Organoids in high-throughput and high-content screenings

Organoids are self-organized three-dimensional (3D) multicellular tissue cultures which derive from cancerous and healthy stem cells, sharing a highly similarity to the corresponding in vivo organs. Since their introduction in 2009, they have emerged as a valuable model for studying early embryogenesis, organ and tissue development, as well as tools in drug screening, disease modeling and personalized therapy. Organoids can now be established for various tissues, including brain, retina, thyroid, gastrointestinal, lung, liver, pancreas, and kidney. These micro-tissues resemble the native organ in terms of gene expression, protein expression, tissue architecture and cell-cell interactions. Despite the success of organoid-based research and the advances in patient-derived organoid culture, important challenges remain. In this review, we briefly showcase the evolution from the primary 3D systems to complex, multilayered 3D structures such as assembloids, gastruloids and ETiX embryoids. We discuss current developments in organoid research and highlight developments in organoid culturing systems and analysis tools which make organoids accessible for high-throughput and high-content screening. Finally, we summarize the potential of machine learning and computational modeling in conjunction with organoid systems

Organoids in high-throughput and high-content screenings

Organoids are self-organized three-dimensional (3D) multicellular tissue cultures which derive from cancerous and healthy stem cells, sharing a highly similarity to the corresponding in vivo organs. Since their introduction in 2009, they have emerged as a valuable model for studying early embryogenesis, organ and tissue development, as well as tools in drug screening, disease modeling and personalized therapy. Organoids can now be established for various tissues, including brain, retina, thyroid, gastrointestinal, lung, liver, pancreas, and kidney. These micro-tissues resemble the native organ in terms of gene expression, protein expression, tissue architecture and cell-cell interactions. Despite the success of organoid-based research and the advances in patient-derived organoid culture, important challenges remain. In this review, we briefly showcase the evolution from the primary 3D systems to complex, multilayered 3D structures such as assembloids, gastruloids and ETiX embryoids. We discuss current developments in organoid research and highlight developments in organoid culturing systems and analysis tools which make organoids accessible for high-throughput and high-content screening. Finally, we summarize the potential of machine learning and computational modeling in conjunction with organoid systems.ISSN:2673-271

TCF/LEF dependent and independent transcriptional regulation of Wnt/β-catenin target genes

During canonical Wnt signalling, the activity of nuclear β-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view, we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones lacking all four TCF/LEF genes. By performing unbiased whole transcriptome sequencing analysis, we found that a subset of β-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that β-catenin occupied specific genomic regions in the absence of TCF/LEF Finally, we revealed the existence of a transcriptional activity of β-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as β-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of β-catenin that bypasses the TCF/LEF transcription factors

Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis.

Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA).Human cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated.We could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed.Here, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM

Gene expression of fibrotic markers and cytokines in human liver microtissues exposed to LPS, TNF-α and TGF-β1.

<p>mRNA was extracted using TRIzol conventional procedure and fold induction were calculated as 2^(-ΔΔCT) for each sample and vehicle control and expressed as mean fold induction ± S.E.M of three replicates with six MTs each. Actin was used as reference gene for each sample. † pool of 16 microtissues analysed as duplicate; no statistical analysis were performed on these samples. ND: no-detected values. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.</p

LPS induces TNF-α production in PMA-treated THP-1 cells.

<p>THP-1 were treated with 0, 5, 10 and 25 ng/mL PMA for 48h prior exposure to 1 μg/mL LPS. The concentration of TNF-α was measured from the 48h culture supernatants by ELISA. These findings indicate that PMA differentiated THP-1 cells are well differentiated and yet respond adequately to a subsequent low-concentration of LPS. Values are presented as duplicates.</p

Immunostaining of formalin fixed paraffin embedded human microtissues after exposure to MTX, TAA and TGF-β1.

<p>Formalin fixed paraffin embedded slides of HepaRG/THP-1 macrophages/hTERT-HSC microtissues were stained with Hematoxylin & Eosin (H&E), vimentin, α-smooth muscle actin (αSMA), collagen I and CD68 after 14 days of treatment with MTX, TAA and TGF-β1. Microtissues were fixed in 4% PFA and embedded in 2% agarose prior paraffinization. Microtissues showed increase in the vimentin, αSMA, collagen I and CD68 positive cells after MTX, TAA and TGF-β1 exposure. Vimentin and CD68 stainings show proliferation of stellate cells and THP-1 macrophages in the microtissues, suggesting the onset of inflammation process, while αSMA and collagen I indicate activation of stellate cells and deposition of collagen. Scale bar: 200μm. Graphics show Quantitative IHC Staining Value (QISV) as mean ± S.D. (N = 5). *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.</p

Albumin production and cell proliferation in liver microtissues.

<p>Formalin fixed paraffin embedded slides of HepaRG/THP-1 macrophages/hTERT-HSC microtissues were stained with Albumin and Ki67 antibodies after 14 days of treatment with MTX, TAA and TGF-β1. Microtissues were fixed in 4% PFA and embedded in 2% agarose prior paraffinization. Microtissues showed decrease in albumin production after MTX, TAA and especially TGF-β1 exposure. Ki67 shows strong induction of cell proliferation in the microtissues after TGF-β1 exposure. Scale bar: 200μm. Graphics show Quantitative IHC Staining Value (QISV) as mean ± S.D. (N = 5). *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.</p

TNF-α and TGF-β1 promote αSMA production in monolayer culture of hTERT-HSC.

<p>hTERT-HSC cells were treated for 2, 5 or 10 days with TNF-α (50ng/mL) or TGF-β1 (1ng/mL). After treatment, the cells were fixed and stained against αSMA (green) and nuclei (DAPI, blue). Pictures taken using fluorescence microscopy. Scale bar: 50μm.</p

Viability response of human liver microtissues treated with pro-fibrotic compounds.

<p>(A) Effect of LPS, TNF-α and TGF-β1 on viability of human liver microtissues was assessed by MTT assay. Microtissues were incubated with 0.5mg/mL MTT solution for 4h after 14 days of exposure to the tested compounds. DMSO and Sörensen buffer were then added into the wells and absorbance was read at 550nm using FlexStation 3. Values are expressed as percentage of negative control. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control. (n = 6, mean ± SD). (B) Effect of MTX, TAA and TGF-β1 on the ATP production. ATP content was measured using CellTiter-Glo^® Luminescent Cell Viability Assay 2.0 after 14 days of exposure to MTX, TAA and TGF-β1. Values are expressed as percentage of negative control. ***; P ≤ 0.001 vs vehicle control (n = 6, mean ± SD).</p