Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools.

Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.1,2.

Differential transcriptional invasion signatures from patient derived organoid models define a functional prognostic tool for head and neck cancer.

Clinical outcome for patients suffering from HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. This is mostly due to highly invasive tumors that cause loco-regional relapses after initial therapeutic intervention and metastatic outgrowth. The molecular pathways governing the detrimental invasive growth modes in HNSCC remain however understudied. Here, we have established HNSCC patient derived organoid (PDO) models that recapitulate 3-dimensional invasion in vitro. Single cell mRNA sequencing was applied to study the differences between non-invasive and invasive conditions, and in a collective versus single cell invading PDO model. Differential expression analysis under invasive conditions in Collagen gels reveals an overall upregulation of a YAP-centered transcriptional program, irrespective of the invasion mode. However, we find that collectively invading HNSCC PDO cells show elevated levels of YAP transcription targets when compared to single cell invasion. Also, collectively invading cells are characterized by increased nuclear translocation of YAP within the invasive strands, which coincides with Collagen-I matrix alignment at the invasive front. Using gene set enrichment analysis, we identify immune cell-like migratory pathways in the single cell invading HNSCC PDO, while collective invasion is characterized by overt upregulation of adhesion and migratory pathways. Lastly, based on clinical head and neck cancer cohorts, we demonstrate that the identified collective invasion signature provides a candidate prognostic platform for survival in HNSCC. By uncoupling collective and single cell invasive programs, we have established invasion signatures that may guide new therapeutic options.

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.

Binucleated human hepatocytes arise through late cytokinetic regression during endomitosis M phase.

Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.

Cell division-dependent dissemination following E-cadherin loss underlies initiation of diffuse-type gastric cancer.

Loss of the cell-cell adhesion protein E-cadherin underlies the development of diffuse-type gastric cancer (DGC), which is characterized by the gradual accumulation of tumor cells originating from the gastric epithelium in the surrounding stroma. How E-cadherin deficiency drives DGC formation remains elusive. Therefore, we investigated the consequences of E-cadherin loss on gastric epithelial organization utilizing a human gastric organoid model and histological analyses of early-stage DGC lesions. E-cadherin depletion from gastric organoids recapitulates DGC initiation, with progressive loss of a single-layered architecture and detachment of individual cells. We found that E-cadherin deficiency in gastric epithelia does not lead to a general loss of epithelial cohesion but disrupts the spindle orientation machinery. This leads to a loss of planar cell division orientation and, consequently, daughter cells are positioned outside of the gastric epithelial layer. Although basally delaminated cells fail to detach and instead reintegrate into the epithelium, apically mispositioned daughter cells can trigger the gradual loss of the single-layered epithelial architecture. This impaired architecture hampers reintegration of mispositioned daughter cells and enables basally delaminated cells to disseminate into the surrounding matrix. Taken together, our findings describe how E-cadherin deficiency disrupts gastric epithelial architecture through displacement of dividing cells and provide new insights in the onset of DGC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7.

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Improved detection of colibactin-induced mutations by genotoxic E. coli in organoids and colorectal cancer.

Co-culture of intestinal organoids with a colibactin-producing pks+E. coli strain (EcC) revealed mutational signatures also found in colorectal cancer (CRC). E. coli Nissle 1917 (EcN) remains a commonly used probiotic, despite harboring the pks operon and inducing double strand DNA breaks. We determine the mutagenicity of EcN and three CRC-derived pks+E. coli strains with an analytical framework based on sequence characteristic of colibactin-induced mutations. All strains, including EcN, display varying levels of mutagenic activity. Furthermore, a machine learning approach attributing individual mutations to colibactin reveals that patients with colibactin-induced mutations are diagnosed at a younger age and that colibactin can induce a specific APC mutation. These approaches allow the sensitive detection of colibactin-induced mutations in ∼12% of CRC genomes and even in whole exome sequencing data, representing a crucial step toward pinpointing the mutagenic activity of distinct pks+E. coli strains.

Derivation of functional thymic epithelial organoid lines from adult murine thymus.

Thymic epithelial cells (TECs) orchestrate T cell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term ex vivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single and double FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire-expressing medullary-like TECs upon RANK ligand + retinoic acid treatment. TEC organoids support T cell development from immature thymocytes in vitro as well as in vivo upon transplantation into athymic nude mice. This organoid-based platform allows in vitro study of TEC biology and offers a potential strategy for ex vivo T cell development.

BEHAV3D: a 3D live imaging platform for comprehensive analysis of engineered T cell behavior and tumor response.

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond.

Polyketide synthase positive Escherichia coli one-time measurement in stool is not informative of colorectal cancer risk in a screening setting.

Environmental factors like the pathogenicity island polyketide synthase positive (pks+) Escherichia coli (E. coli) could have potential for risk stratification in colorectal cancer (CRC) screening. The association between pks+ E. coli measured in fecal immunochemical test (FIT) samples and the detection of advanced neoplasia (AN) at colonoscopy was investigated. Biobanked FIT samples were analyzed for both presence of E. coli and pks+ E. coli and correlated with colonoscopy findings; 5020 CRC screening participants were included. Controls were participants in which no relevant lesion was detected because of FIT-negative results (cut-off ≥15 µg Hb/g feces), a negative colonoscopy, or a colonoscopy during which only a nonadvanced polyp was detected. Cases were participants with AN [CRC, advanced adenoma (AA), or advanced serrated polyp (ASP)]. Existing DNA isolation and quantitative polymerase chain reaction (qPCR) procedures were used for the detection of E. coli and pks+ E. coli in stool. A total of 4542 (90.2%) individuals were E. coli positive, and 1322 (26.2%) were pks+ E. coli positive. The prevalence of E. coli in FIT samples from individuals with AN was 92.9% compared to 89.7% in FIT samples of controls (p = 0.010). The prevalence of pks+ E. coli in FIT samples from individuals with AN (28.6%) and controls (25.9%) was not significantly different (p = 0.13). The prevalences of pks+ E. coli in FIT samples from individuals with CRC, AA, or ASP were 29.6%, 28.3%, and 32.1%, respectively. In conclusion, the prevalence of pks+ E. coli in a screening population was 26.2% and did not differ significantly between individuals with AN and controls. These findings disqualify the straightforward option of using a snapshot measurement of pks+ E. coli in FIT samples as a stratification biomarker for CRC risk. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Multi-omic dataset of patient-derived tumor organoids of neuroendocrine neoplasms.

BACKGROUND: Organoids are 3-dimensional experimental models that summarize the anatomical and functional structure of an organ. Although a promising experimental model for precision medicine, patient-derived tumor organoids (PDTOs) have currently been developed only for a fraction of tumor types. RESULTS: We have generated the first multi-omic dataset (whole-genome sequencing [WGS] and RNA-sequencing [RNA-seq]) of PDTOs from the rare and understudied pulmonary neuroendocrine tumors (n = 12; 6 grade 1, 6 grade 2) and provide data from other rare neuroendocrine neoplasms: small intestine (ileal) neuroendocrine tumors (n = 6; 2 grade 1 and 4 grade 2) and large-cell neuroendocrine carcinoma (n = 5; 1 pancreatic and 4 pulmonary). This dataset includes a matched sample from the parental sample (primary tumor or metastasis) for a majority of samples (21/23) and longitudinal sampling of the PDTOs (1 to 2 time points), for a total of n = 47 RNA-seq and n = 33 WGS. We here provide quality control for each technique and the raw and processed data as well as all scripts for genomic analyses to ensure an optimal reuse of the data. In addition, we report gene expression data and somatic small variant calls and describe how they were generated, in particular how we used WGS somatic calls to train a random forest classifier to detect variants in tumor-only RNA-seq. We also report all histopathological images used for medical diagnosis: hematoxylin and eosin-stained slides, brightfield images, and immunohistochemistry images of protein markers of clinical relevance. CONCLUSIONS: This dataset will be critical to future studies relying on this PDTO biobank, such as drug screens for novel therapies and experiments investigating the mechanisms of carcinogenesis in these understudied diseases.

Chromatin Remodeling in Patient-Derived Colorectal Cancer Models.

Patient-Derived Organoids (PDO) and Xenografts (PDX) are the current gold standards for patient-derived models of cancer (PDMC). Nevertheless, how patient tumor cells evolve in these models and the impact on drug response remains unclear. Herein, the transcriptomic and chromatin accessibility landscapes of matched colorectal cancer (CRC) PDO, PDX, PDO-derived PDX (PDOX), and original patient tumors (PT) are compared. Two major remodeling axes are discovered. The first axis delineates PDMC from PT, and the second axis distinguishes PDX and PDO. PDOX are more similar to PDX than PDO, indicating the growth environment is a driving force for chromatin adaptation. Transcription factors (TF) that differentially bind to open chromatins between matched PDO and PDOX are identified. Among them, KLF14 and EGR2 footprints are enriched in PDOX relative to matched PDO, and silencing of KLF14 or EGR2 promoted tumor growth. Furthermore, EPHA4, a shared downstream target gene of KLF14 and EGR2, altered tumor sensitivity to MEK inhibitor treatment. Altogether, patient-derived CRC cells undergo both common and distinct chromatin remodeling in PDO and PDX/PDOX, driven largely by their respective microenvironments, which results in differences in growth and drug sensitivity and needs to be taken into consideration when interpreting their ability to predict clinical outcome.

Hallmarks of stemness in mammalian tissues.

All adult tissues experience wear and tear. Most tissues can compensate for cell loss through the activity of resident stem cells. Although the cellular maintenance strategies vary greatly between different adult (read: postnatal) tissues, the function of stem cells is best defined by their capacity to replace lost tissue through division. We discuss a set of six complementary hallmarks that are key enabling features of this basic function. These include longevity and self-renewal, multipotency, transplantability, plasticity, dependence on niche signals, and maintenance of genome integrity. We discuss these hallmarks in the context of some of the best-understood adult stem cell niches.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

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.

Tubuloid differentiation to model the human distal nephron and collecting duct in health and disease.

Organoid technology is rapidly gaining ground for studies on organ (patho)physiology. Tubuloids are long-term expanding organoids grown from adult kidney tissue or urine. The progenitor state of expanding tubuloids comes at the expense of differentiation. Here, we differentiate tubuloids to model the distal nephron and collecting ducts, essential functional parts of the kidney. Differentiation suppresses progenitor traits and upregulates genes required for function. A single-cell atlas reveals that differentiation predominantly generates thick ascending limb and principal cells. Differentiated human tubuloids express luminal NKCC2 and ENaC capable of diuretic-inhibitable electrolyte uptake and enable disease modeling as demonstrated by a lithium-induced tubulopathy model. Lithium causes hallmark AQP2 loss, induces proliferation, and upregulates inflammatory mediators, as seen in vivo. Lithium also suppresses electrolyte transport in multiple segments. In conclusion, this tubuloid model enables modeling of the human distal nephron and collecting duct in health and disease and provides opportunities to develop improved therapies.