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Comparative proteomics between 2D and 3D (neurospheres) cultured NSCs

ABSTRACT: Neurospheres are a relevant 3D model with many applications, but it lacks sufficient omics studies. Here, we performed a comparative proteomics experiment to investigate what distinguishes a 2D from a 3D culture of Neural Stem Cells (NSCs). In both cases, NSCs were iPSC-derived.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell, Cell Culture

SUBMITTER: Pedro Muniz

LAB HEAD: Magno Rodrigues Junqueira

PROVIDER: PXD063185 | Pride | 2026-01-20

REPOSITORIES: Pride

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Project description:Purpose: The aim of this study is (1) to identify the chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain; (2) to profile key epigenetic marks H3K4me3, H3K27me3 and DNA methylation in wild type and Smchd1 null NSCs; (3) to identify the chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs. Methods: Chromatin immunoprecipitation for Smchd1, H3K4me3, H3K27me3 and Ctcf was performed essentially as in (Nelson et al. 2006). Briefly, nuclei were isolated from formaldehyde crosslinked NSCs and chromatin was fragmented by sonication. Chromatin immunoprecipitation was performed with corresponding antibodies for Smchd1, H3K4me3 and H3K27me3. DNA was extracted from the immunoprecipitated fraction following reverse-crosslinking. Isolated DNA was used to generate sequencing libraries with Illumina's TruSeq DNA Sample Preparation Kit or Ovation Ultralow system (NuGen) according to manufacturer's instruction. Libraries were pooled and sequenced on the Illumina HiSeq 2000 platform for 100 bp single-end reads. Image analysis was performed in real time by the HiSeq Control Software (HCS) v1.4.8 and Real Time Analysis (RTA) v1.12.4.2, running on the instrument computer. Real-time base calling on the HiSeq instrument computer was performed with the RTA software. Illumina CASAVA1.8 pipeline was used to generate the sequence data. To examine the level of DNA methylation, genomic DNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen) and methylated DNA was isolated via binding to the methyl-CpG binding domain of human MBD2 protein coupled beads using the MethylMiner methylated DNA enrichment kit (Life Technologies) according to the manufacturer’s instructions. Isolated DNA was used to generate sequencing libraries as for the ChIP-seq experiment with Illumina’s TruSeq DNA Sample Preparation Kit according to manufacturer's instruction and sequenced on the Illumina HiSeq 2000 platform for 49 bp single-end reads. Sequencing analysis was performed as described for the ChIP-seq experiments. Chromatin occupancy of the epigenetic regulator Smchd1 in neural stem cells (NSCs) derived from E14.5 mouse brain was determined by Smchd1 ChIP-seq. Enrichment of H3K4me3 and H3K27me3 in wild type and Smchd1 null NSCs were assessed by H3K4me3 and H3K27me3 ChIP-seq, respectively. DNA methylation in wild type and Smchd1 null NSCs was assessed by MBD-seq. Chromatin occupancy of Ctcf in wild type and Smchd1 null NSCs was determined by by Ctcf ChIP-seq.

Project description:A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur prior to any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Neural stem cells derived from mouse embryonic stem cells were differentiated into neurons and FACS purified based on RedStar fluorescence driven by the Tau promoter. Chromatin was prepared from NSCs and neurons (n=1), sonicated to roughly 300bp and immunoprecipitated with antibodies against H3K4me3, H3K27me3, total Histone H3 and total IgG, alongside a 5% input sample. K4/K27 and corresponding input samples were analysed by ChIPSeq

			Action	DRS
	Cell_Type.xlsx	Xlsx
	GO_Biological_Process.xlsx	Xlsx
	GO_Celular_Component.xlsx	Xlsx
	Identifications.mzTab	Mztab
	Kegg.xlsx	Xlsx

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Comparative proteomics between 2D and 3D (neurospheres) cultured NSCs

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