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3D ECM-rich environment sustains the identity of naïve human iPSCs [Bulk RNA-seq in triplicate after 1, 4, and 7 days of culture]

ABSTRACT: The establishment of in vitro naïve human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. In the last 5 years the signalling environment promoting human naïve pluripotency has been extensively investigated. However, little is known about the role exerted by the extracellular matrix and three-dimensional organization in naïve maintenance. Here, by using an unbiased and integrated approach we found that naïve, but not primed, hiPSC colonies are characterized by an extracellular matrix-rich microenvironment, and by a specific extracellular matrix fingerprint at transcriptional, proteomic and secretome levels. Based on this, we developed a 3D culture system which supports robust long-term feeder-free expansion of naïve hiPSCs. In this same 3D ECM-rich environment, we can recapitulate timely developmental morphogenesis simply by modulating the signalling environment. Our study opens new perspectives for future applications of naïve hiPSCs to study critical stages of human development in three-dimensions starting from a single cell.

ORGANISM(S): Homo sapiens

PROVIDER: GSE161717 | GEO | 2021/11/11

REPOSITORIES: GEO

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Project description:Full title: Comprehensive Characterization of Three-Dimensional Models for Prostate Cancer Growth and Invasion in Laminin-rich Extracellular Matrix Prostate Cancer (PrCa) cells undergo acinar morphogenesis and spheroid formation in three-dimensional (3D) culture, supported by laminin-rich extracellular matrix (lrECM, Matrigel). We developed miniaturized 3D model systems that facilitate investigation of morphogenesis and invasion of normal and PrCa cell lines in lrECM. Primary and non-transformed cell lines formed round structures with strong cell-cell contacts and epithelial polarization, lumen and a complete basal lamina (BL). In contrast, most PrCa cell lines formed either defective, “mass” spheroids with incomplete BL, or invasive “stellate” structures. The bioinformatic analyses of genome-wide mRNA expression data revealed massive alteration of key functional and signaling pathways in 3D cultures, with lipid and steroid metabolism, epigenetic reprogramming, and differentiation-related transcription factors induced across all cell lines by lrECM. In invasive cells, AKT, PI3Kinase, mTOR, and hedgehog signaling pathways were most highly activated, validated by small molecule inhibitors compounds specifically targeting key regulatory molecules. Compounds against AKT and PI3kinase pathways were significantly more effective in invasive cells, compared to mass or round/normal phenotype spheroids, and monolayer culture. A severe morphologic conversion was observed in PC-3 and PC-3M cells, transforming initially round, normal-appearing epithelial spheroids into rapidly invading cell masses. Markers for EMT (epithelial-mesenchymal transition) were highly expressed already in early stage, round spheroids prior to invasive conversion, and were not further increased in invasive cells. This indicates that PrCa cells can display extraordinary plasticity. EMT may be involved in providing a metastable genotype that allows morphological transformation, but is not be required for invasive processes themselves. Total RNA was obtained from non-transformed prostate epithelial cells and prostate cancer cells cultured in monolayer and three-dimensional laminin-rich extracellular matrix (growth factor-reduced Matrigel).

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3D ECM-rich environment sustains the identity of naïve human iPSCs [Bulk RNA-seq in triplicate after 1, 4, and 7 days of culture]

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