemicon MAB5326, IF 1:200); α OCT4 (Santa Cruz sc‐5279, IF 1:100; Santa Cruz sc‐8629, IF 1:100, WB 1:5000; Santa Cruz sc‐8628 X, ChIP 2 μg); α OTX2 (R&D AF1979, IF 1:100; Abcam ab21990, IF 1:500, WB 1:5000, ChIP 2 μg); α PAX6 (Covance PRB‐278P, IF 1:500, WB 1:5000); α Phospho‐MAPK (Cell Signaling Technology 9106, WB 1:2000); α SMA (DakoCytomation M0851, IF 1:100); α β‐III Tubulin (Sigma T8660, IF 1:1000; Covance PRB‐435P IF 1:2000; BD Pharmingen 560381, FACS 20 μl per sample); α TH (Millipore AB152, IF 1:250); α VGLUT2 (Millipore MAB5504, IF 1:50). Secondary Alexa 488, 568, and 594 labelled antibodies were diluted 1:1000 (catalogue numbers A21468, A21206, A11078, A11008, A11001, A11057, A11079, A11061, A11011—all from Invitrogen). Unless otherwise stated, representative pictures are shown.
Gene silencing and overexpression
For overexpression, open reading frames were PCR amplified from hESC or neuroectoderm cDNA using primers in Supplementary Table SIII, cloned into pCRII‐TOPO (Invitrogen), sequence‐validated, and subcloned into the PmeI and SpeI sites of pLVTHM (Addgene plasmid 12247). The OTX2 ORF contained the insert sequence shown in Supplementary Figure S3C. Lentiviral particles were produced using 293T cells grown in MEF‐conditioned medium for 24 h, following cotransfection of the pLVTHM construct and Addgene plasmids 12260 and 12259 at a mass ratio of 4:3:1 using Lipofectamine 2000 (Invitrogen). hESCs were infected in MEF‐conditioned medium 1 day after splitting with Accutase or upon replating. Virus supernatants were adjusted to result in around >50% infection efficiency, as judged based on GFP controls. Defined medium was used from the day after infection onwards. OTX2 RNAi sequences were designed using the Invitrogen Stealth RNAi design tool. By following the accompanying guidelines, silencing constructs were prepared by annealing and cloning short‐hairpin oligonucleotides shown in Supplementary Table SIII into pLKO.1 puro (Addgene plasmid 8453). Plasmid inserts were sequence‐validated and lentiviruses were produced as above. At 48 h after infection, cells were treated as described in text and concomitantly selected using 1 μg/ml puromycin. Virus supernatants with three different shRNA constructs were pooled as this tended to yield slightly higher knockdown efficiencies. Addgene plasmid 1864 expressing scrambled shRNA served as control. The NANOG RNAi vector used was LL‐NANOGi (Zaehres et al, 2005).
Recordings were performed in culture dishes at room temperature and at 33°C. Cell cultures were superfused with a solution containing 120 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM HEPES, 2 mM MgSO4, 2 mM CaCl2, 10 mM dextrose, 25 μM Alexa 594 hydrazide; pH: 7.25 with HCl; osmolality: 305 mOsmol/kg. Electrodes for whole‐cell recordings with a resistance of 6–8 MΩ were pulled from borosilicate glass capillaries. Electrodes were filled with a saline solution containing 95 mM k‐gluconate, 20 mM K3‐citrate, 10 mM NaCl, 10 mM HEPES, 1 mM MgCl2, 0.5 mM CaCl2, 3 mM BAPTA, 3 mM Mg‐ATP, 0.5 mM Na‐GTP; pH: 7.25 with KOH; osmolality: 295 mOsm/kg. Electrodes were connected to an EPC‐10 amplifier (HEKA Elektronik, Lamprecht, Germany). After gaining access to the cell, dyes were allowed to equilibrate by diffusion for at least 5 min. Access resistances were in the range of 5–20 MΩ. Experiments were controlled by PatchMaster software (HEKA Elektronik). Liquid junction potential was not corrected for. TTX (0.5–1 μM) was applied via bath perfusion to block Na+ channels and abolish action potentials.
Human iPSCs were produced from foreskin fibroblasts (ATCC #CRL‐2097) by following Melton's protocol (Huangfu et al, 2008) with three factors (OCT4, SOX2, and KLF4). Retroviruses were produced in 293T cells using Fugene 6 (Roche) and Addgene plasmids 8454, 8449, 17217, 17218, and 17219 (Takahashi et al, 2007). Two lines showing typical hESC morphology and growth characteristics were further characterized according to the standard assays (Supplementary Figure S4D). Bisulphite sequencing was carried out using an EpiTect Bisulfite Kit (Qiagen), followed by PCR cloning using primers listed in Supplementary Table SIII and sequencing of inserts.
Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Table SI
Supplementary Table SII
Supplementary Table SIII
We thank Sandra Heising for technical assistance, Dr Martin Stehling for FACS analysis, Dr David Eccles for analysing RNA‐seq data, and Dr Holm Zaehres for sharing the NANOG knockdown construct. This work was supported by the Max Planck Society.
Author contributions: BG, PC, H‐CP, and HRS designed the experiments. BG, PC, MZ, and DWH performed the experiments. BG, PC, AJMM, MJA‐B, MZ, SM, and SF analysed the data. BG and HRS wrote the manuscript.
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