U.S. Department of Health and Human ServicesHHS National Institutes of HealthNIH National Center for Advancing Translational SciencesNCATS

Time-course and dose-dependent transcriptome profiling reveal key regulators for neural conversion of human iPSCs under chemically defined conditions

Posted on August 27th, 2019 by claire.malley@nih.gov

Members of SCTL presented a poster at the ISSCR 2019 conference. The poster is entitled: Time-course and dose-dependent transcriptome profiling reveal key regulators for neural conversion of human iPSCs under chemically defined conditions.

Authors (underlined, presenting): Pei-Hsuan Chu, Claire Malley, John Braisted, Carlos A. Tristan, Ruili Huang, Yuhong Wang, Pinar Ormanoglu, Christopher P. Austin, Anton Simeonov, Ilyas Singeç

Download the full size poster here.

More information about ISSCR 2019.


The systematic study of cell differentiation provides deeper insights into developmental pathways and how they control complex genetic programs. Neural induction of human pluripotent cells can be used as a model system to investigate the interplay between pathway manipulation and gene activation/silencing in chemically defined E6 medium. Small molecule based inhibition of bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF-β) pathways (dual SMAD inhibition) is a widely used approach to convert pluripotent cells into neuroectoderm and neural crest. While previous studies characterized human neural induction in bulk cultures, high-resolution analysis should capture the dynamic molecular changes more comprehensively. Here, we performed 7-day time-course single-cell sequencing (scRNA-Seq) to reconstruct differentiation trajectories induced by blocking BMP and TGF-β pathways either separately or in combination. Using dual SMAD inhibitors at single concentrations (0.1 ­M LDN-193189, 2 ­M A83-01), we identified distinct transition stages characterized by transiently expressed genes (e.g. SIX3, HESX1, and LMO1), which led to expression of PAX6, DLK1, TPBG, TMSB15A, HES4, IGFBP5, FOXG1, SOX11. Next, to systematically investigate gene expression dynamics, RASL-Seq was used for gene expression profiling upon BMP inhibition and TGF-β inhibition alone or in combination across seven different small molecule concentrations. During BMP inhibition, FOXG1, SOX1, FZD5, ZIC4, HEXS1, SIX3, PTN, HES4 were strongly upregulated while PAX3, FOXD3, SOX10, SNAI2, S100B were antagonized in a dose-dependent manner. Conversely, TGF-β inhibition alone positively regulated PAX3 and SOX10, suggesting that this strategy favors the induction of neural crest. In summary, modulation of cell signaling pathways via high-throughput gene expression profiling and small molecule titration can control expression of transcription factors determining cell specification. This strategy should help to optimize cell differentiation protocols using precisely calibrated small molecule combinations to produce functional phenotypes for clinical therapies.