Author response: Imaging-based chemical screening reveals activity-dependent neural differentiation of pluripotent stem cells (2013)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Mammalian pluripotent stem cells (PSCs) represent an important venue for understanding basic principles regulating tissue-specific differentiation and discovering new tools that may facilitate clinical applications. Mechanisms that direct neural differentiation of PSCs involve growth factor signaling and transcription regulation. However, it is unknown whether and how electrical activity influences this process. Here we report a high throughput imaging-based screen, which uncovers that selamectin, an anti-helminthic therapeutic compound with reported activity on invertebrate glutamate-gated chloride channels, promotes neural differentiation of PSCs. We show that selamectin's pro-neurogenic activity is mediated by γ2-containing GABAA receptors in subsets of neural rosette progenitors, accompanied by increased proneural and lineage-specific transcription factor expression and cell cycle exit. In vivo, selamectin promotes neurogenesis in developing zebrafish. Our results establish a chemical screening platform that reveals activity-dependent neural differentiation from PSCs. Compounds identified in this and future screening might prove therapeutically beneficial for treating neurodevelopmental or neurodegenerative disorders. https://doi.org/10.7554/eLife.00508.001 eLife digest Pluripotent stem cells have the potential to become most of the cell types that make up an organism. However, the signals that trigger these cells to turn into neurons rather than lung cells or muscle cells, for example, are not fully understood. Proteins called growth factors are known to have a role in this process, as are transcription factors, but it is not clear if other factors are also involved. In an attempt to identify additional mechanisms that could contribute to the formation of neurons, Sun et al. screened more than 2,000 small molecules for their ability to transform mouse pluripotent stem cells into neurons in cell culture. Surprisingly, they found that a compound called selamectin, which is used to treat parasitic flatworm infections, also triggered stem cells to turn into neurons. Selamectin works by blocking a particular type of ion channel in flatworms, but this ion channel is not found in vertebrates, which means that selamectin must be promoting the formation of neurons in mice via a different mechanism. Given that a drug related to selamectin is known to act on a subtype of receptors for the neurotransmitter GABA, Sun et al. wondered whether these receptors—known as GABAA receptors—might also underlie the effects of selamectin. Consistent with this idea, drugs that increased GABAA activity stimulated the formation of neurons, whereas drugs that reduced GABAA function blocked the effects of selamectin. In addition, Sun et al. showed that selamectin triggers human embryonic stem cells to become neurons, and that it also promotes the formation of new neurons in developing zebrafish in vivo. As well as revealing an additional mechanism for the formation of neurons from stem cells, the screening technique introduced by Sun et al. could help to identify further pro-neuronal molecules, which could aid the treatment of neurodevelopmental and neurodegenerative disorders. https://doi.org/10.7554/eLife.00508.002 Introduction Mouse embryonic stem cells (mESCs), capable of generating most cell types that constitute the entire organism, have made important contributions to our understanding of mammalian biology (Smith, 2001). How mESCs differentiate into neural lineages is a fascinating question that remains incompletely understood (Okano and Temple, 2009; Gaspard and Vanderhaeghen, 2010). Neural induction, the first step in neural differentiation of mESCs, requires active FGF signaling (Ying et al., 2003) and inhibition of the BMP/TGF-beta pathway (Chambers et al., 2009). Subsequent regional identity and lineage-guided differentiation are further regulated by the presence or absence of various morphogens or transcription factors (Lee et al., 2000; Wichterle et al., 2002; Andersson et al., 2006; Martinat et al., 2006). However, it is unknown whether mechanisms additional to growth factors and transcription regulators direct the differentiation of mESCs into neural lineages. Effective means for perturbing a complex biological system are key to gaining new insights into the underlying molecular and cellular mechanisms. Small organic molecules have proven to be invaluable tools for probing biological mechanisms, owing to their versatile nature and ease of application and removal from the system under study (Stockwell, 2004; Zon and Peterson, 2005). These features also make bioactive small molecules highly attractive for therapeutic applications. One critical challenge in small molecule discovery is that the chemical space is infinite, thereby requiring high throughput screening for speed and bioassays that are of sufficient specificity and sensitivity to distinguish active small molecules from background noise. Here we report an imaging-based screen of ∼2000 bioactive compounds in mESC monolayer cultures labeled with the anti-tyrosine hydroxylase (TH) antibody (a marker for dopaminergic, noradrenergic, and adrenergic neurons). We identified small molecules that increased the appearance of TH+ neurons in the assay, including those with known neurotrophic activity and those that are functionally novel. Notably, we show that the anti-parasitic compound selamectin, with reported activity on invertebrate glutamate-gated chloride channels, increased not only TH+ neurons, but also multiple other neural types including the serotonergic (5-HT), GABAergic, and Islet+ motor neurons as well as Olig2+ oligodendrocytes. We further reveal, through pharmacology, genetics, single-cell electrophysiological recordings and clonal analyses, that selamectin acts by enhancing GABAA receptor signaling, increasing the expression of proneural and lineage-specific transcription factors, and promoting cell cycle exit and differentiation of neural progenitors. We also demonstrate that selamectin can increase neuronal differentiation in human ESCs and induced pluripotent stem cells (iPSCs) as well as in vivo in the developing zebrafish. Results High throughput chemical screen In order to apply chemistry to probe the basic biology of neural differentiation from pluripotent stem cells (PSCs), we designed a high content screen to isolate small molecules that can increase the total number of TH+ neurons derived from mESC monolayer cultures. The system was chosen for several reasons: First, the mESC culture system is an established model for understanding neural development, with much insight gained in recent years (Okano and Temple, 2009; Gaspard and Vanderhaeghen, 2010). Second, mESCs can be cultured in large quantities and in multi-well plates in a high throughput manner. Finally, our adaptation of the mESC monolayer culture and differentiation method (Ying and Smith, 2003) showed that a relatively low and consistent number of TH+ neurons were detected in the culture system (Figure 1). Figure 1 Download asset Open asset High-throughput screening. (A) Scheme of the three-stage mESC neuronal differentiation-based chemical screening. (B) E14 mESC cultures express neural progenitor makers (Sox2, Lmx1A, and Nestin) after 7-day stage one culture. 46C mESC cultures, in which GFP is driven by the sox1 promoter, are GFP positive after 7-day stage one culture. (C) The quantification analysis of TH+ neurons among total cells using the INCell Developer software. (D) Summary of coefficient of variation (C.V.) of the DMSO control. (E) Representative images of immunostaining in control (left, treated with DMSO) and a hit compound (right). (F) A schematic summary of the chemical screen. Scale bar, 10 μm. https://doi.org/10.7554/eLife.00508.003 A three-stage protocol was devised ('Materials and methods' for details) (Figure 1A). The mESCs of both E14 and 46C lines (Ying and Smith, 2003) were used, the latter of which expresses GFP reporter under the control of sox1 promoter. During stage one, undifferentiated mESCs were cultured on a gelatin-coated surface and in the media without LIF, resulting in neural progenitors that express Sox2, Lmx1a, Nestin, and Sox1 (Figure 1B). At stage two, neural progenitors were plated into multi-well plates and treated with chemicals for three days. Finally, chemical treatment was withdrawn and cells were cultured for additional three days before immunostaining with anti-TH antibody (Stage three). This protocol was further subjected to automation at multiple steps, including cell dispensing into 96-well plate using Thermo Matrix Well Plate, compound distribution into wells using Biomek FXP Laboratory Automation Workstation, immunostaining using Thermo Matrix PlateMate Plus, image capture using GE INCell 1000/2000, and image quantification using INCell Developer software ('Materials and methods' for details). We then screened a library containing 2080 biologically active and structurally diverse compounds, including many FDA approved and currently marketed drugs. Compounds were screened at a final concentration of 1 μM in a volume of 120 μl per well containing 0.67% DMSO (vol/vol). After automated immunostaining, image acquisition, and image analysis, the percentage of TH+ cells in each well was calculated (Figure 1C). We did not use actual cell count (as cells in the well are not well separated, making 'cell count' inaccurate); instead, we calculated the area of each segmented target. The percentage of TH signal in each well was expressed as a ratio of TH-covered area over DNA-covered area. The final readout was calculated as fold change compared to the DMSO-treated control. The cut-off for selecting primary hits was set as fold change > mean + 3 S.D. relative to DMSO control, which is a rather stringent selection criteria based on previous studies (Borowiak et al., 2009). To assess assay performance, the coefficient of variation (C.V.) of DMSO control was calculated for each of the twenty-six 96-well plates screened, and all C.V.s but one were smaller than 20%, suggesting an acceptable variation during this cell-based screen (Figure 1D). Out of 2080 chemicals screened, 26 led to a fold change of TH+ cells larger than mean + 3 S.D. (1.16%) (Figure 1E for an example), and 20 out of the 26 were neither cytotoxic nor auto-fluorescent (Figure 1F). After two rounds of validation, two compounds were selected as hits, yielding an overall hit rate of 0.09%. One identified molecule is Dihydrodeoxygedunin (DOG), which is a natural product with known neurotrophic activity via activating the TrkB receptor and its downstream signaling cascades (Jang et al., 2010a). Both DOG and 7,8-dihydroxyflavone (DHF, another selective TrkB agonist [Jang et al., 2010b]) increased TH+ cells in mESC cultures, albeit modestly (Figure 2). This data suggest that our screen is capable of identifying compounds with neuronal promoting activity. Figure 2 Download asset Open asset The neurotrophin receptor TrkB agonists [Dihydrodeoxygedunin (DOG) and 7,8-dihydroxyflavone (DHF)] increases TH+ cells in mESC cultures. (A) Structure of DHF and DOG. (B) DHF and DOG increase TH% in mESCs (t test, p<0.05, n = 4). https://doi.org/10.7554/eLife.00508.004 Selamectin increases the differentiation of multiple neural lineages from mESCs The other hit from our screen is selamectin, whose role in promoting ESC differentiation into TH+ neurons is novel, and was selected for further study. We first determined whether selamectin-induced increase of TH+ neurons is selective for these subtypes by immunocytochemistry with the pan-neuronal marker NeuN. Treatment with selamectin increased the percentage of total neurons, compared to the DMSO-treated control (Figure 3A–B). This result suggests that the effect of selamectin is not specific to TH+ neuronal subtypes. Further analysis showed that selamectin also significantly increased the production of 5-HT neurons (Figure 3C), GABAergic neurons (Figure 3D), and Islet+ motor neurons (Figure 3E). The increase of 5-HT neurons was remarkably high (∼sevenfold), suggesting that selamectin might have a preferential activity for inducing 5-HT neurons. Figure 3 Download asset Open asset Selamectin increases mESC differentiation into multiple neural lineages. (A) Representative images of TH and NeuN staining in control (DMSO) and selamectin-treated cultures. (B) Quantification shows increased production of both TH+ and total neurons by selamectin (t-test, n = 4, p<0.001). (C–F) Selamectin treatment also increases the production of 5-HT neurons (C), GABAergic neurons (D), islet+ motor neurons (E), and Olig2+ oligodendocyte precursors (F) (t-test, n = 4, *p<0.05, **p<0.01, ***p<0.001). https://doi.org/10.7554/eLife.00508.005 Since neuronal differentiation precedes that of gliogenesis in the vertebrate central nervous system (Alvarez-Buylla et al., 2001; Mehler, 2002), we wondered whether the increased neuronal production by selamectin is at a cost of later-born glial cells. As our culturing protocol largely favors the differentiation of neurons, we were able to observe only a small numbers of Oligo2+ cells at a much later stage (Day 18), suggesting that they were likely to be oligodendrocytes or their precursors. After treatment with selamectin, the percentage of Oligo2+ cells also increased compared with the DMSO-treated control (Figure 3F), suggesting that the increase of neuronal production is not at the expense of glial production. Taken together, selamectin appears capable of promoting mESC differentiation into both neuronal and oligodendrocyte lineages. Dose response and time course of selamectin action To determine the dose response and time course of selamectin action, we tested a wide range of selamectin concentrations (from 32 nM to 500 nM). The result showed that selamectin functions in a dose-dependent manner with an effector concentration for half-maximum response EC50 = 293 nM (Figure 4A). When the concentration of selamectin was higher than 500 nM, it became toxic to cells. Figure 4 Download asset Open asset Dose response and time course of selamectin's action in mESC cultures. (A) Dose response curve of selamectin (based on TH immunostaining). Cells were treated from Day 8 to Day 11. Selamectin is toxic above 500 nM. The EC50 value and curve fitting were performed with GraphPad Prism using a Sigmoidal dose-response (variable slope). Data are presented as mean ± S.D., n = 4. (B) Time course of selamectin's action. Only cells treated with selamectin from Day 7 to Day 10 show significant increase of TH+ neurons (t-test, n = 4). https://doi.org/10.7554/eLife.00508.006 Importantly, analysis of the temporal response showed that when cells were treated with selamectin at different stages, its effect was quite different. Treatment during Stage one (from Day 4 to Day 7, mainly composed of undifferentiated ESCs) suppressed cell proliferation, resulting in insufficient number of cells on Day 7 for further evaluation. Treatment during Stage two (from Day 7 to Day 10, mainly composed of neural progenitors) led to a significant increase of neurons evaluated on Day 13 (Figure 4B). Shortening the treatment regimen to one day (i.e., from Day 7 to Day 8) or treatment during Stage three (from Day 10 to Day 13, mainly composed of differentiating neurons) showed no significant effect of selamectin (Figure 4B). These results suggest that selamectin's proneurogenic activity is likely due to its action on mESC-derived neural progenitors. Pharmacological evidence points to an action of selamectin on GABAA receptors Selamectin belongs to a chemical family of macrocyclic lactones used to treat nematode infections that cause onchocerciasis (also known as the river blindness) in humans (Goa et al., 1991) and as topical or oral parasiticide and antihelminthic on dogs and cats (Bishop et al., 2000). This class of drugs disables parasites by displacing glutamate in their muscle synapses through acting on the glutamate-gated chloride (GluCl) channels (Bloomquist, 2003), which have no orthologues in vertebrates. A better-studied example of this family is avermectin (for the structures of selamectin and avermectin, Figure 5A), which, when tested for its activity in promoting neuronal differentiation, showed a less potent effect than selamectin with a marginal significance achieved at 0.25 μM (Figure 5B). Figure 5 Download asset Open asset Pharmacological evidence indicates that selamectin's proneurogenic activity is mediated by GABAA receptors. (A) Selamectin and avermectin belong to a chemical family of macrocyclic lactones and have the same structural backbone. (B) Avermectin has less potent proneurogenic activity than selamectin. Only 250 nM avermectin shows a significant effect, which was much less potent compared to that of 250 nM selamectin (t-test, n = 4, p=0.034 for Avermetin vs p=0.002 for selamectin). (C) Taurine, the most abundant endogenous ligand for glycine receptors during neocortical development has no proneurogenic activity in mESC cultures. (D) Muscimol, a GABAA receptor agonist, has a significant proneurogenic activity (t-test, n = 4, p<0.001). (E) Chlordiazepoxide (CDZ), a positive allosteric modulator of GABAA receptor, also has a significant proneurogenic activity (t-test, p<0.05), but there was no obvious additive effect when cells were treated with both selamectin and CDZ (t-test, p=0.480). (F) The GABAA receptor antagonist bicucullin and non-competitive blockers picrotoxin and pentylenetetrazol alone had no effect on neuronal production (white columns, control group were normalized to fold change = 1, displayed as the red dot line). However, when tested together with selamectin, the effect of selamectin was blocked (gray columns). In contrast, the glycine receptor inhibitor strychnine does not block the effect of selamectin. Final concentration: Bicuculline (Bicu) = 100 μM; Picrotoxin (PTX) = 500 μM; Pentylenetetrazol (PTZ) = 5 mM; Strychnine (STY) = 100 μM; Selamectin (Sela) = 0.3 μM; Muscimol (Musci) = 10 μM (t-test, n = 4). https://doi.org/10.7554/eLife.00508.007 Since GluCl channels are not found in vertebrates, selamectin must act on other protein targets in mESC cultures. Based on gene structure and phylogenetic analyses, vertebrate glycine channels are thought to be orthologous to the invertebrate GluCl channels (Vassilatis et al., 1997). It has been reported that ivermectin (an avermectin derivative) can act as an agonist of the glycine receptor (Shan et al., 2001). Moreover, non-synaptically released taurine can activate the endogenous glycine receptor during neocortical development (Flint et al., 1998). Therefore, we first tested whether taurine, the most abundant endogenous ligand for glycine receptors during neocortical development (Agrawal et al., 1971), the deficiency of which affects cortical development in kittens (Palackal et al., 1986), has the same proneurogenic effect as selamectin. We treated cells from Day 7 to Day 11 with a wide range of taurine concentrations, none of which, however, showed any effect in increasing TH% among total cells (Figure 5C). This result suggests that selamectin does not act through glycine receptors to promote neuronal differentiation from mESCs. Next we turned to another potential candidate, the GABAA receptor, since some reports in the literature suggest that avermectin can bind to the GABAA receptor in rat brain membranes or cultured cerebellar neuronal assays (Pong et al., 1982; Huang and Casida, 1997), and avermectin exhibits an anticonvulsant action in a mouse seizure model (Dawson et al., 2000). We first tested the effect of the GABAA receptor agonist Muscimol at a wide range of concentrations (from 1 μM to 100 μM). Remarkably, all of them had proneurogenic activity like selamectin (Figure 5D). We also tested the effect of Chlordiazepoxide (CDZ), a positive allosteric modulator of the GABAA receptor. When cells were treated alone with 10 μM Chlordiazepoxide or 0.3 μM Selamectin, significant proneurogenic activity was observed as compared to the DMSO control (p<0.05, t-test, Figure 5E). When cells were treated with both Selamectin (0.3 μM) and CDZ (10 μM), no significant additive effect was observed (p=0.480, t-test, Figure 5F), suggesting a lack of synergistic or cooperative action between these two compounds. To determine whether the proneurogenic activity of selamectin is indeed mediated through the GABAA receptor, we whether it could be blocked by GABAA receptor picrotoxin or pentylenetetrazol (PTZ) blockers of the GABAA When cells were treated alone with these no effect on differentiation of TH+ neurons was suggesting that at this stage of the mESC might not be released in sufficient quantities to neuronal differentiation (Figure columns, all control were normalized to fold change = 1, displayed as the red line). When cells were treated with selamectin together with these or blockers μM 5 or 100 μM the effect of selamectin was to no significant between the treated group and control (Figure columns). In contrast, the glycine receptor inhibitor strychnine 100 μM) to block the effect of selamectin (Figure these data suggest that selamectin promotes neuronal differentiation from mESCs through the of GABAA receptor The of GABAA receptor is for the proneurogenic activity of selamectin To determine whether GABAA receptor is to the effects of selamectin, we used an to the activity of various GABAA receptor The mammalian expresses GABAA receptor the of which a molecular and of GABAA receptor subtypes and 2005). the expression of all receptor in mESC cultures, were identified to high expression in mESC-derived neural progenitors not and were chosen for of on Day and analysis on Day (Figure showed that the by the and were highly in the of (Figure treatment with the the effect of selamectin in inducing and TH+ neurons (Figure Treatment with the and did not block the effect of selamectin, suggesting that the of is not sufficient to their gene activity or these not the effect of selamectin. these results evidence that selamectin acts through the γ2-containing GABAA receptor to promote neuronal Figure Download asset Open asset evidence indicates that selamectin's proneurogenic activity is mediated by the GABAA receptor. (A) Scheme of the and cell for (B) shows fold change of the expression of different GABAA receptor after vs control, vs GFP control. (C) Scheme of the to identify the GABAA receptor that selamectin's activity. Representative images of NeuN and TH staining in DMSO and selamectin-treated cultures of (D), (E), and Quantification shows of but not and the effect of selamectin in increasing neurons = 4, **p<0.01, vs Quantification shows of but not and the effect of selamectin in increasing TH+ neurons neurons = 4, **p<0.01, vs Scale bar, 100 μm. A of neural rosette cells in mESC cultures to and selamectin to its in neurons, signaling mediated by the of GABAA receptors can cells in the of the rat embryonic et al., neural cells in the mouse et al., or et al., due to the concentrations in progenitor cells and neurons and 2002; 2006; et al., et al., Based on this we to identify the cell types in mESC cultures that might be to and selamectin by single-cell electrophysiological recordings of neural activity. The mESC cell 46C that expresses GFP driven by Sox1 was Based on the observed temporal effect of selamectin in mESC cultures, we our on neural a functionally type of neural stem cells to represent the stage in vivo et al., Neural are of their and the of neural we also their GFP signals (Figure In addition, we some cells with the of neurons (Figure a of cells we tested both and selamectin-induced Figure 7 Download asset Open asset and selamectin in neural and cells. (A) image and protein signal of neural rosette cells. of the cells in the are neural rosette cells, and one of them with is by a red (B) example of of μM) induced by μM). (C) an example of neural rosette cell μM) induced that also displayed selamectin induced an example of neural rosette cell μM) induced that did not selamectin induced μM Selamectin DMSO containing was used as the control as in the (D) A of the numbers of the of neural rosette cell based on whether they displayed and selamectin induced The The number of the cells in each group is in the the group which is (E) image and protein signal of cells. 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