Human liver cancer research currently lacks in vitro models that faithfully recapitulate the pathophysiology of the original tumour. We recently described a novel, near-physiological organoid culture system, where primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here, we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumours. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumour, allowing discrimination between different tumour tissues and subtypes, even after long term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumourogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug screening testing and lead to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized medicine approaches for the disease.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions leading to cancer. As for most cancer types, however, understanding of the earliest phases of colorectal neoplastic change, which may occur in morphologically normal tissue, is comparatively limited. Here, we whole genome sequenced hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed, some ubiquitous and continuous, others only found in some individuals, in some crypts or during certain periods of life. Likely driver mutations were present in ~1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially elevated mutation burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancerdriver mutations, which conceivably are morphologically indistinguishable from normal cells, are similarly unclear. In large part, these deficiencies are due to the technical challenge of identifying somatic mutations in normal tissues, which are composed of myriad microscopic cell clones. Several different approaches have been adopted to address this 4-14 , revealing signatures of common somatic mutational processes in normal cells of the small and large intestine, liver, blood, skin, and nervous system. Thus far, however, studies have not been of sufficient scale to characterise variation in signature activity or detect less frequent processes 4-14. Remarkably high proportions of normal skin, oesophageal, and endometrial epithelial cells have been shown to be members of clones already carrying driver mutations 10,11,15,16 , and large mutant clones have been detected in blood 17-20. The extent of this phenomenon in the colon, an organ with a high cancer incidence, has not been investigated. Colonic epithelium is a contiguous cell sheet organised into ~15,000,000 crypts each composed of ~2,000 cells 21. Towards the base of each crypt resides a small number of stem cells ancestral to the maturing and differentiated cells in the crypt 22. These stem cells stochastically replace one another through a process of neutral drift 23,24 such that all stem cells, and thus all cells, in a crypt derive from a single ancestor stem cell that existed in recent years 25-27. The somatic mutations that were present in this ancestor are thus found in all ~2,000 descendant cells and can be revealed by DNA sequencing of an individual crypt. These stem cells are thought to be the cells of origin of colorectal cancers 28. To characterise the earliest stages of colorectal carcinogenesis, somatic mutation burdens, mutational signatures, clonal dynamics, and the frequency of driver mutations in normal colorectal epithelium were explored by sequencing individual colorect...

26The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for 27 understanding the successive somatic genetic changes and consequent clonal expansions 28 leading to cancer. As for most cancer types, however

BackgroundThe Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design.ResultsThis study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types.We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific.ConclusionsIn conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.