Project description:Comparison of differentiation of human iPSC derived alveolar epitheilal type 2 cells (iAT2s) into AT1-like cells (iAT1s) using a defined "LDCI" medium vs. lentiviral forced over-expression of activated nuclear YAP (YAP5SA)
Project description:Breast cancer cells reprogram the oncogenic lncRNAs/mRNAs co-expression networks in three- dimensional microenvironment To have a more functional approach, organotypic 3D cell cultures that more accurately mimic the characteristics of solid tumors in vivo and the tumor microenvironment are required. In this study, DNA microarrays were employed to deline the changes in lncRNAs expression patterns of breast cancer cells, cultured in 3D and 2D conditions from BT-474 cell line. Furthermore, potential lncRNAs/mRNAs pairs co-expressed in 3D cultures exhibit a high degree of similarity with those found in luminal B breast cancer patients suggesting that they could be adequate pre-clinical tools to identify, not only biomarkers related to endocrine therapy response and PCR, but to understand the biological behavior of cancer cells in 3D microenvironments, which point towards an important contribution of the roles of lncRNAs in organotypic 3D cultures.
Project description:Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells Three-dimensional (3D) cell cultures have several advantages over conventional monolayer two-dimensional (2D) cultures as they can better mimic tumor biology. This study delineated the changes in microRNA (miRNA) expression patterns of breast cancer cells cultured in 3D and 2D conditions. 3D organotypic cultures showed morphological changes such as cell–cell and cell–extracellular matrix interactions associated with a loss of polarity and reorganization on bulk structures in both basal Hs578T and luminal T47D breast cancer cells. Data indicate that down-regulated miRNAs in Hs578T 3D cultures, relative to the 2D condition, contribute to a positive regulation of biological processes such as response to hypoxia and focal adhesion, whereas over-expressed miRNAs were related to negative regulation of the cell cycle. Remarkably, the repro-gramming of miRNAs’ transcriptional profiles was accompanied by changes in the expression of key miRNA/mRNA coregulation networks, such as miR-935/HIF-1A, which correlated with the expression found in clinical breast tumors and predicted poor patient outcomes. These data have implications in our understanding of cancer biology and impact the miRNA/mRNA regulatory axes of cells grown in 3D cultures. Our data represent a guide for novel miRNA candidates for functional analysis, including the response to therapy and biomarker discovery in breast cancer.
Project description:Brain microenvironment plays an important role in neurodevelopment and function, where extracellular matrix (ECM) components and soluble factors modulate cellular features, as migration, proliferation survival and neuronal function. Disruption of microenvironment’s homeostasis is often related to pathological conditions. Here, we addressed the microenvironment remodeling occurring during in vitro differentiation of human neural stem cells (NSC) in a three-dimensional (3D) culture system. Proteome and transcriptome dynamics revealed significant changes namely at cell membrane and ECM composition during 3D differentiation, diverging significantly from the profile of monolayer cultures (2D). Structural proteoglycans typically found in brain ECM were enriched during 3D differentiation, while 2D cultures presented increased levels of basement membrane constituents (e.g., laminins, collagens and fibrillins). Moreover, higher expression levels of synaptic machinery and ion transport machinery constituents observed for 3D cultures, both at mRNA and protein levels, suggested a higher degree of neuronal maturation and organization relative to 2D differentiation. This work demonstrated that neural cellular and extracellular features can be recapitulated in the presented 3D neural cell model, highlighting its value to address molecular defects in cell-ECM interactions associated with neurological disorders. <html><head>Associated GEO dataset is available at</head><body><a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi">GSE102139</a></body></html>
Project description:Premature birth disrupts normal lung development and places infants at risk for bronchopulmonary dysplasia (BPD), a disease increasing in incidence which disrupts lung health throughout the lifespan. The TGFβ superfamily has been implicated in BPD pathogenesis, however, what cell lineage it impacts remains unclear. We show that the primary TGFβ receptor, Tgfbr2, is critical for AT1 cell fate maintenance and function. Loss of Tgfbr2 in AT1 cells during late lung development leads to AT1-AT2 cell reprogramming and altered pulmonary architecture, which persists into adulthood. Restriction of fetal lung stretch and associated AT1 cell spreading through a model of oligohydramnios enhances AT1-AT2 reprogramming. Transcriptomic and proteomic analysis reveal that expression of extracellular matrix components by AT1 cells is attenuated with loss of Tgfbr2. Cell spreading assays shows that TGFβ signaling regulates integrin transcription to alter AT1 cell morphology, which further impacts ECM expression through changes in mechanotransduction. These data reveal the cell intrinsic necessity of TGFβ signaling to maintain AT1 fate and define this cell lineage as a major orchestrator of the alveolar matrisome, which, if altered, may predispose to BPD.
Project description:3D spheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. However, the 3D spheroids are maintained under different conditions, with possible cofounding results. Here we performed an in-depth analysis of how various culture conditions influence 3D spheroids. Our aim was to find optimal conditions for the maintenance of a normal PHH phenotype.
Project description:The principal organization of mammalian neuromuscular junctions (NMJs) shares essential features across species, yet human NMJs (hNMJs) exhibit distinct structural and physiological properties. While recent advances in stem cell-based systems have significantly improved in vitro modeling of hNMJs, the extent to which these models recapitulate in vivo development remains unclear. Here, we performed spatial transcriptomic analysis of human 3D neuromuscular co-cultures, composed of iPSC-derived motoneurons and 3D skeletal muscle engineered from primary myoblasts. We found the transcriptomic analysis follows a temporally coordinated gene expression program underlying NMJ maturation. The model recapitulates key transcriptional features of NMJ development, including early myoblast fusion, sequential upregulation of presynaptic and postsynaptic maturation genes, and late-stage induction of embryonic AChR subunits. Importantly, consistent transcriptional dynamics across two independent hiPSC lines and differentiation protocols confirm the reproducibility and robustness of this system. This study provides a valuable transcriptomic resource and demonstrates that human 3D neuromuscular co-cultures are a robust and physiologically relevant model for investigating human NMJ development, function, and disease.