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.

One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids.

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.

Rapid tissue prototyping with micro-organospheres.

In vitro tissue models hold great promise for modeling diseases and drug responses. Here, we used emulsion microfluidics to form micro-organospheres (MOSs), which are droplet-encapsulated miniature three-dimensional (3D) tissue models that can be established rapidly from patient tissues or cells. MOSs retain key biological features and responses to chemo-, targeted, and radiation therapies compared with organoids. The small size and large surface-to-volume ratio of MOSs enable various applications including quantitative assessment of nutrient dependence, pathogen-host interaction for anti-viral drug screening, and a rapid potency assay for chimeric antigen receptor (CAR)-T therapy. An automated MOS imaging pipeline combined with machine learning overcomes plating variation, distinguishes tumorspheres from stroma, differentiates cytostatic versus cytotoxic drug effects, and captures resistant clones and heterogeneity in drug response. This pipeline is capable of robust assessments of drug response at individual-tumorsphere resolution and provides a rapid and high-throughput therapeutic profiling platform for precision medicine.

Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids.

Prime editing is a recently reported genome editing tool using a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. Here, we explore the use of prime editing in human organoids. Common TP53 mutations can be correctly modeled in human adult stem cell-derived colonic organoids with efficiencies up to 25% and up to 97% in hepatocyte organoids. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9-mediated homology-directed repair and adenine base editing on the CFTR-R785* mutation. Whole-genome sequencing of prime editing-repaired organoids revealed no detectable off-target effects. Despite encountering varying editing efficiencies and undesired mutations at the target site, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.

Murine Endometrial Organoids to Model Chlamydia Infection.

The obligate intracellular bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. Once internalized in host cells, C. trachomatis undergoes a biphasic developmental cycle within a membrane-bound compartment, known as the inclusion. Successful establishment of the intracellular niche relies on bacterial Type III effector proteins, such as Inc proteins. In vitro and in vivo systems have contributed to elucidating the intracellular lifestyle of C. trachomatis, but additional models combining the archetypal environment of infection with the advantages of in vitro systems are needed. Organoids are three-dimensional structures that recapitulate the microanatomy of an organ's epithelial layer, bridging the gap between in vitro and in vivo systems. Organoids are emerging as relevant model systems to study interactions between bacterial pathogens and their hosts. Here, we took advantage of recently developed murine endometrial organoids (EMOs) and present a C. trachomatis-murine EMO infection model system. Confocal microscopy of EMOs infected with fluorescent protein-expressing bacteria revealed that inclusions are formed within the cytosol of epithelial cells. Moreover, infection with a C. trachomatis strain that allows for the tracking of RB to EB transition indicated that the bacteria undergo a full developmental cycle, which was confirmed by harvesting infectious bacteria from infected EMOs. Finally, the inducible gene expression and cellular localization of a Chlamydia Inc protein within infected EMOs further demonstrated that this model is compatible with the study of Type III secreted effectors. Altogether, we describe a novel and relevant system for the study of Chlamydia-host interactions.

Human-Derived Model Systems in Gynecological Cancer Research.

The human female reproductive tract (FRT) is a complex system that combines series of organs, including ovaries, fallopian tubes, uterus, cervix, vagina, and vulva; each of which possesses unique cellular characteristics and functions. This versatility, in turn, allows for the development of a wide range of epithelial gynecological cancers with distinct features. Thus, reliable model systems are required to better understand the diverse mechanisms involved in the regional pathogenesis of the reproductive tract and improve treatment strategies. Here, we review the current human-derived model systems available to study the multitude of gynecological cancers, including ovarian, endometrial, cervical, vaginal, and vulvar cancer, and the recent advances in the push towards personalized therapy.

Organoids can be established reliably from cryopreserved biopsy catheter-derived endometrial tissue of infertile women.

RESEARCH QUESTION: Can organoids be established from endometrial tissue of infertile women and does tissue cryopreservation allow for establishment of organoids comparable to organoids derived from freshly biopsied endometrial tissue? DESIGN: Endometrial tissue was obtained from six infertile women through minimally invasive biopsy using a Pipelle catheter and subjected to organoid development, immediately after biopsy as well as after tissue cryopreservation. Organoid formation efficiency, morphology, expandability potential, endometrial marker expression (immunostaining and reverse transcription quantitative real-time polymerase chain reaction) and hormonal responsiveness (after oestradiol and progesterone treatment) were assessed. RESULTS: Organoids established from both fresh and frozen tissue at comparable efficiency could be passaged long-term and showed similar morphology, i.e. cystic with a central lumen lined by a single epithelial cell layer. They also exhibited comparable expression of endometrial markers and proliferative activity (Ki67 expression). Finally, organoids from freshly biopsied and cryopreserved endometrial tissue showed similar responses to oestradiol and progesterone treatment. CONCLUSIONS: Organoids can be established from cryopreserved endometrial tissue of infertile women and cryopreservation of the biopsy does not affect organoid formation and overall organoid characteristics. Cryopreservation of biopsies for later organoid development facilitates sample collection from any fertility clinic, not just the ones near an organoid laboratory.

Developing Organoids from Ovarian Cancer as Experimental and Preclinical Models.

Ovarian cancer (OC) represents the most dismal gynecological cancer. Pathobiology is poorly understood, mainly due to lack of appropriate study models. Organoids, defined as self-developing three-dimensional in vitro reconstructions of tissues, provide powerful tools to model human diseases. Here, we established organoid cultures from patient-derived OC, in particular from the most prevalent high-grade serous OC (HGSOC). Testing multiple culture medium components identified neuregulin-1 (NRG1) as key factor in maximizing OC organoid development and growth, although overall derivation efficiency remained moderate (36% for HGSOC patients, 44% for all patients together). Established organoid lines showed patient tumor-dependent morphology and disease characteristics, and recapitulated the parent tumor's marker expression and mutational landscape. Moreover, the organoids displayed tumor-specific sensitivity to clinical HGSOC chemotherapeutic drugs. Patient-derived OC organoids provide powerful tools for the study of the cancer's pathobiology (such as importance of the NRG1/ERBB pathway) as well as advanced preclinical tools for (personalized) drug screening and discovery.

Coordination of germ layer lineage choice by TET1 during primed pluripotency.

Gastrulation in the early postimplantation stage mammalian embryo begins when epiblast cells ingress to form the primitive streak or develop as the embryonic ectoderm. The DNA dioxygenase Tet1 is highly expressed in the epiblast and yet continues to regulate lineage specification during gastrulation when its expression is diminished. Here, we show how Tet1 plays a pivotal role upstream of germ layer lineage bifurcation. During the transition from naive pluripotency to lineage priming, a global reconfiguration redistributes Tet1 from Oct4-cobound promoters to distal regulatory elements at lineage differentiation genes, which are distinct from high-affinity sites engaged by Oct4. An altered chromatin landscape in Tet1-deficient primed epiblast-like cells is associated with enhanced Oct4 expression and binding to Nodal and Wnt target genes, resulting in collaborative signals that enhance mesendodermal and inhibit neuroectodermal gene expression during lineage segregation. A permissive role for Tet1 in neural fate induction involves Zic2-dependent engagement at neural target genes at lineage priming, is dependent on the signaling environment during gastrulation, and impacts neural tube closure after gastrulation. Our findings provide mechanistic information for epigenetic integration of pluripotency and signal-induced differentiation cues.

Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening.

Endometrial disorders represent a major gynaecological burden. Current research models fail to recapitulate the nature and heterogeneity of these diseases, thereby hampering scientific and clinical progress. Here we developed long-term expandable organoids from a broad spectrum of endometrial pathologies. Organoids from endometriosis show disease-associated traits and cancer-linked mutations. Endometrial cancer-derived organoids accurately capture cancer subtypes, replicate the mutational landscape of the tumours and display patient-specific drug responses. Organoids were also established from precancerous pathologies encompassing endometrial hyperplasia and Lynch syndrome, and inherited gene mutations were maintained. Endometrial disease organoids reproduced the original lesion when transplanted in vivo. In summary, we developed multiple organoid models that capture endometrial disease diversity and will provide powerful research models and drug screening and discovery tools.

Functional expression of the mechanosensitive PIEZO1 channel in primary endometrial epithelial cells and endometrial organoids.

Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. Knowledge regarding molecular candidates involved in this early communication process is inadequate due to limited access to primary human endometrial epithelial cells (EEC). Since pseudo-pregnancy in rodents can be induced by mechanical scratching of an appropriately primed uterus, this study aimed to investigate the expression of mechanosensitive ion channels in EEC. Poking of EEC provoked a robust calcium influx and induced an increase in current densities, which could be blocked by an inhibitor of mechanosensitive ion channels. Interestingly, RNA expression studies showed high expression of PIEZO1 in EEC of mouse and human. Additional analysis provided further evidence for the functional expression of PIEZO1 since stimulation with Yoda1, a chemical agonist of PIEZO1, induced increases in intracellular calcium concentrations and current densities in EEC. Moreover, the ion channel profile of human endometrial organoids (EMO) was validated as a representative model for endometrial epithelial cells. Mechanical and chemical stimulation of EMO induced strong calcium responses supporting the hypothesis of mechanosensitive ion channel expression in endometrial epithelial cells. In conclusion, EEC and EMO functionally express the mechanosensitive PIEZO1 channel that could act as a potential target for the development of novel treatments to further improve successful implantation processes.

Organoids from pituitary as a novel research model toward pituitary stem cell exploration.

The pituitary is the master endocrine gland, harboring stem cells of which the phenotype and role remain poorly characterized. Here, we established organoids from mouse pituitary with the aim to generate a novel research model to study pituitary stem cell biology. The organoids originated from the pituitary cells expressing the stem cell marker SOX2 were long-term expandable, displayed a stemness phenotype during expansive culture and showed specific hormonal differentiation ability, although limited, after subrenal transplantation. Application of the protocol to transgenically injured pituitary harboring an activated stem cell population, resulted in more numerous organoids. Intriguingly, these organoids presented with a cystic morphology, whereas the organoids from undamaged gland were predominantly dense and appeared more limited in expandability. Transcriptomic analysis revealed distinct epithelial phenotypes and showed that cystic organoids more resembled the pituitary phenotype, at least to an immature state, and displayed in vitro differentiation, although yet moderate. Organoid characterization further exposed facets of regulatory pathways of the putative stem cells of the pituitary and advanced new injury-activated markers. Taken together, we established a novel organoid research model revealing new insights into the identity and regulation of the putative pituitary stem cells. This organoid model may eventually lead to an interesting tool to decipher pituitary stem cell biology in both healthy and diseased gland.

Major depletion of SOX2+ stem cells in the adult pituitary is not restored which does not affect hormonal cell homeostasis and remodelling.

The pituitary gland contains SOX2-expressing stem cells. However, their functional significance remains largely unmapped. We investigated their importance by depleting SOX2+ cells through diphtheria toxin (DT)-mediated ablation. DT treatment of adult Sox2CreERT2/+;R26iDTR/+ mice (after tamoxifen-induced expression of DT receptor in SOX2+ cells) resulted in 80% obliteration of SOX2+ cells in the endocrine pituitary, coinciding with reduced pituisphere-forming activity. Counterintuitively for a stem cell population, the SOX2+ cell compartment did not repopulate. Considering the more active phenotype of the stem cells during early-postnatal pituitary maturation, SOX2+ cell ablation was also performed in 4- and 1-week-old animals. Ablation grade diminished with decreasing age and was accompanied by a proliferative reaction of the SOX2+ cells, suggesting a rescue attempt. Despite this activation, SOX2+ cells did also not recover. Finally, the major SOX2+ cell depletion in adult mice did not affect the homeostatic maintenance of pituitary hormonal cell populations, nor the corticotrope remodelling response to adrenalectomy challenge. Taken together, our study shows that pituitary SOX2+ fail to regenerate after major depletion which does not affect adult endocrine cell homeostasis and remodelling. Thus, pituitary SOX2+ cells may constitute a copious stem cell reserve or may have other critical role(s) still to be clearly defined.

Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability.

The endometrium, which is of crucial importance for reproduction, undergoes dynamic cyclic tissue remodeling. Knowledge of its molecular and cellular regulation is poor, primarily owing to a lack of study models. Here, we have established a novel and promising organoid model from both mouse and human endometrium. Dissociated endometrial tissue, embedded in Matrigel under WNT-activating conditions, swiftly formed organoid structures that showed long-term expansion capacity, and reproduced the molecular and histological phenotype of the tissue's epithelium. The supplemented WNT level determined the type of mouse endometrial organoids obtained: high WNT yielded cystic organoids displaying a more differentiated phenotype than the dense organoids obtained in low WNT. The organoids phenocopied physiological responses of endometrial epithelium to hormones, including increased cell proliferation under estrogen and maturation upon progesterone. Moreover, the human endometrial organoids replicated the menstrual cycle under hormonal treatment at both the morpho-histological and molecular levels. Together, we established an organoid culture system for endometrium, reproducing tissue epithelium physiology and allowing long-term expansion. This novel model provides a powerful tool for studying mechanisms underlying the biology as well as the pathology of this key reproductive organ.