Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

Drug Metabolism of Hepatocyte-like Organoids and Their Applicability in In Vitro Toxicity Testing.

Emerging advances in the field of in vitro toxicity testing attempt to meet the need for reliable human-based safety assessment in drug development. Intrahepatic cholangiocyte organoids (ICOs) are described as a donor-derived in vitro model for disease modelling and regenerative medicine. Here, we explored the potential of hepatocyte-like ICOs (HL-ICOs) in in vitro toxicity testing by exploring the expression and activity of genes involved in drug metabolism, a key determinant in drug-induced toxicity, and the exposure of HL-ICOs to well-known hepatotoxicants. The current state of drug metabolism in HL-ICOs showed levels comparable to those of PHHs and HepaRGs for CYP3A4; however, other enzymes, such as CYP2B6 and CYP2D6, were expressed at lower levels. Additionally, EC50 values were determined in HL-ICOs for acetaminophen (24.0−26.8 mM), diclofenac (475.5−>500 µM), perhexiline (9.7−>31.5 µM), troglitazone (23.1−90.8 µM), and valproic acid (>10 mM). Exposure to the hepatotoxicants showed EC50s in HL-ICOs comparable to those in PHHs and HepaRGs; however, for acetaminophen exposure, HL-ICOs were less sensitive. Further elucidation of enzyme and transporter activity in drug metabolism in HL-ICOs and exposure to a more extensive compound set are needed to accurately define the potential of HL-ICOs in in vitro toxicity testing.

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

BACKGROUND AND AIMS: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. APPROACH AND RESULTS: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSIONS: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

Snf2 controls pulcherriminic acid biosynthesis and antifungal activity of the biocontrol yeast Metschnikowia pulcherrima.

Metschnikowia pulcherrima synthesises the pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wild-type strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wild-type and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator gene SNF2 in the mutant. Complementation of the mutant strain with the wild-type SNF2 gene restored pigmentation and recovered the strong antifungal activity. Mass spectrometry (UPLC HR HESI-MS) proved the presence of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and identified new precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wild-type and complemented strain, but were undetectable in the pigmentless snf2 mutant strain. These results thus identify Snf2 as a regulator of antifungal activity and pulcherriminic acid biosynthesis in M. pulcherrima and provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast.

A Chemically Defined Hydrogel for Human Liver Organoid Culture.

End-stage liver diseases are an increasing health burden, and liver transplantations are currently the only curative treatment option. Due to a lack of donor livers, alternative treatments are urgently needed. Human liver organoids are very promising for regenerative medicine; however, organoids are currently cultured in Matrigel, which is extracted from the extracellular matrix of the Engelbreth-Holm-Swarm mouse sarcoma. Matrigel is poorly defined, suffers from high batch-to-batch variability and is of xenogeneic origin, which limits the clinical application of organoids. Here, a novel hydrogel based on polyisocyanopeptides (PIC) and laminin-111 is described for human liver organoid cultures. PIC is a synthetic polymer that can form a hydrogel with thermosensitive properties, making it easy to handle and very attractive for clinical applications. Organoids in an optimized PIC hydrogel proliferate at rates comparable to those observed with Matrigel; proliferation rates are stiffness-dependent, with lower stiffnesses being optimal for organoid proliferation. Moreover, organoids can be efficiently differentiated toward a hepatocyte-like phenotype with key liver functions. This proliferation and differentiation potential maintain over at least 14 passages. The results indicate that PIC is very promising for human liver organoid culture and has the potential to be used in a variety of clinical applications including cell therapy and tissue engineering.

An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking.

Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear. To address the specific role of MYO5B in the intestine, we generated an intestine-specific conditional Myo5b-deficient (Myo5bfl/fl;Vil-CreERT2) mouse model. We analyzed intestinal tissues and cultured organoids of Myo5bfl/fl;Vil-CreERT2 mice by electron microscopy, immunofluorescence, and immunohistochemistry. Our data showed that Myo5bfl/fl;Vil-CreERT2 mice developed severe diarrhea within 4 d after tamoxifen induction. Periodic Acid Schiff and alkaline phosphatase staining revealed subapical accumulation of intracellular vesicles in villus enterocytes. Analysis by electron microscopy confirmed an almost complete absence of apical microvilli, the appearance of microvillus inclusions, and enlarged intercellular spaces in induced Myo5bfl/fl;Vil-CreERT2 intestines. In addition, we determined that MYO5B is involved not only in apical but also basolateral trafficking of proteins. The analysis of the intestine during the early onset of the disease revealed that subapical accumulation of secretory granules precedes occurrence of microvillus inclusions, indicating involvement of MYO5B in early differentiation of epithelial cells. By comparing our data with a novel MVID patient, we conclude that our mouse model completely recapitulates the intestinal phenotype of human MVID. This includes severe diarrhea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules. Thus, loss of MYO5B disturbs both apical and basolateral trafficking of proteins and causes MVID in mice.

Biofilm Formation Potential of Heat-Resistant Escherichia coli Dairy Isolates and the Complete Genome of Multidrug-Resistant, Heat-Resistant Strain FAM21845.

We tested the biofilm formation potential of 30 heat-resistant and 6 heat-sensitive Escherichia coli dairy isolates. Production of curli and cellulose, static biofilm formation on polystyrene (PS) and stainless steel surfaces, biofilm formation under dynamic conditions (Bioflux), and initial adhesion rates (IAR) were evaluated. Biofilm formation varied greatly between strains, media, and assays. Our results highlight the importance of the experimental setup in determining biofilm formation under conditions of interest, as correlation between different assays was often not a given. The heat-resistant, multidrug-resistant (MDR) strain FAM21845 showed the strongest biofilm formation on PS and the highest IAR and was the only strain that formed significant biofilms on stainless steel under conditions relevant to the dairy industry, and it was therefore fully sequenced. Its chromosome is 4.9 Mb long, and it harbors a total of five plasmids (147.2, 54.2, 5.8, 2.5, and 1.9 kb). The strain carries a broad range of genes relevant to antimicrobial resistance and biofilm formation, including some on its two large conjugative plasmids, as demonstrated in plate mating assays.IMPORTANCE In biofilms, cells are embedded in an extracellular matrix that protects them from stresses, such as UV radiation, osmotic shock, desiccation, antibiotics, and predation. Biofilm formation is a major bacterial persistence factor of great concern in the clinic and the food industry. Many tested strains formed strong biofilms, and especially strains such as the heat-resistant, MDR strain FAM21845 may pose a serious issue for food production. Strong biofilm formation combined with diverse resistances (some encoded on conjugative plasmids) may allow for increased persistence, coselection, and possible transfer of these resistance factors. Horizontal gene transfer may conceivably occur in the food production setting or the gastrointestinal tract after consumption.

Loss of syntaxin 3 causes variant microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.

Adult stem cells in the small intestine are intrinsically programmed with their location-specific function.

Differentiation and specialization of epithelial cells in the small intestine are regulated in two ways. First, there is differentiation along the crypt-villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine, or M cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum, and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location-specific functional properties are intrinsically programmed within stem cells or if continuous signaling from mesenchymal cells is necessary to maintain the location-specific identity of the small intestine. Using the pure epithelial organoid technique, we show that region-specific gene expression profiles are conserved throughout long-term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4-regulated gene expression is only allowed in absence of WNT signaling. These data show that location-specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location-specific extracellular signals. In light of the potential future clinical application of small intestine-derived organoids, our data imply that it is important to generate GATA4-positive and GATA4-negative cultures to regenerate all essential functions of the small intestine.

Toward Transplantation of Liver Organoids: From Biology and Ethics to Cost-effective Therapy.

Liver disease is a common cause of morbidity and mortality, and many patients would benefit from liver transplantation. However, because of a shortage of suitable donor livers, even of those patients who are placed on the donor liver waiting list, many do not survive the waiting time for transplantation. Therefore, alternative treatments for end-stage liver disease need to be explored. Recent advances in organoid technology might serve as a solution to overcome the donor liver shortage in the future. In this overview, we highlight the potential of organoid technology for cell therapy and tissue engineering approaches. Both organoid-based approaches could be used as treatment for end-stage liver disease patients. Additionally, organoid-based cell therapy can also be used to repair liver grafts ex vivo to increase the supply of transplantable liver tissue. The potential of both approaches to become clinically available is carefully assessed, including their clinical, ethical, and economic implications. We provide insight into what aspects should be considered further to allow alternatives to donor liver transplantation to be successfully clinically implemented.

Hydrogels derived from decellularized liver tissue support the growth and differentiation of cholangiocyte organoids.

Human cholangiocyte organoids are promising for regenerative medicine applications, such as repair of damaged bile ducts. However, organoids are typically cultured in mouse tumor-derived basement membrane extracts (BME), which is poorly defined, highly variable and limits the direct clinical applications of organoids in patients. Extracellular matrix (ECM)-derived hydrogels prepared from decellularized human or porcine livers are attractive alternative culture substrates. Here, the culture and expansion of human cholangiocyte organoids in liver ECM(LECM)-derived hydrogels is described. These hydrogels support proliferation of cholangiocyte organoids and maintain the cholangiocyte-like phenotype. The use of LECM hydrogels does not significantly alter the expression of selected genes or proteins, such as the cholangiocyte marker cytokeratin-7, and no species-specific effect is found between human or porcine LECM hydrogels. Proliferation rates of organoids cultured in LECM hydrogels are lower, but the differentiation capacity of the cholangiocyte organoids towards hepatocyte-like cells is not altered by the presence of tissue-specific ECM components. Moreover, human LECM extracts support the expansion of ICO in a dynamic culture set up without the need for laborious static culture of organoids in hydrogel domes. Liver ECM hydrogels can successfully replace tumor-derived BME and can potentially unlock the full clinical potential of human cholangiocyte organoids.

Bioprinting of Human Liver-Derived Epithelial Organoids for Toxicity Studies.

There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte-like cells derived from organoids are generated using extrusion-based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88-107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non-printed controls. Exposure to acetaminophen, a well-known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21-51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR-122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver-derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology- and disease-models.

Linking Comparative Genomics of Nine Potato-Associated Pseudomonas Isolates With Their Differing Biocontrol Potential Against Late Blight.

For plants, the advantages of associating with beneficial bacteria include plant growth promotion, reduction of abiotic and biotic stresses and enhanced protection against various pests and diseases. Beneficial bacteria rightly equipped for successful plant colonization and showing antagonistic activity toward plant pathogens seem to be actively recruited by plants. To gain more insights into the genetic determinants responsible for plant colonization and antagonistic activities, we first sequenced and de novo assembled the complete genomes of nine Pseudomonas strains that had exhibited varying antagonistic potential against the notorious oomycete Phytophthora infestans, placed them into the phylogenomic context of known Pseudomonas biocontrol strains and carried out a comparative genomic analysis to define core, accessory (i.e., genes found in two or more, but not all strains) and unique genes. Next, we assessed the colonizing abilities of these strains and used bioassays to characterize their inhibitory effects against different stages of P. infestans' lifecycle. The phenotype data were then correlated with genotype information, assessing over three hundred genes encoding known factors for plant colonization and antimicrobial activity as well as secondary metabolite biosynthesis clusters predicted by antiSMASH. All strains harbored genes required for successful plant colonization but also distinct arsenals of antimicrobial compounds. We identified genes coding for phenazine, hydrogen cyanide, 2-hexyl, 5-propyl resorcinol and pyrrolnitrin synthesis, as well as various siderophores, pyocins and type VI secretion systems. Additionally, the comparative genomic analysis revealed about a hundred accessory genes putatively involved in anti-Phytophthora activity, including a type II secretion system (T2SS), several peptidases and a toxin. Transcriptomic studies and mutagenesis are needed to further investigate the putative involvement of the novel candidate genes and to identify the various mechanisms involved in the inhibition of P. infestans by different Pseudomonas strains.

Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine.

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.

Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson's disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Hydrogels for Liver Tissue Engineering.

Bioengineered livers are promising in vitro models for drug testing, toxicological studies, and as disease models, and might in the future be an alternative for donor organs to treat end-stage liver diseases. Liver tissue engineering (LTE) aims to construct liver models that are physiologically relevant. To make bioengineered livers, the two most important ingredients are hepatic cells and supportive materials such as hydrogels. In the past decades, dozens of hydrogels have been developed to act as supportive materials, and some have been used for in vitro models and formed functional liver constructs. However, currently none of the used hydrogels are suitable for in vivo transplantation. Here, the histology of the human liver and its relationship with LTE is introduced. After that, significant characteristics of hydrogels are described focusing on LTE. Then, both natural and synthetic materials utilized in hydrogels for LTE are reviewed individually. Finally, a conclusion is drawn on a comparison of the different hydrogels and their characteristics and ideal hydrogels are proposed to promote LTE.

Intestinal epithelial cell polarity defects in disease: lessons from microvillus inclusion disease.

The intestinal epithelium is a highly organized tissue. The establishment of epithelial cell polarity, with distinct apical and basolateral plasma membrane domains, is pivotal for both barrier formation and for the uptake and vectorial transport of nutrients. The establishment of cell polarity requires a specialized subcellular machinery to transport and recycle proteins to their appropriate location. In order to understand and treat polarity-associated diseases, it is necessary to understand epithelial cell-specific trafficking mechanisms. In this Review, we focus on cell polarity in the adult mammalian intestine. We discuss how intestinal epithelial polarity is established and maintained, and how disturbances in the trafficking machinery can lead to a polarity-associated disorder, microvillus inclusion disease (MVID). Furthermore, we discuss the recent developments in studying MVID, including the creation of genetically manipulated cell lines, mouse models and intestinal organoids, and their uses in basic and applied research.

Bioengineered bile ducts recapitulate key cholangiocyte functions.

Investigation of diseases of the bile duct system and identification of potential therapeutic targets are hampered by the lack of tractable in vitro systems to model cholangiocyte biology. Here, we show a step-wise method for the differentiation of murine Lgr5+ liver stem cells (organoids) into cholangiocyte-like cells (CLCs) using a combination of growth factors and extracellular matrix components. Organoid-derived CLCs display key properties of primary cholangiocytes, such as expressing cholangiocyte markers, forming primary cilia, transporting small molecules and responding to farnesoid X receptor agonist. Integration of organoid-derived cholangiocytes with collagen-coated polyethersulfone hollow fiber membranes yielded bioengineered bile ducts that morphologically resembled native bile ducts and possessed polarized bile acid transport activity. As such, we present a novel in vitro model for studying and therapeutically modulating cholangiocyte function.

Converging biofabrication and organoid technologies: the next frontier in hepatic and intestinal tissue engineering?

Adult tissue stem cells can form self-organizing 3D organoids in vitro. Organoids resemble small units of their organ of origin and have great potential for tissue engineering, as well as models of disease. However, current culture technology limits the size, architecture and complexity of organoids. Here, we review the establishment of intestinal and hepatic organoids and discuss how the convergence of organoids and biofabrication technologies can help overcome current limitations, and thereby further advance the translational application of organoids in tissue engineering and regenerative medicine.

Novel approaches: Tissue engineering and stem cells--In vitro modelling of the gut.

In many intestinal diseases, the function of the epithelial lining is impaired. In this review, we describe the recent developments of in vitro intestinal stem cell cultures. When these stem cells are grown in 3D structures (organoids), they provide a model of the intestinal epithelium, which is closely similar to the growth and development of the in vivo gut. This model provides a new tool to study various diseases of malabsorption in functional detail and therapeutic applications, which could not be achieved with traditional cell lines. First, we describe the organization and function of the healthy small intestinal epithelium. Then, we discuss the establishment of organoid cultures and how these structures represent the healthy epithelium. Finally, we discuss organoid cultures as a tool for studying intrinsic properties of the epithelium, as a model for intestinal disease, and as a possible source for stem cell transplantations.