RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

Distinct metabolic programs established in the thymus control effector functions of γδ T cell subsets in tumor microenvironments.

Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy.

An Intestinal Organ Culture System Uncovers a Role for the Nervous System in Microbe-Immune Crosstalk.

Investigation of host-environment interactions in the gut would benefit from a culture system that maintained tissue architecture yet allowed tight experimental control. We devised a microfabricated organ culture system that viably preserves the normal multicellular composition of the mouse intestine, with luminal flow to control perturbations (e.g., microbes, drugs). It enables studying short-term responses of diverse gut components (immune, neuronal, etc.). We focused on the early response to bacteria that induce either Th17 or RORg+ T-regulatory (Treg) cells in vivo. Transcriptional responses partially reproduced in vivo signatures, but these microbes elicited diametrically opposite changes in expression of a neuronal-specific gene set, notably nociceptive neuropeptides. We demonstrated activation of sensory neurons by microbes, correlating with RORg+ Treg induction. Colonic RORg+ Treg frequencies increased in mice lacking TAC1 neuropeptide precursor and decreased in capsaicin-diet fed mice. Thus, differential engagement of the enteric nervous system may partake in bifurcating pro- or anti-inflammatory responses to microbes.

Reverse Engineering Human Pathophysiology with Organs-on-Chips.

While studies of cultured cells have led to new insights into biological control, greater understanding of human pathophysiology requires the development of experimental systems that permit analysis of intercellular communications and tissue-tissue interactions in a more relevant organ context. Human organs-on-chips offer a potentially powerful new approach to confront this long-standing problem.

Long-term culture of genome-stable bipotent stem cells from adult human liver.

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

Organoid models of human and mouse ductal pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Prospective derivation of a living organoid biobank of colorectal cancer patients.

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.

Identification of multipotent luminal progenitor cells in human prostate organoid cultures.

The prostate gland consists of basal and luminal cells arranged as pseudostratified epithelium. In tissue recombination models, only basal cells reconstitute a complete prostate gland, yet murine lineage-tracing experiments show that luminal cells generate basal cells. It has remained challenging to address the molecular details of these transitions and whether they apply to humans, due to the lack of culture conditions that recapitulate prostate gland architecture. Here, we describe a 3D culture system that supports long-term expansion of primary mouse and human prostate organoids, composed of fully differentiated CK5+ basal and CK8+ luminal cells. Organoids are genetically stable, reconstitute prostate glands in recombination assays, and can be experimentally manipulated. Single human luminal and basal cells give rise to organoids, yet luminal-cell-derived organoids more closely resemble prostate glands. These data support a luminal multilineage progenitor cell model for prostate tissue and establish a robust, scalable system for mechanistic studies.

Human neural tube morphogenesis in vitro by geometric constraints.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

An organoid-based organ-repurposing approach to treat short bowel syndrome.

The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.

Three-dimensional organotypic culture: experimental models of mammalian biology and disease.

Mammalian organs are challenging to study as they are fairly inaccessible to experimental manipulation and optical observation. Recent advances in three-dimensional (3D) culture techniques, coupled with the ability to independently manipulate genetic and microenvironmental factors, have enabled the real-time study of mammalian tissues. These systems have been used to visualize the cellular basis of epithelial morphogenesis, to test the roles of specific genes in regulating cell behaviours within epithelial tissues and to elucidate the contribution of microenvironmental factors to normal and disease processes. Collectively, these novel models can be used to answer fundamental biological questions and generate replacement human tissues, and they enable testing of novel therapeutic approaches, often using patient-derived cells.

DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12-CXCR4 signaling axis.

Sufficient blood flow to tissues relies on arterial blood vessels, but the mechanisms regulating their development are poorly understood. Many arteries, including coronary arteries of the heart, form through remodeling of an immature vascular plexus in a process triggered and shaped by blood flow. However, little is known about how cues from fluid shear stress are translated into responses that pattern artery development. Here, we show that mice lacking endothelial Dach1 had small coronary arteries, decreased endothelial cell polarization, and reduced expression of the chemokine Cxcl12 Under shear stress in culture, Dach1 overexpression stimulated endothelial cell polarization and migration against flow, which was reversed upon CXCL12/CXCR4 inhibition. In vivo, DACH1 was expressed during early arteriogenesis but was down in mature arteries. Mature artery-type shear stress (high, uniform laminar) specifically down-regulated DACH1, while the remodeling artery-type flow (low, variable) maintained DACH1 expression. Together, our data support a model in which DACH1 stimulates coronary artery growth by activating Cxcl12 expression and endothelial cell migration against blood flow into developing arteries. This activity is suppressed once arteries reach a mature morphology and acquire high, laminar flow that down-regulates DACH1. Thus, we identified a mechanism by which blood flow quality balances artery growth and maturation.

Direct diffusion of anti-Müllerian hormone from both the cranial and caudal regions of the testis during early gonadal development in mice.

BACKGROUND: The Müllerian duct (MD), the primordium of the female reproductive tract, is also formed in males during the early stage of development, then regresses due to the anti-Müllerian hormone (AMH) secreted from the testes. However, the detailed diffusion pathway of AMH remains unclear. We herein investigated the mechanism by which AMH reaches the middle region of the MD using an organ culture system. RESULTS: Injection of recombinant human AMH into the testis around the start of MD regression induced diffuse immunoreactivity in the mesonephros near the injection site. When the testis and mesonephros were cultured separately, the diameters of both cranial and middle MDs were significantly increased compared to the control. In the testis-mesonephros complex cultured by inhibiting the diffusion of AMH through the cranial region, the cranial MD diameter was significantly increased compared to the control, and there was no difference in middle MD diameter. CONCLUSIONS: These results indicate that AMH, which infiltrates from the testis through the cranial region at physiological concentrations, induces regression of the cranial MD at the start of MD regression. They also indicate that AMH infiltrating through the caudal regions induces regression of the middle MD.

Krüppel-like factor 12 regulates aging ovarian granulosa cell apoptosis by repressing SPHK1 transcription and sphingosine-1-phosphate (S1P) production.

Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.

New uses for an old technique: live imaging on the slice organ culture to study reproductive processes†.

Reproductive processes are dynamic and involve extensive morphological remodeling and cell-cell interactions. Live imaging of organs enhances our understanding of how biological processes occur in real time. Slice culture is a type of organ culture where thick slices are collected from an organ and cultured for several days. Slice culture is a useful and easy-to-implement technique for live imaging of reproductive events at cellular resolution. Here we describe a pipeline of live imaging on slice culture to visualize the process of urethra closure in mouse embryonic penis as a proof of principle. In combination with genetic reporter mice, nuclear stains, and exposure experiments, we demonstrate the feasibility of slice culture on a reproductive organ. We also provide a step-by-step protocol and troubleshooting guide to facilitate the adoption of slice culture with live imaging in other reproductive organs. Lastly, we discuss potential utilities and experiments that could be implemented with slice culture in reproductive sciences.

Deep Anterior Lamellar Keratoplasty Using Dehydrated versus Standard Organ Culture-Stored Donor Corneas: Prospective Randomized Trial.

PURPOSE: To compare the outcomes of deep anterior lamellar keratoplasty (DALK) using dehydrated versus standard organ culture-stored donor corneas for eyes with keratoconus. DESIGN: Prospective, randomized, single-center trial conducted in Italy. PARTICIPANTS: Adult patients (age ≥ 18 years) with keratoconus scheduled for elective DALK. METHODS: Patients undergoing successful type 1 bubble pneumatic dissection using a standard DALK technique were randomized during surgery to receive either dehydrated (n = 30) or standard organ culture-stored (n = 30) donor corneas. MAIN OUTCOME MEASURES: The primary study outcome was best spectacle-corrected visual acuity (BSCVA) 12 months after surgery. Secondary outcomes were refractive astigmatism (RA), endothelial cell density (ECD), and complication rates. RESULTS: Postoperative BSCVA did not significantly differ between groups at both time points: mean difference at 6 months was 0.030 logarithm of the minimum angle of resolution (logMAR; 95% confidence interval [CI], -0.53 to 0.10 logMAR; P = 0.471) and at 12 months was -0.013 logMAR (95% CI, -0.10 to 0.08 logMAR; P = 0.764). No significant differences between groups were observed in terms of postoperative RA and ECD at all time points. In the first 3 days after DALK, an epithelial defect was present in 10 patients (33%) in the organ culture cornea group and in 29 patients (97%) in the dehydrated cornea group. Complete re-epithelialization was achieved by day 7 in all patients (100%) in both groups. CONCLUSIONS: The study provides evidence that the use of dehydrated corneas is noninferior to the use of standard organ culture donor corneas for DALK. Corneal tissue dehydration represents a viable solution that can allow long-term cornea preservation and avoid wastage of unused corneas. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

An untargeted metabolomics approach to study changes of the medium during human cornea culture.

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.

Editorial for special issue-human precision cut lung slices: an Ex vivo platform for therapeutic target discovery and drug testing in lung disease.

Publications utilizing precision cut lung slices (PCLS) steadily increased from the 1970's, with a significant increase in 2010, to tripling by 2023. PCLS have been used to study a vast array of pulmonary diseases and exposures to pathogens and toxicants to understand pathogenesis of disease but also to examine basic cellular mechanisms that underly lung biology. This Special Issue will highlight new, exciting, and novel research using PCLS, while acknowledging the substantial fund of knowledge that has been gained using this platform.

Precision cut lung slices: an integrated ex vivo model for studying lung physiology, pharmacology, disease pathogenesis and drug discovery.

Precision Cut Lung Slices (PCLS) have emerged as a sophisticated and physiologically relevant ex vivo model for studying the intricacies of lung diseases, including fibrosis, injury, repair, and host defense mechanisms. This innovative methodology presents a unique opportunity to bridge the gap between traditional in vitro cell cultures and in vivo animal models, offering researchers a more accurate representation of the intricate microenvironment of the lung. PCLS require the precise sectioning of lung tissue to maintain its structural and functional integrity. These thin slices serve as invaluable tools for various research endeavors, particularly in the realm of airway diseases. By providing a controlled microenvironment, precision-cut lung slices empower researchers to dissect and comprehend the multifaceted interactions and responses within lung tissue, thereby advancing our understanding of pulmonary pathophysiology.

'Speed-ageing' of human skin in serum-free organ culture ex vivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5-dimethylpyrazine.

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.