Volume 21, Issue 3, Pages 399-410.e7 (September 2017) A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages  Jason Tchieu, Bastian Zimmer, Faranak Fattahi, Sadaf Amin, Nadja Zeltner, Shuibing Chen, Lorenz Studer  Cell Stem Cell  Volume 21, Issue 3, Pages 399-410.e7 (September 2017) DOI: 10.1016/j.stem.2017.08.015 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2017 21, 399-410.e7DOI: (10.1016/j.stem.2017.08.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 BMP Signaling Is Necessary to Obtain NNE (A) Diagram of differentiation strategies by replacement of KSR for E6. (B) Representative immunofluorescence staining of particular lineage markers during the differentiation of hPSCs into the ectodermal lineages. (C) Representation of the neural plate border model and important signaling pathways that influence particular cell fates. (D) Immunofluorescence staining of differentiating cells treated with various concentrations of BMP4 for 3 days. (E) Quantification of TFAP2A+ cells at various BMP4 concentrations after 3 days of treatment. Values represent mean ± SEM. (F) The derivation of TFAP2A+, PAX6−, SIX1−, and SOX10− NNE is achieved by using a high concentration of BMP4 (20 ng/ml). (G) Immunofluorescence staining of keratinocyte markers K18 and K14 upon further differentiation of the NNE at two different time points. Scale bars, 50 μm. Cell Stem Cell 2017 21, 399-410.e7DOI: (10.1016/j.stem.2017.08.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 A BMP Gradient Is Sufficient to Derive NC and CP Cells (A) The expression of SIX1::GFP+ placode using a gradient of BMP4. Each bar within the group represents an independent replicate. (B) Quantification of SIX::GFP after treatment of cells with FGF2 or FGF8 during the differentiation. (C) Quantitative PCR of anterior markers PAX6 and SIX3 during two different time points along the differentiation. Values represent mean ± SEM. (D) Immunofluorescence staining of CRYAA and CRYAB in lens placode cultures on day 30. (E) Immunofluorescence staining of PAX6+ lens placode in the absence of WNT and PAX3+ trigeminal placodes after the addition of WNT signals. (F) The expression of SOX10::GFP+ NC using a gradient of BMP4. Each bar within the group represents an independent replicate. (G) Immunofluorescence staining of differentiating cells treated with various concentrations of BMP4 with or without 600nM CHIR for 3 days. (H) Immunofluorescence staining of spontaneously differentiated NC cells for their ability to generate ASCL1 and ISL1 neurons representing autonomic and sensory neurons, respectively. (I) Calcium imaging was performed on differentiated sensory and autonomic neurons for a response to glutamate. Values represent mean ± SEM. Scale bars, 50 μm. Cell Stem Cell 2017 21, 399-410.e7DOI: (10.1016/j.stem.2017.08.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Novel Differentiation Strategies Are Applicable to a Range of Human Embryonic and Induced PSCs (A) Representative images used for high-content imaging of validated antibodies to mark the different ectodermal lineages. (B) Quantification of the percentage of positive cells during a particular differentiation. Biological replicates (n = 4) and technical replicates (n = 2 per biological replicate) were performed and quantified. Scale bars, 50 μm. Cell Stem Cell 2017 21, 399-410.e7DOI: (10.1016/j.stem.2017.08.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 RNA Sequencing of Purified Cell Populations (A) Heatmap and unbiased clustering depicting the differences in gene expression between the four ectodermal lineages. (B) Principle component analysis of all samples represented in (A). (C) Dendrogram and heatmap of genes with 2-fold differential expression compared to NE. Clusters were then classified as all upregulated, all downregulated, NC-specific, placode-specific and NNE-specific. (D) Highest enriched gene ontology (GO) terms in all of the classified groups. (E) Expression of genes specific to all ectodermal lineages and genes specific to non-CNS formation. (F) Expression level of novel genes associated with NE, NC, CP, or NNE. Cell Stem Cell 2017 21, 399-410.e7DOI: (10.1016/j.stem.2017.08.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 TFAP2A Promotes the Acquisition of Non-CNS Ectodermal Cells (A) Immunofluorescence of TFAP2A in wild-type and TFAP2A KO differentiating cells treated with BMP4 for 3 days. (B) Analysis of CDH1 expression during the differentiation of the four ectodermal lineages in wild-type and TFAP2A KO lines. (C) Immunofluorescence staining of PAX6 and SOX1 in wild-type and TFAP2A KO cells during the differentiation of the ectodermal lineages. (D) Quantitative PCR of the differentiation of wild-type and TFAP2A KO cells into the four ectodermal lineages. Asterisks indicate that no Ct values were generated. Scale bars, 50 μm. Cell Stem Cell 2017 21, 399-410.e7DOI: (10.1016/j.stem.2017.08.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Chemical Screen Identifies Phenanthroline as a Compound that Promotes the Derivation of CP (A) A schematic representation of the small-molecule screen. (B) All compounds with two different concentrations are plotted. Those compounds that enriched the SIX1::GFP signal were used for subsequent validation. (C) Dosage gradient of Phenanthroline treatment indicate a narrow window of efficacy that supports SIX1 expression. (D) Quantitative PCR analysis of lineage markers SIX1, SOX10, T, MYOD, and SOX17 with the addition of Phenanthroline. Values represent mean ± SEM. (E) Quantification of SIX1::GFP+ placode treated with DMSO, Phenanthroline, FGF2, and Phenanthroline plus FGF2 after 12 days. Black spots represent each independent experiment. Cell Stem Cell 2017 21, 399-410.e7DOI: (10.1016/j.stem.2017.08.015) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Improved Efficiency and Strategy in the Generation of the Four Ectodermal Lineages (A) Quantification of all data points generated in this study. CP formation was quantified with the presence of FGF2. Each dot represents independent experiments. (B) General schematic of all the differentiation protocols to induce the ectoderm. Yellow demonstrates the duration of the BMP signal and purple, the duration of the WNT signal during the various protocols. Cell Stem Cell 2017 21, 399-410.e7DOI: (10.1016/j.stem.2017.08.015) Copyright © 2017 Elsevier Inc. Terms and Conditions