A Modular Platform for Differentiation of Human PSCs into All Major Ectodermal Lineages

Abstract

Directing the fate of human pluripotent stem cells (hPSCs) into different lineages requires variable starting conditions and components with undefined activities, introducing inconsistencies that confound reproducibility and assessment of specific perturbations. Here we introduce a simple, modular protocol for deriving the four main ectodermal lineages from hPSCs. By precisely varying FGF, BMP, WNT, and TGFβ pathway activity in a minimal, chemically defined medium, we show parallel, robust, and reproducible derivation of neuroectoderm, neural crest (NC), cranial placode (CP), and non-neural ectoderm in multiple hPSC lines, on different substrates independently of cell density. We highlight the utility of this system by interrogating the role of TFAP2 transcription factors in ectodermal differentiation, revealing the importance of TFAP2A in NC and CP specification, and performing a small-molecule screen that identified compounds that further enhance CP differentiation. This platform provides a simple stage for systematic derivation of the entire range of ectodermal cell types.

Keywords: TFAP2A; chemical screen; chemically defined Ectoderm; cortical neurons; cranial placode; directed differentiation; human pluripotent stem cells; neural crest; non-neural ectoderm.

Figure 1. BMP signaling is necessary to obtain non-neural ectoderm

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 positive cells at various BMP4 concentrations after 3 days of treatment. F. The derivation of TFAP2A positive, PAX6 negative, SIX1 negative, and SOX10 negative non-neural ectoderm is achieved by using a high concentration of BMP4 (20ng/ml). G. Immunofluorescence staining of keratinocyte markers K18 and K14 upon further differentiation of the NNE at two different time points. Scale bars 50μm.

Figure 2. A BMP gradient is sufficient to derive neural crest and cranial placode cells

A. The expression of SIX1::GFP positive placode using a gradient of BMP4 each bar within the group represents an independent replicate. B. Quantification of SIX::GFP after treating 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. D. Immunofluorescence staining of CRYAA and CRYAB in lens placode cultures on day 30. E. Immunofluorescence staining of PAX6 positive lens placode in the absence of WNT and PAX3 positive trigeminal placodes after the addition of WNT signals. F. The expression of SOX10::GFP positive neural crest 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 neural crest cells for the 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. Scale bars 50μm.

Figure 3. Novel differentiation strategies are applicable to a range of human embryonic and induced pluripotent stem cells

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 50μm.

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 two-fold differential expression compared to neuroectoderm. Clusters were then classified as all upregulated, all downregulated, neural crest specific, placode specific and non-neural ectoderm 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. G. Expression level of novel genes associated with NE, NC, CP or NNE.

Figure 5. TFAP2A promotes the acquisition of non-CNS ectodermal cells

A. Immunofluorescence of TFAP2A in wildtype and TFAP2A KO differentiating cells treated with BMP4 for 3 days. B. Analysis of E-cadherin expression during the differentiation of the four ectodermal lineages in wildtype and TFAP2A knockout lines. C. Immunofluorescence staining of PAX6 and SOX1 in wildtype and TFAP2A knockout cells during the differentiation of the ectodermal lineages. D. Quantitative PCR of the differentiation of wildtype and TFAP2A KO cells into the four ectodermal lineages. Asterisks indicate no Ct values were generated. Scale bars 50μm.

Figure 6. Chemical screen identifies Phenanthroline as a compound that promotes the derivation of cranial placode

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. E. Quantification of SIX1::GFP positive placode treated with DMSO, Phenanthroline, FGF2, and Phenanthroline plus FGF2 after 12 days. Black spots represent each independent experiment.

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.