The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The ''oval cell'' response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.

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Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.

Adult neurogenesis in the murine dentate gyrus occurs in a specialized microenvironment that sustains the generation of neurons during life. To fully understand adult neurogenesis, it is essential to determine the neural stem cell (NSC) and progenitor developmental stages, their molecular determinants, and the niche cellular and molecular composition. We report on a single-cell RNA sequencing study of the hippocampal niche, performed by isolating all the non-neuronal cell populations. Our analysis provides a comprehensive description of the dentate gyrus cells, and it allows the identification of exclusive cell-type-specific markers. We define the developmental stages and transcriptional dynamics of NSCs and progenitors, and we find that, while NSCs represent a heterogeneous cellular continuum, progenitors can be grouped into distinct subtypes. We determine the oligodendrocyte lineage and transcriptional dynamics, and we describe the microglia transcriptional profile and activation state. The combined data constitute a valuable resource to understand regulatory mechanisms of adult neurogenesis.

Size and folding of the cerebral cortex increased massively during mammalian evolution leading to the current diversity of brain morphologies. Various subtypes of neural stem and progenitor cells have been proposed to contribute differently in regulating thickness or folding of the cerebral cortex during development, but their specific roles have not been demonstrated. We report that the controlled expansion of unipotent basal progenitors in mouse embryos led to megalencephaly, with increased surface area of the cerebral cortex, but not to cortical folding. In contrast, expansion of multipotent basal progenitors in the naturally gyrencephalic ferret was sufficient to drive the formation of additional folds and fissures. In both models, changes occurred while preserving a structurally normal, six-layered cortex. Our results are the first experimental demonstration of specific and distinct roles for basal progenitor subtypes in regulating cerebral cortex size and folding during development underlying the superior intellectual capability acquired by higher mammals during evolution.

Enteroendocrine cells (EECs) control a wide range of physiological processes linked to metabolism. We show that EEC hormones are differentially expressed between crypts (for example, Glp1) and villi (for example, secretin). As demonstrated by single-cell mRNA sequencing using murine Lgr5 cell-derived organoids, BMP4 signals alter the hormone expression profiles of individual EECs to resemble those found in the villus. Accordingly, BMP4 induces hormone switching of EECs migrating up the crypt-villus axis in vivo. Our findings imply that EEC lineages in the small intestine exhibit a more flexible hormone repertoire than previously proposed. We also describe a protocol to generate human EECs in organoids and demonstrate a similar regulation of hormone expression by BMP signalling. These findings establish alternative strategies to target EECs with therapeutically relevant hormone production through BMP modulation.

Highlights d A human organoid biobank combines hormone labeling and enteroendocrine cell generation d Transcriptomic profiling of human enteroendocrine cells uncovers differences with mice d Functional validation of EEC receptors and transcription factors d Secretome analysis reveals the repertoire of enteroendocrine secreted products

The deubiquitinating enzyme BAP1 is a tumor suppressor, among others involved in cholangiocarcinoma. BAP1 has many proposed molecular targets, while its Drosophila homolog is known to deubiquitinate histone H2AK119. We introduce BAP1 loss-of-function by CRISPR/Cas9 in normal human cholangiocyte organoids. We find that BAP1 controls the expression of junctional and cytoskeleton components by regulating chromatin accessibility. Consequently, we observe loss of multiple epithelial characteristics while motility increases. Importantly, restoring the catalytic activity of BAP1 in the nucleus rescues these cellular and molecular changes. We engineer human liver organoids to combine four common cholangiocarcinoma mutations (TP53, PTEN, SMAD4, and NF1). In this genetic background, BAP1 loss results in acquisition of malignant features upon xenotransplantation. Thus, control of epithelial identity through the regulation of chromatin accessibility appears to be a key aspect of BAP1's tumor suppressor function. Organoid technology combined with CRISPR/Cas9 provides an experimental platform for mechanistic studies of cancer gene function in a human context.