Patient Derived Colonoids as Drug Testing Platforms–Critical Importance of Oxygen Concentration

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology.

Download Full-text

A Micro-engineered Human Colon Intestine-Chip Platform to Study Leaky Barrier

10.1101/2020.08.28.271759 ◽

2020 ◽

Cited By ~ 1

Author(s):

Athanasia Apostolou ◽

Rohit A. Panchakshari ◽

Antara Banerjee ◽

Dimitris V. Manatakis ◽

Maria D. Paraskevopoulou ◽

...

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelium ◽

Unmet Need ◽

Intestinal Barrier ◽

Human Colon ◽

Epithelial Barrier ◽

Host Immune System ◽

Intestinal Epithelial ◽

Barrier Disruption ◽

Interleukin 22

ABSTRACTThe intestinal epithelial barrier supports the symbiotic relationship between the microbiota colonizing the intestinal epithelium and the host immune system to maintain homeostasis. Leaky barrier is increasingly recognized as part of the pathogenesis of a number of chronic conditions in addition to inflammatory and infectious diseases. As our understanding on the regulation of the barrier remains limited, effective therapeutic targeting for the compromised barrier is still an unmet need. Here we combined advancements on the organoids and Organ-on-Chip technologies to establish a micro-engineered Colon Intestine-Chip for studying development and regulation of the human intestinal barrier. Our data demonstrate the significance of the endothelium in co-culture with the epithelial cells within a tissue-relevant microenvironment for the establishment of a tight epithelial barrier of polarized cells. Pathway analysis of the RNA sequencing (RNA-Seq), revealed significant upregulation of mechanisms relevant to the maturation of the intestinal epithelium in organoid-derived epithelial cells in co-culture with endothelium as compared to organoids maintained in suspension. We provide evidence that the Colon Intestine-Chip platform responds to interferon gamma (IFNγ), a prototype cytokine utilized to model inflammation-induced barrier disruption, by induction of apoptosis and reorganization of the apical junctional complexes as shown with other systems. We also describe the mechanism of action of interleukin 22 (IL-22) on mature, organoid-derived intestinal epithelial cells that is consistent with barrier disruption. Overall we propose the Colon Intestine-Chip as a promising human organoid-derived platform to decipher mechanisms driving the development of leaky gut in patients and enable their translation for this unmet medical need.

Download Full-text

Development of fetal rat intestine in organ and monolayer culture.

The Journal of Cell Biology ◽

10.1083/jcb.100.5.1611 ◽

1985 ◽

Vol 100 (5) ◽

pp. 1611-1622 ◽

Cited By ~ 51

Author(s):

A Quaroni

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelium ◽

Monolayer Culture ◽

Primary Cultures ◽

Organ Cultures ◽

Functional Changes ◽

Fetal Rat ◽

Extracellular Matrix Components ◽

Fetal Intestine

Maturation and differentiation of intestinal epithelial cells was demonstrated in segments of fetal rat small intestine, maintained for more than a month in suspension organ culture, by ultrastructural, biochemical, and immunological criteria. Over a 5-7 d period, fragments of fetal intestine evolved into globular structures covered with a single columnar epithelium ultrastructurally similar to suckling villus cells. Loose mesenchymal cells, cellular debris, and collagen were present inside the structures. After 6 d in culture, goblet cells, not present in the fetal intestine at day 18, were numerous and well developed. Intestinal endocrine cells were also observed. Immunofluorescence studies employing monoclonal antibodies specific for villus and crypt cells in vivo, and various enzyme assays, have demonstrated a level of differentiation and maturation of the cultured epithelial cells similar but not identical to that of suckling intestinal mucosa in vivo. Crypts and crypt cell markers were not observed in the the cultures. Addition of glucocorticoids to the culture medium resulted in the induction of sucrase-isomaltase but failed to promote most of the functional changes characteristic of the intestinal epithelium at weaning in vivo. Epithelial cells were identified in explants derived from the organ cultures by their specific expression of intestinal cytokeratin. Differentiation-specific markers, present in the epithelial cells in primary cultures, were lost upon selection and subculturing of pure epithelial cell populations. These results suggest a requirement for mesenchymal and/or extracellular matrix components in the maintenance of the differentiated state of the epithelial cells. The fetal intestinal organ cultures described here present significant advantages over traditional organ and monolayer culture techniques for the study of the cellular and molecular interactions involved in the development and differentiation of the intestinal epithelium.

Download Full-text

Epidermal growth factor reduces autophagy in intestinal epithelium and in the rat model of necrotizing enterocolitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00076.2010 ◽

2010 ◽

Vol 299 (3) ◽

pp. G614-G622 ◽

Cited By ~ 42

Author(s):

Andrew A. Maynard ◽

Katerina Dvorak ◽

Ludmila Khailova ◽

Holly Dobrenen ◽

Kelly M. Arganbright ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Epithelial Cells ◽

Necrotizing Enterocolitis ◽

Intestinal Epithelium ◽

Beclin 1 ◽

Ultrastructural Changes ◽

Epidermal Growth

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Epidermal growth factor (EGF) is one of the most promising candidates in NEC prophylaxis. Autophagy regulates cell homeostasis, but uncontrolled activation of autophagy may lead to cellular injury. The aim was to evaluate the effects of EGF on intestinal autophagy in epithelial cells and in the rat NEC model and measure autophagy in NEC patients. Intestinal epithelial cells (IEC-6) and the rat NEC model were used to study the effect of EGF on intestinal autophagy. Protein levels of Beclin 1 and LC3II were measured in the intestinal epithelium in both in vivo and in vitro models. Ultrastructural changes in intestinal epithelium were studied by electron microscopy. Expression of Beclin 1, LC3II, and p62 protein was evaluated in biopsies from NEC patients. Autophagy was induced in IEC-6 cells and inhibited by adding EGF into the culture. In the rat NEC model, EGF treatment of NEC reduced expression of Beclin 1 and LC3II in ileal epithelium. Morphologically, typical signs of autophagy were observed in the epithelium of the NEC group, but not in the EGF group. A strong signal for Beclin 1 and LC3II was detected in the intestine from patients with NEC. Autophagy is activated in the intestinal epithelium of NEC patients and in the ileum of NEC rats. Supplementation of EGF blocks intestinal autophagy in both in vivo and in vitro conditions. Results from this study indicate that EGF-mediated protection against NEC injury is associated with regulation of intestinal autophagy.

Download Full-text

A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

mSphere ◽

10.1128/msphere.00030-15 ◽

2015 ◽

Vol 1 (1) ◽

Cited By ~ 25

Author(s):

Coyne G. Drummond ◽

Cheryl A. Nickerson ◽

Carolyn B. Coyne

Keyword(s):

Cell Culture ◽

Epithelial Cells ◽

Intestinal Epithelium ◽

Intestinal Epithelial Cells ◽

Three Dimensional ◽

Intestinal Epithelial ◽

Cell Culture Model ◽

Culture Model ◽

Gastrointestinal Epithelium

ABSTRACT Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the epithelium lining the gastrointestinal tract early in infection. The lack of suitable in vivo and in vitro models to study CVB infection of the gastrointestinal epithelium has limited our understanding of the events that surround infection of these specialized cells. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of human intestinal epithelial cells that better models the gastrointestinal epithelium in vivo. By applying this 3-D model, which recapitulates many aspects of the gastrointestinal epithelium in vivo, to the study of CVB infection, our work provides a new cell system to model the mechanisms by which CVB infects the intestinal epithelium, which may have a profound impact on CVB pathogenesis. Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the epithelium lining the gastrointestinal tract early in infection. The lack of suitable in vivo and in vitro models to study CVB infection of the gastrointestinal epithelium has limited our understanding of the events that surround infection of these specialized cells. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of human intestinal epithelial cells that better models the gastrointestinal epithelium in vivo. By applying this 3-D model, which recapitulates many aspects of the gastrointestinal epithelium in vivo, to the study of CVB infection, our work provides a new cell system to model the mechanisms by which CVB infects the intestinal epithelium, which may have a profound impact on CVB pathogenesis. Podcast: A podcast concerning this article is available.

Download Full-text

Epithelial cells of the intestine acquire cell-intrinsic inflammation signatures during ageing

10.1101/2021.12.19.473357 ◽

2021 ◽

Author(s):

Maja C Funk ◽

Jan G Gleixner ◽

Florian Heigwer ◽

Erica Valentini ◽

Zeynep Aydin ◽

...

Keyword(s):

Epithelial Cells ◽

Mhc Class Ii ◽

Ex Vivo ◽

Cell Types ◽

Chromatin Accessibility ◽

Extrinsic Factors ◽

Intestinal Organoids ◽

Persistent Inflammation ◽

A Cell

During ageing, cell-intrinsic and extrinsic factors lead to the decline of tissue function and organismal health. Disentangling these factors is important for developing effective strategies to prolong organismal healthspan. Here, we addressed this question in the mouse intestinal epithelium, which forms a dynamic interface with its microenvironment and receives extrinsic signals affecting its homeostasis and tissue ageing. We systematically compared transcriptional profiles of young and aged epithelial cells in vivo and ex vivo in cultured intestinal organoids. We found that all cell types of the aged epithelium exhibit an inflammation phenotype, which is marked by MHC class II upregulation and most pronounced in enterocytes. This was accompanied by elevated levels of the immune tolerance markers PD-1 and PD-L1 in the aged tissue microenvironment, indicating dysregulation of immunological homeostasis. Intestinal organoids from aged mice still showed an inflammation signature after weeks in culture, which was concurrent with increased chromatin accessibility of inflammation-associated loci. Our results reveal a cell-intrinsic, persistent inflammation phenotype in aged epithelial cells, which might contribute to systemic inflammation observed during ageing.

Download Full-text

Use of transgenic mice to study the routing of secretory proteins in intestinal epithelial cells: analysis of human growth hormone compartmentalization as a function of cell type and differentiation.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.3231 ◽

1989 ◽

Vol 109 (6) ◽

pp. 3231-3242 ◽

Cited By ~ 31

Author(s):

J F Trahair ◽

M R Neutra ◽

J I Gordon

Keyword(s):

Growth Hormone ◽

Epithelial Cells ◽

Transgenic Mice ◽

Intestinal Epithelium ◽

Secretory Granules ◽

Human Growth ◽

Goblet Cells ◽

Secretory Proteins ◽

Intermediate Cells

The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.

Download Full-text

Patient-Derived Acute Myeloid Leukemia (AML) Ex Vivo platform for Evaluation of Novel Molecular Targeted Therapeutic Agents

Blood ◽

10.1182/blood-2019-126127 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5146-5146

Author(s):

Bhavna Verma ◽

Nicolett Biel ◽

Bruce Ruggeri ◽

Amy Wesa

Keyword(s):

Flow Cytometry ◽

Drug Discovery ◽

Drug Testing ◽

Ex Vivo ◽

Unmet Need ◽

Therapeutic Agents ◽

Myelogenous Leukemia ◽

Culture Period ◽

Term Survival

Acute myelogenous leukemia (AML) is the most common acute leukemia in adults. A hematologic cancer, the disease is highly heterogeneous, with multiple subtypes. Despite advances in treatment, the long-term survival for AML remains poor and the development of novel treatments is an unmet need. Due to the highly divergent subtypes and mutation profiles in AML, the use of patient-derived models may improve drug discovery and development. To address this, we have established a short-term culture system that supports the growth of primary AML cells ex vivo to permit the evaluation and/or screening of candidate therapeutic agents. Our AML bank is comprised of patient-derived specimens across a range of subtypes (which includes M1, M2, M4, M5, NOS and others), and includes models with common mutations in FLT3 (ITD), IDH1/IDH2 and NPM. Primary AML specimens were characterized for common mutations by TruSight sequencing and for surface marker expression by flow cytometry. The ex vivo assay system was evaluated for the ability to support the survival and expansion of primary AML specimens. Among these, the majority of the models showed evidence of proliferation, a few models had no net expansion, and some failed to survive. Extension of the culture period for up to 14 days was feasible, with most models having equal or increased cell numbers by the end of the culture period (8 of 10 models evaluated). All models stably expressed CD33 throughout the assay. To verify the applicability of this system for drug testing, a standard of care agent cytarabine (Ara-C) was assessed for each of the AML models. A small cohort of models with distinct mutations were tested for sensitivity towards small molecule inhibitors venetoclax, gilteritnib, glasdegib, panobinostat and Ibrutinib. Cell growth/viability was assessed using Cell titer-Glo assay. Concentration-dependent responses to Ara-C were observed across multiple models (IC50 10 nM to 150 nM), indicating a range of relatively sensitive to resistant AML models. Heterogeneous concentration-dependent responses were observed across multiple patient-derived ex vivo models when treated with venetoclax, glasdegib, gilteritnib, ibrutinib and panobinostat, with both sensitive and resistant models identified. A cohort of models were evaluated in vivo for sensitivity to Ara-C. Systemic engraftment of the patient-derived xenograft AML models (into NOG or NOG-EXL mice) was evaluated by flow cytometry. When engrafted was confirmed, AML-bearing mice were randomized into Ara-C or vehicle control groups. Two weeks later, mice were evaluated for the presence of human CD45+CD33+AML cells. Models that were relatively sensitive to Ara C ex vivo (IC50 < 30 nM) generally showed a greater in vivo response as evidenced by a significant reduction in the mean circulating AML cells versus models with IC50 values >100 nM that had no response to Ara C in vivo. Over thirty distinct patient-derived primary, unpassaged models of AML have been studied both in ex vivo culture and as in vivo systemic models. The diversity in these AML models is reflective of patient diversity, enhancing their utility in the evaluation of novel therapeutic candidates. These data indicate the feasibility of utilization of these primary models, ex vivo as well as in vivo, for drug discovery for AML, from screening to preclinical efficacy modeling. Disclosures Verma: Champions Oncology: Employment. Biel:Champions Oncology: Employment. Ruggeri:Champions Oncology: Consultancy. Wesa:Champions Oncology: Employment, Other: stock options.

Download Full-text

In vivo feasibility study of the use of porous polyhedral oligomeric silsesquioxane implants in partial laryngeal reconstruction

10.1101/587691 ◽

2019 ◽

Author(s):

Martin A. Birchall ◽

Peggy Herrmann ◽

Paul Sibbons

Keyword(s):

Epithelial Cells ◽

Thyroid Cartilage ◽

Unmet Need ◽

Polyhedral Oligomeric Silsesquioxane ◽

Present Formulation ◽

Post Surgery ◽

Oligomeric Silsesquioxane ◽

Post Implantation

AbstractBackgroundLoss of substantial volumes of laryngeal tissue after trauma or cancer significantly impairs quality of life. We hypothesised that repair of laryngeal defects with a candidate biomaterial, seeded with mesenchymal stem cells (MSC) and epithelial cells, may offer a therapeutic approach to this unmet need.MethodMoulded porous polyhedral oligomeric silsesquioxane polycarbonate-urea (POSS-PCU) scaffolds were seeded with human-derived MSC and epithelial cells, were implanted orthotopically into a defect created in the thyroid cartilage in eight pigs and monitoredin vivofor 2 months.In vivoassessments were performed at 1, 2, 4 and 8 weeks post implantation. Histology was performed following termination.ResultsImplant operations were uncomplicated. One pig was terminated early (2 weeks post-implantation) following expectoration of its implant. No other mortality or morbidity was observed. Endoscopy showed partial extrusion of implants at two weeks and complete extrusion of all implants by termination.ConclusionsPOSS-PCU moulded laryngeal implants, in the present formulation, are extruded from the site of implantation between two- and eight-weeks post-surgery in pigs. In its present formulation and with the present, one-stage, protocol, this material does not appear to provide a suitable scaffold and vehicle for cells intended for partial laryngeal replacement in pigs.

Download Full-text

Separable mechanisms drive local and global polarity establishment in the C. elegans intestinal epithelium

10.1101/2021.11.01.466827 ◽

2021 ◽

Author(s):

Melissa A Pickett ◽

Maria D. Sallee ◽

Victor F. Naturale ◽

Deniz Akpinaroglu ◽

Joo Lee ◽

...

Keyword(s):

Epithelial Cells ◽

Small Groups ◽

Intestinal Epithelium ◽

Protein Complex ◽

Specific Protein ◽

Dependent Manner ◽

Epithelial Polarity ◽

C Elegans ◽

Polarity Establishment

Apico-basolateral polarization is essential for epithelial cells to function as selective barriers and transporters, and to provide mechanical resiliency to organs. Epithelial polarity is established locally, within individual cells to establish distinct apical, junctional, and basolateral domains, and globally, within a tissue where cells coordinately orient their apico-basolateral axes. Using live imaging of endogenously tagged proteins and tissue specific protein depletion in the C. elegans embryonic intestine, we found that local and global polarity establishment are temporally and genetically separable. Local polarity is initiated prior to global polarity and is robust to perturbation. PAR-3 is required for global polarization across the intestine but is not required for local polarity establishment as small groups of cells are able to correctly establish polarized domains in PAR-3 depleted intestines in an HMR-1/E-cadherin dependent manner. Despite belonging to the same apical protein complex, we additionally find that PAR-3 and PKC-3/aPKC have distinct roles in the establishment and maintenance of local and global polarity. Together, our results indicate that different mechanisms are required for local and global polarity establishment in vivo.

Download Full-text