Project description:Single cell RNA seq to compare the gene expression profile of hepatocytes cultured in expansion or differentiation media to primary hepatocytes
Project description:We have established a scaffold-free protocol to generate multicellular, beating and self-organized human organotypic cardiac microtissues (hOCMTs) in vitro from human induced pluripotent stem cells (hiPSCs) that can be cultured for long term. This is achieved by differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs containing multiple cell types that physiologically compose the heart, gradually self-organize and beat without external stimuli for more than 50 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and maturation as compared to standard monolayer cardiac differentiation in 2D. We also confirmed the functionality of hOCMTs by metabolic flux analysis and their response to cardioactive and cardiotoxic drugs in long term culture. This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.
Project description:In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.
Project description:The aim of the project was the analysis of the bulk proteome and phosphoproteome in 3D organoids (tubuloids) from the adult mouse kidney. Proteome (nearly 9000 proteins) and phosphoproteome (nearly 16000 phospho sites) were compared in three biological (independent) replicates of epithelial organoids (early passage and long-term, 3-month cultures) and freshly isolated, MAC-sorted, Epcam-positive kidney epithelial cells. After collection of the organoids, Matrigel was removed using a non-enzymatic solution.
Project description:Recent studies have characterized the genomic structures of many eukaryotic cells, often with a focus on their relation to gene expression. So far, these studies have largely only investigated cells grown in 2D culture, although the transcriptomes of 3D cultured cells are generally closer to their in vivo phenotype. To examine the effects of spatial constraints on chromosome conformation, we investigated the genomic architecture of mouse hepatocytes grown in 2D and 3D cultures using in situ Hi-C. Our results reveal significant differences in long-range genomic interactions, notably in compartment identity and strength as well as in TAD-TAD interactions, but only minor differences at the TAD level. RNA-seq analysis reveals an up-regulation in the 3D cultured cells of those genes involved in physiological hepatocyte functions. We find that these genes are associated with only a subset of the structural changes, suggesting that the differences in genomic structure are indeed critically important for transcriptional regulation but also that there are major structural differences owing to other functions than gene expression. Overall, our results indicate that growth in 3D significantly alters longer-range genomic interactions, which may be consequential for a subset of genes that are important for the physiological functioning of the cell.
Project description:Studying primary Chronic Lymphocytic Leukemia cells in vitro is very challenging due to their short survival in culture, and also due to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited three-dimensional (3D) bioprinting to advance in vitro models for CLL. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings.