Enhanced Cell-Cell Contact and Planarian Tissue Extract increase DNA replication and cell viability for neoblast cultivation
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PROVIDER: PRJNA1294330 | ENA |
REPOSITORIES: ENA
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Project description:The development of an in vitro culture system for planarian neoblasts remains at an early stage. Although modified neoblast enrichment method and basic culture conditions have been established, it is still unclear whether mimicking the in vivo micro- and macro-environments can functionally improve neoblast behavior in vitro. Neoblasts tend to aggregate in culture, but when cultured as aggregates in the U-bottom plate or on feeder layers, they show increased DNA replication, potentially due to improved cell-cell interactions that mimic the in vivo microenvironment. At the macroenvironment level, the addition of planarian extract to the culture medium reduces cell death, increases lipid droplet formation, and decreases the intracellular reactive oxygen species (ROS) levels. An optimized culture condition has thus been established using U-bottom plates supplemented with planarian extract for the first three days, effectively recapitulating aspects of both the in vivo microenvironment and macroenvironment. Although the culture period remains limited, this system enabled gene knockdown via dsRNA in culturing neoblasts. By modulating both micro- and macro-environmental factors, this study provides a foundation for further optimization at the metabolic and genetic levels in subsequent studies.
2025-07-25 | GSE303204 | GEO
Project description:The complexity of cell types and states revealed by scRNAseq cell atlases presents a challenge for the systematic analysis of fate determinants using traditional screening methodologies. Differentiation in the planarian Schmidtea mediterranea exemplifies this problem, as these animals continuously produce over 100 differentiated cell types for homeostasis and regeneration using adult pluripotent stem cells termed neoblasts. The signaling factors enabling neoblast self-renewal and selective differentiation of these many fates are still incompletely understood. We developed a method using high-throughput expression profiling by qPCR and whole-animal RNAseq to simultaneously assess numerous cell fate markers as the phenotypic readout in large-scale RNAi screens. Applying this method, we performed an RNAi screen of 400 kinases, receptors, and other regulatory molecules to reveal specific functions for 30 previously unknown factors in neoblast biology. 17 genes were required for neoblast maintenance, including factors likely involved in cell-cycle regulation, nutrient sensing, and chromatin modification. Multidimensional expression information additionally revealed several specific regulators of other neoblast activities, including a mink1 kinase regulating global neoblast differentiation, the energy responsive kinase adenylate kinase-2 regulating intestine specification within the neoblast population, an RNA acetyl transferase nat10 regulating epidermal differentiation, and a pak1 kinase that restricts neoblast localization to prevent tissue outgrowths. These results identify several new regulators of neoblast activities and demonstrate the applicability of expression-based screening for systematic analysis of stem cell phenotypes in whole animals.
2023-08-31 | GSE212136 | GEO
Project description:Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for piRNA expression in planarians. Nevertheless, the full complement of piRNAs, and microRNAs (miRNAs) in this organism has yet to be defined. Here, we report on the large scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs uncovered ten miRNAs enriched in planarian stem cells. These miRNAs are down-regulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals. Small RNAs were sequenced from FACS-sorted neoblast stem cells, untreated planarians and irradiated planarians, depleted of neoblasts
2009-06-30 | E-GEOD-16159 | biostudies-arrayexpress
Project description:Neoblasts are adult stem cells (ASCs) in planarians which sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving the postmitotic fate remain poorly defined. Here we used transcriptional profiling of irradiation-sensitive and -insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers, which distinguish two epithelial progenitor populations, and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 is required for generating progenitors of epithelial, neural, eye, pharyngeal, and protonephridial lineages, and restricting expansion of the stem cell compartment. We also demonstrate the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results show that mex3-1 promotes differentiation in multiple, if not all, lineages, and maintains the balance between ASC self-renewal and commitment. Sorted and irradiated animals were collected as previously described (Labbe, et al., 2012) and RNAi conditions were triplicated, RNA was purified independently, then each replicate was pooled in equal amounts for sequencing at the timepoint: 1 feed day 12 for control RNAi and mex3-1 RNAi.
2015-06-15 | E-GEOD-68581 | biostudies-arrayexpress
Project description:PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Planarian PIWI proteins thus act as both nucleases and RNA-binding proteins, enabling planarians to repurpose piRNAs for critical roles in neoblast mRNA turnover.
2019-09-04 | GSE122199 | GEO
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.
2025-06-15 | GSE274282 | GEO
Project description:Neoblasts are adult stem cells (ASCs) in planarians which sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving the postmitotic fate remain poorly defined. Here we used transcriptional profiling of irradiation-sensitive and -insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers, which distinguish two epithelial progenitor populations, and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 is required for generating progenitors of epithelial, neural, eye, pharyngeal, and protonephridial lineages, and restricting expansion of the stem cell compartment. We also demonstrate the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results show that mex3-1 promotes differentiation in multiple, if not all, lineages, and maintains the balance between ASC self-renewal and commitment.
2015-06-15 | GSE68581 | GEO
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.
2025-06-15 | GSE274281 | GEO
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.
2025-06-15 | GSE274280 | GEO
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.
2025-06-15 | GSE299320 | GEO