Project description:Stem cell differentiation underlies development and regeneration in multicellular organisms. Cell type identity is determined by the expression of specific transcription factors (TFs) regulating target genes (TGs) via binding to open chromatin regions (OCRs). The regulatory logic of differentiation includes factors specific to one or multiple cell types, functioning in a combinatorial fashion. Single cell transcriptomics has emerged as a revolutionary approach to study gene expression with cell type resolution, but integrating it with single cell analysis techniques to study chromatin remains challenging. Planarians, with their pluripotent neoblast stem cells continuously giving rise to all cell types, offer an ideal model to attempt this integration. Despite extensive single cell transcriptomic studies, the transcriptional and chromatin regulation at the cell type level remains unexplored. Here, we investigate the regulatory logic of planarian stem cell differentiation by obtaining an organism-level integration of single cell transcriptomics and single cell ATAC-seq. We identify specific open chromatin profiles for major differentiated cell types and analyse their transcriptomic landscape, revealing distinct gene modules expressed in individual types and combinations of them. Integrated analysis unveils gene networks reflecting known TF interactions in each type and identifies TFs potentially driving differentiation across multiple cell types. To validate our predictions, we combined TF knockdown RNAi experiments with single cell transcriptomics. We focus on hnf4, a TF known to be expressed in gut phagocytes, and confirm its influence on other types, including parenchymal cells. Our results demonstrate high overlap between predicted targets and experimentally-validated differentially-regulated genes. Overall, our study integrates TFs, TGs and OCRs to reveal the regulatory logic of planarian stem cell differentiation, showcasing the potential of single cell methods for characterising differentiation trajectories and their regulation.

Project description:A network of transcription factors (TFs) determines cell identity, but identity can be altered by overexpressing a combination of TFs. In principle, this opens the possibility of achieving one of the goals of regenerative medicine generating the desired differentiated cells from pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, choosing and verifying combinations of TFs for specific cell differentiation has been daunting due to a large number of possible combinations of ~2,000 TFs. Here we report an unbiased systematic approach identifying individual TFs that can direct efficient and rapid specific lineage differentiation. We start from a correlation matrix of global gene expression responses to the induction of single TFs and global gene expression profiles of a variety of tissues and organs. Based on the correlation matrix, we select TFs as examples and show that their overexpression differentiates ES cells into cells of specific organs, as predicted: Sfpi1 for blood cells, Hnf4a or Foxa1 for hepatocytes, and Ascl1 for neurons. Furthermore, we show that transfection of synthetic mRNAs of Sfpi1, Hnf4a, or Ascl1 generate correspondingly specific target cells. These results demonstrate both the wide-ranging utility of this approach to identify potent TFs for cell differentiation, and also the unanticipated capacity of single TFs to directly guide differentiation to specific lineage fates. Previously, we generated the global gene expression profiles obtained by overexpressing single TFs using the NIA mouse ES cell bank, which consists of 137 mouse ES cell lines carrying a doxycycline-regulatable TF. We then generated the correlation matrix by comparing TF-induced gene expression profiles and the expression profiles of a variety of cell types. TFs predicted by the correlation matrix for specific cell differentiation were selected and subjected to the detailed differentiation assays. ES cell lines were cultured in the GMEM with 1% FBS, 10% knockout serum (invitrogen), and LIF (Millipore). To induce differentiation, ES cell lines were cultured in the differentiation medium (DM) [(alpha minimal essential medium, aMEM) supplemented with 10% fetal calf serum and 5 × 10–5 M 2-mercaptoethanol] with or without doxycycline (1 g/ml). The following organ-specific cell culture media were also used: StemPro-34 Serum Free Media (invitrogen) for blood cells, Hepatocyte culture media kit (BD Biosciences) for hepatocytes, and NeuroCult differentiation kit (StemCell Technologies Inc) for neurons.

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The Regulatory Logic of Planarian Stem Cell Differentiation [RNA-Seq]

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