Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.

Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids.

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.

Identification of novel human Wnt target genes using adult endodermal tissue-derived organoids.

Canonical Wnt signaling plays a key role during organ development, homeostasis and regeneration and these processes are conserved between invertebrates and vertebrates. Mutations in Wnt pathway components are commonly found in various types of cancer. Upon activation of canonical Wnt signaling, ß-catenin binds in the nucleus to members of the TCF-LEF family and activates the transcription of target genes. Multiple Wnt target genes, including Lgr5/LGR5 and Axin2/AXIN2, have been identified in mouse models and human cancer cell lines. Here we set out to identify the transcriptional targets of Wnt signaling in five human tissues using organoid technology. Organoids are derived from adult stem cells and recapitulate the functionality as well as the structure of the original tissue. Since the Wnt pathway is critical to maintain the organoids from the human intestine, colon, liver, pancreas and stomach, organoid technology allows us to assess Wnt target gene expression in a human wildtype situation. We performed bulk mRNA sequencing of organoids immediately after inhibition of Wnt pathway and identified 41 genes as commonly regulated genes in these tissues. We also identified large numbers of target genes specific to each tissue. One of the shared target genes is TEAD4, a transcription factor driving expression of YAP/TAZ signaling target genes. In addition to TEAD4, we identified a variety of genes which encode for proteins that are involved in Wnt-independent pathways, implicating the possibility of direct crosstalk between Wnt signaling and other pathways. Collectively, this study identified tissue-specific and common Wnt target gene signatures and provides evidence for a conserved role for these Wnt targets in different tissues.

Use and application of 3D-organoid technology.

The capacity of the 3D-organoid cultures to resemble a near-physiological tissue organization and to mimic - to a certain degree - organ functionality, make organoids an excellent model for applications spanning from basic developmental/stem cell research to personalized medicine. Here, we review key findings achieved through organoid technology, and we discuss applications such as disease - and tumour modelling, correction of genetic mutations and understanding gene - and cell functions. Finally, we discuss future developments in the field.

Tox: a multifunctional transcription factor and novel regulator of mammalian corticogenesis.

Major efforts are invested to characterize the factors controlling the proliferation of neural stem cells. During mammalian corticogenesis, our group has identified a small pool of genes that are transiently downregulated in the switch of neural stem cells to neurogenic division and reinduced in newborn neurons. Among these switch genes, we found Tox, a transcription factor with hitherto uncharacterized roles in the nervous system. Here, we investigated the role of Tox in corticogenesis by characterizing its expression at the tissue, cellular and temporal level. We found that Tox is regulated by calcineurin/Nfat signalling. Moreover, we combined DNA adenine methyltransferase identification (DamID) with deep sequencing to characterize the chromatin binding properties of Tox including its motif and downstream transcriptional targets including Sox2, Tbr2, Prox1 and other key factors. Finally, we manipulated Tox in the developing brain and validated its multiple roles in promoting neural stem cell proliferation and neurite outgrowth of newborn neurons. Our data provide a valuable resource to study the role of Tox in other tissues and highlight a novel key player in brain development.

Regulation of cerebral cortex size and folding by expansion of basal progenitors.

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.

Lentiviruses allow widespread and conditional manipulation of gene expression in the developing mouse brain.

Generation of transgenic mice, in utero electroporation and viral injection are common approaches to manipulate gene expression during embryonic development of the mammalian brain. While very powerful in many contexts, these approaches are each characterized by their own limitations: namely, that generation of transgenic mice is time-consuming and electroporation only allows the targeting of a small area of the brain. Similarly, viral injection has been predominantly characterized by using retroviruses or adenoviruses that are limited by a relatively low infectivity or lack of integration, respectively. Here we report the use of integrating lentiviral vectors as a system to achieve widespread and efficient infection of the whole brain after in utero injection in the telencephalic ventricle of mouse embryos. In addition, we explored the use of Cre-mediated recombination of loxP-containing lentiviral vectors to achieve spatial and temporal control of gene expression of virtually any transgene without the need for generation of additional mouse lines. Our work provides a system to overcome the limitations of retroviruses and adenoviruses by achieving widespread and high efficiency of transduction. The combination of lentiviral injection and site-specific recombination offers a fast and efficient alternative to complement and diversify the current methodologies to acutely manipulate gene expression in developing mammalian embryos.

Efficient transient genetic manipulation in vitro and in vivo by prototype foamy virus-mediated nonviral RNA transfer.

Vector systems based on different retroviruses are widely used to achieve stable integration and expression of transgenes. More recently, transient genetic manipulation systems were developed that are based on integration- or reverse transcription-deficient retroviruses. Lack of viral genome integration is desirable not only for reducing tumorigenic potential but also for applications requiring transient transgene expression such as reprogramming or genome editing. However, all existing transient retroviral vector systems rely on virus-encoded encapsidation sequences for the transfer of heterologous genetic material. We discovered that the transient transgene expression observed in target cells transduced by reverse transcriptase-deficient foamy virus (FV) vectors is the consequence of subgenomic RNA encapsidation into FV particles. Based on this initial observation, we describe here the establishment of FV vectors that enable the efficient transient expression of various transgenes by packaging, transfer, and de novo translation of nonviral RNAs both in vitro and in vivo. Transient transgene expression levels were comparable to integrase-deficient vectors but, unlike the latter, declined to background levels within a few days. Our results show that this new FV vector system provides a useful, novel tool for efficient transient genetic manipulation of target tissues by transfer of nonviral RNAs.

Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth.

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.

Temporal morphogen gradient-driven neural induction shapes single expanded neuroepithelium brain organoids with enhanced cortical identity.

Pluripotent stem cell (PSC)-derived human brain organoids enable the study of human brain development in vitro. Typically, the fate of PSCs is guided into subsequent specification steps through static medium switches. In vivo, morphogen gradients are critical for proper brain development and determine cell specification, and associated defects result in neurodevelopmental disorders. Here, we show that initiating neural induction in a temporal stepwise gradient guides the generation of brain organoids composed of a single, self-organized apical-out neuroepithelium, termed ENOs (expanded neuroepithelium organoids). This is at odds with standard brain organoid protocols in which multiple and independent neuroepithelium units (rosettes) are formed. We find that a prolonged, decreasing gradient of TGF-ß signaling is a determining factor in ENO formation and allows for an extended phase of neuroepithelium expansion. In-depth characterization reveals that ENOs display improved cellular morphology and tissue architectural features that resemble in vivo human brain development, including expanded germinal zones. Consequently, cortical specification is enhanced in ENOs. ENOs constitute a platform to study the early events of human cortical development and allow interrogation of the complex relationship between tissue architecture and cellular states in shaping the developing human brain.

Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss.

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.

Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis.

The lack of registered drugs for nonalcoholic fatty liver disease (NAFLD) is partly due to the paucity of human-relevant models for target discovery and compound screening. Here we use human fetal hepatocyte organoids to model the first stage of NAFLD, steatosis, representing three different triggers: free fatty acid loading, interindividual genetic variability (PNPLA3 I148M) and monogenic lipid disorders (APOB and MTTP mutations). Screening of drug candidates revealed compounds effective at resolving steatosis. Mechanistic evaluation of effective drugs uncovered repression of de novo lipogenesis as the convergent molecular pathway. We present FatTracer, a CRISPR screening platform to identify steatosis modulators and putative targets using APOB-/- and MTTP-/- organoids. From a screen targeting 35 genes implicated in lipid metabolism and/or NAFLD risk, FADS2 (fatty acid desaturase 2) emerged as an important determinant of hepatic steatosis. Enhancement of FADS2 expression increases polyunsaturated fatty acid abundancy which, in turn, reduces de novo lipogenesis. These organoid models facilitate study of steatosis etiology and drug targets.

A comprehensive transcriptomic comparison of hepatocyte model systems improves selection of models for experimental use.

The myriad of available hepatocyte in vitro models provides researchers the possibility to select hepatocyte-like cells (HLCs) for specific research goals. However, direct comparison of hepatocyte models is currently challenging. We systematically searched the literature and compared different HLCs, but reported functions were limited to a small subset of hepatic functions. To enable a more comprehensive comparison, we developed an algorithm to compare transcriptomic data across studies that tested HLCs derived from hepatocytes, biliary cells, fibroblasts, and pluripotent stem cells, alongside primary human hepatocytes (PHHs). This revealed that no HLC covered the complete hepatic transcriptome, highlighting the importance of HLC selection. HLCs derived from hepatocytes had the highest transcriptional resemblance to PHHs regardless of the protocol, whereas the quality of fibroblasts and PSC derived HLCs varied depending on the protocol used. Finally, we developed and validated a web application (HLCompR) enabling comparison for specific pathways and addition of new HLCs. In conclusion, our comprehensive transcriptomic comparison of HLCs allows selection of HLCs for specific research questions and can guide improvements in culturing conditions.

The interaction with HMG20a/b proteins suggests a potential role for beta-dystrobrevin in neuronal differentiation.

alpha and beta dystrobrevins are cytoplasmic components of the dystrophin-associated protein complex that are thought to play a role as scaffold proteins in signal transduction and intracellular transport. In the search of new insights into the functions of beta-dystrobrevin, the isoform restricted to non-muscle tissues, we performed a two-hybrid screen of a mouse cDNA library to look for interacting proteins. Among the positive clones, one encodes iBRAF/HMG20a, a high mobility group (HMG)-domain protein that activates REST (RE-1 silencing transcription factor)-responsive genes, playing a key role in the initiation of neuronal differentiation. We characterized the beta-dystrobrevin-iBRAF interaction by in vitro and in vivo association assays, localized the binding region of one protein to the other, and assessed the kinetics of the interaction as one of high affinity. We also found that beta-dystrobrevin directly binds to BRAF35/HMG20b, a close homologue of iBRAF and a member of a co-repressor complex required for the repression of neural specific genes in neuronal progenitors. In vitro assays indicated that beta-dystrobrevin binds to RE-1 and represses the promoter activity of synapsin I, a REST-responsive gene that is a marker for neuronal differentiation. Altogether, our data demonstrate a direct interaction of beta-dystrobrevin with the HMG20 proteins iBRAF and BRAF35 and suggest that beta-dystrobrevin may be involved in regulating chromatin dynamics, possibly playing a role in neuronal differentiation.

Establishment of human fetal hepatocyte organoids and CRISPR-Cas9-based gene knockin and knockout in organoid cultures from human liver.

The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR-Cas9 in both human fetal hepatocyte and adult liver ductal organoid systems. Using a CRISPR-Cas9 and homology-independent organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte organoid cultures takes ~1-2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2-3 months.

CRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids.

CRISPR-Cas technology has revolutionized biological research and holds great therapeutic potential. Here, we review CRISPR-Cas systems and their latest developments with an emphasis on application to human cells. We also discuss how different CRISPR-based strategies can be used to accomplish a particular genome engineering goal. We then review how different CRISPR tools have been used in genome engineering of human stem cells in vitro, covering both the pluripotent (iPSC/ESC) and somatic adult stem cell fields and, in particular, 3D organoid cultures. Finally, we discuss the progress and challenges associated with CRISPR-based genome editing of human stem cells for therapeutic use.

Author Correction: Increasing neurogenesis refines hippocampal activity rejuvenating navigational learning strategies and contextual memory throughout life.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing.

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.