Publications by authors named "Kim B Jensen"

53 Publications

A Semi-automated Organoid Screening Method Demonstrates Epigenetic Control of Intestinal Epithelial Differentiation.

Front Cell Dev Biol 2020 21;8:618552. Epub 2021 Jan 21.

Centre of Molecular Inflammation Research (CEMIR), Department of Clinical and Molecular Medicine (IKOM), NTNU - Norwegian University of Science and Technology, Trondheim, Norway.

Intestinal organoids are an excellent model to study epithelial biology. Yet, the selection of analytical tools to accurately quantify heterogeneous organoid cultures remains limited. Here, we developed a semi-automated organoid screening method, which we applied to a library of highly specific chemical probes to identify epigenetic regulators of intestinal epithelial biology. The role of epigenetic modifiers in adult stem cell systems, such as the intestinal epithelium, is still undefined. Based on this resource dataset, we identified several targets that affected epithelial cell differentiation, including HDACs, EP300/CREBBP, LSD1, and type I PRMTs, which were verified by complementary methods. For example, we show that inhibiting type I PRMTs, which leads enhanced epithelial differentiation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are powerful tools to study intestinal epithelial biology and may have therapeutic potential.
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http://dx.doi.org/10.3389/fcell.2020.618552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872100PMC
January 2021

A Nickel(II)-Mediated Thiocarbonylation Strategy for Carbon Isotope Labeling of Aliphatic Carboxamides.

Chemistry 2021 Jan 16. Epub 2021 Jan 16.

Carbon Dioxide Activation Center (CADIAC), Department of, Chemistry and the Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark.

A series of pharmaceutically relevant small molecules and biopharmaceuticals bearing aliphatic carboxamides have been successfully labeled with carbon-13. Key to the success of this novel carbon isotope labeling technique is the observation that C-labeled Ni -acyl complexes, formed from a CO insertion step with Ni -alkyl intermediates, rapidly react in less than one minute with 2,2'-dipyridyl disulfide to quantitatively form the corresponding 2-pyridyl thioesters. Either the use of C-SilaCOgen or C-COgen allows for the stoichiometric addition of isotopically labeled carbon monoxide. Subsequent one-pot acylation of a series of structurally diverse amines provides the desired C-labeled carboxamides in good yields. A single electron transfer pathway is proposed between the Ni -acyl complexes and the disulfide providing a reactive Ni -acyl sulfide intermediate, which rapidly undergoes reductive elimination to the desired thioester. By further optimization of the reaction parameters, reaction times down to only 11 min were identified, opening up the possibility of exploring this chemistry for carbon-11 isotope labeling. Finally, this isotope labeling strategy could be adapted to the synthesis of C-labeled liraglutide and insulin degludec, representing two antidiabetic drugs.
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http://dx.doi.org/10.1002/chem.202005261DOI Listing
January 2021

A bioengineering perspective on modelling the intestinal epithelial physiology in vitro.

Nat Commun 2020 12 7;11(1):6244. Epub 2020 Dec 7.

BRIC - Biotech Research and Innovation Centre, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The small intestine is a specialised organ, essential for nutrient digestion and absorption. It is lined with a complex epithelial cell layer. Intestinal epithelial cells can be cultured in three-dimensional (3D) scaffolds as self-organising entities with distinct domains containing stem cells and differentiated cells. Recent developments in bioengineering provide new possibilities for directing the organisation of cells in vitro. In this Perspective, focusing on the small intestine, we discuss how studies at the interface between bioengineering and intestinal biology provide new insights into organ function. Specifically, we focus on engineered biomaterials, complex 3D structures resembling the intestinal architecture, and micro-physiological systems.
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http://dx.doi.org/10.1038/s41467-020-20052-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721730PMC
December 2020

Personalized B cell response to the probiotic in healthy human subjects: a randomized trial.

Gut Microbes 2020 Nov;12(1):1-14

Biotech Research and Innovation Centre, University of Copenhagen , Copenhagen N, Denmark.

The specific effects of administering live probiotics in the human gut are not well characterized. To this end, we investigated the immediate effect of GG (LGG) in the jejunum of 27 healthy volunteers 2 h after ingestion using a combination of global RNA sequencing of human biopsies and bacterial DNA sequencing in a multi-visit, randomized, cross-over design (ClinicalTrials.gov number NCT03140878). While LGG was detectable in jejunum after 2 h in treated subjects, the gene expression response vs. placebo was subtle if assessed across all subjects. However, clustering analysis revealed that one-third of subjects exhibited a strong and consistent LGG response involving hundreds of genes, where genes related to B cell activation were upregulated, consistent with prior results in mice. Immunohistochemistry and single cell-based deconvolution analyses showed that this B cell signature likely is due to activation and proliferation of existing B cells rather than B cell immigration to the tissue. Our results indicate that the LGG strain has an immediate effect in the human gut in a subpopulation of individuals. In extension, our data strongly suggest that studies on probiotic effects in humans require large cohorts and must take individual variation into account.
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http://dx.doi.org/10.1080/19490976.2020.1854639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722709PMC
November 2020

Intestinal Organoids: A Tool for Modelling Diet-Microbiome-Host Interactions.

Trends Endocrinol Metab 2020 11 9;31(11):848-858. Epub 2020 Mar 9.

CIBIO - Department of Cellular, Computational, and Integrative Biology, University of Trento, Via Sommarive 9, Trento, Italy.

Dietary patterns, microbiome dysbiosis, and gut microbial metabolites (GMMs) have a pivotal role in the homeostasis of intestinal epithelial cells and in disease progression, such as that of colorectal cancer (CRC). Although GMMs and microorganisms have crucial roles in many biological activities, models for deciphering diet-microbiome-host relationships are largely limited to animal models. Thus, intestinal organoids (IOs) have provided unprecedented opportunities for the generation of in vitro platforms with the sufficient level of complexity to model physiological and pathological diet-microbiome-host conditions. Overall, IO responses to GMM metabolites and microorganisms can provide new insights into the mechanisms by which those agents may prevent or trigger diseases, significantly extending our knowledge of diet-microbiome-host interactions.
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http://dx.doi.org/10.1016/j.tem.2020.02.004DOI Listing
November 2020

LSD1 represses a neonatal/reparative gene program in adult intestinal epithelium.

Sci Adv 2020 Sep 11;6(37). Epub 2020 Sep 11.

CEMIR-Centre of Molecular Inflammation Research, Department of Clinical and Molecular Medicine, NTNU-Norwegian University of Science and Technology, 7491 Trondheim, Norway.

Intestinal epithelial homeostasis is maintained by adult intestinal stem cells, which, alongside Paneth cells, appear after birth in the neonatal period. We aimed to identify regulators of neonatal intestinal epithelial development by testing a small library of epigenetic modifier inhibitors in Paneth cell-skewed organoid cultures. We found that lysine-specific demethylase 1A () is absolutely required for Paneth cell differentiation. -deficient crypts, devoid of Paneth cells, are still able to form organoids without a requirement of exogenous or endogenous Wnt. Mechanistically, we find that LSD1 enzymatically represses genes that are normally expressed only in fetal and neonatal epithelium. This gene profile is similar to what is seen in repairing epithelium, and we find that -deficient epithelium has superior regenerative capacities after irradiation injury. In summary, we found an important regulator of neonatal intestinal development and identified a druggable target to reprogram intestinal epithelium toward a reparative state.
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http://dx.doi.org/10.1126/sciadv.abc0367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486101PMC
September 2020

Tracing the cellular dynamics of sebaceous gland development in normal and perturbed states.

Nat Cell Biol 2019 08 29;21(8):924-932. Epub 2019 Jul 29.

BRIC-Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark.

The sebaceous gland (SG) is an essential component of the skin, and SG dysfunction is debilitating. Yet, the cellular bases for its origin, development and subsequent maintenance remain poorly understood. Here, we apply large-scale quantitative fate mapping to define the patterns of cell fate behaviour during SG development and maintenance. We show that the SG develops from a defined number of lineage-restricted progenitors that undergo a programme of independent and stochastic cell fate decisions. Following an expansion phase, equipotent progenitors transition into a phase of homeostatic turnover, which is correlated with changes in the mechanical properties of the stroma and spatial restrictions on gland size. Expression of the oncogene KrasG12D results in a release from these constraints and unbridled gland expansion. Quantitative clonal fate analysis reveals that, during this phase, the primary effect of the Kras oncogene is to drive a constant fate bias with little effect on cell division rates. These findings provide insight into the developmental programme of the SG, as well as the mechanisms that drive tumour progression and gland dysfunction.
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http://dx.doi.org/10.1038/s41556-019-0362-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978139PMC
August 2019

Tissue-Engineering the Intestine: The Trials before the Trials.

Cell Stem Cell 2019 06;24(6):855-859

Specialist Neonatal and Paediatric Surgery Unit, Great Ormond Street Hospital, London, UK; Stem Cells & Regenerative Medicine Section, NIHR Biomedical Research Center, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. Electronic address:

Building complex tissues requires the development of innovative interdisciplinary engineering solutions. In this Forum, the INTENS Consortium discuss experimental considerations and challenges for generating a tissue-engineered intestine for the treatment of short bowel syndrome, taking into account cell source, scaffold choice, and design strategy for achieving proper assembly and function.
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http://dx.doi.org/10.1016/j.stem.2019.04.018DOI Listing
June 2019

Fluorescence-based tracing of transplanted intestinal epithelial cells using confocal laser endomicroscopy.

Stem Cell Res Ther 2019 05 27;10(1):148. Epub 2019 May 27.

Department of Gastroenterology, Herlev Hospital, University of Copenhagen, 2730, Herlev, Denmark.

Background: Intestinal stem cell transplantation has been shown to promote mucosal healing and to engender fully functional epithelium in experimental colitis. Hence, stem cell therapies may provide an innovative approach to accomplish mucosal healing in patients with debilitating conditions such as inflammatory bowel disease. However, an approach to label and trace transplanted cells, in order to assess engraftment efficiency and to monitor wound healing, is a key hurdle to overcome prior to initiating human studies. Genetic engineering is commonly employed in animal studies, but may be problematic in humans due to potential off-target and long-term adverse effects.

Methods: We investigated the applicability of a panel of fluorescent dyes and nanoparticles to label intestinal organoids for visualization using the clinically approved imaging modality, confocal laser endomicroscopy (CLE). Staining homogeneity, durability, cell viability, differentiation capacity, and organoid forming efficiency were evaluated, together with visualization of labeled organoids in vitro and ex vivo using CLE.

Results: 5-Chloromethylfluorescein diacetate (CMFDA) proved to be suitable as it efficiently stained all organoids without transfer to unstained organoids in co-cultures. No noticeable adverse effects on viability, organoid growth, or stem cell differentiation capacity were observed, although single-cell reseeding revealed a dose-dependent reduction in organoid forming efficiency. Labeled organoids were easily identified in vitro using CLE for a duration of at least 3 days and could additionally be detected ex vivo following transplantation into murine experimental colitis.

Conclusions: It is highly feasible to use fluorescent dye-based labeling in combination with CLE to trace intestinal organoids following transplantation to confirm implantation at the intestinal target site.
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http://dx.doi.org/10.1186/s13287-019-1246-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6537188PMC
May 2019

Tracing the origin of adult intestinal stem cells.

Nature 2019 06 15;570(7759):107-111. Epub 2019 May 15.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

Adult intestinal stem cells are located at the bottom of crypts of Lieberkühn, where they express markers such as LGR5 and fuel the constant replenishment of the intestinal epithelium. Although fetal LGR5-expressing cells can give rise to adult intestinal stem cells, it remains unclear whether this population in the patterned epithelium represents unique intestinal stem-cell precursors. Here we show, using unbiased quantitative lineage-tracing approaches, biophysical modelling and intestinal transplantation, that all cells of the mouse intestinal epithelium-irrespective of their location and pattern of LGR5 expression in the fetal gut tube-contribute actively to the adult intestinal stem cell pool. Using 3D imaging, we find that during fetal development the villus undergoes gross remodelling and fission. This brings epithelial cells from the non-proliferative villus into the proliferative intervillus region, which enables them to contribute to the adult stem-cell niche. Our results demonstrate that large-scale remodelling of the intestinal wall and cell-fate specification are closely linked. Moreover, these findings provide a direct link between the observed plasticity and cellular reprogramming of differentiating cells in adult tissues following damage, revealing that stem-cell identity is an induced rather than a hardwired property.
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http://dx.doi.org/10.1038/s41586-019-1212-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986928PMC
June 2019

IL-17R-EGFR axis links wound healing to tumorigenesis in Lrig1 stem cells.

J Exp Med 2019 01 21;216(1):195-214. Epub 2018 Dec 21.

Department of Inflammation and Immunity, Cleveland Clinic, Cleveland, OH

Lrig1 marks a distinct population of stem cells restricted to the upper pilosebaceous unit in normal epidermis. Here we report that IL-17A-mediated activation of EGFR plays a critical role in the expansion and migration of Lrig1 stem cells and their progenies in response to wounding, thereby promoting wound healing and skin tumorigenesis. Lrig1-specific deletion of the IL-17R adaptor Act1 or EGFR in mice impairs wound healing and reduces tumor formation. Mechanistically, IL-17R recruits EGFR for IL-17A-mediated signaling in Lrig1 stem cells. While TRAF4, enriched in Lrig1 stem cells, tethers IL-17RA and EGFR, Act1 recruits c-Src for IL-17A-induced EGFR transactivation and downstream activation of ERK5, which promotes the expansion and migration of Lrig1 stem cells. This study demonstrates that IL-17A activates the IL-17R-EGFR axis in Lrig1 stem cells linking wound healing to tumorigenesis.
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http://dx.doi.org/10.1084/jem.20171849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314525PMC
January 2019

Lrig1 marks a population of gastric epithelial cells capable of long-term tissue maintenance and growth in vitro.

Sci Rep 2018 10 15;8(1):15255. Epub 2018 Oct 15.

BRIC - Biotech Research & Innovation Centre, University of Copenhagen, DK-2200, Copenhagen N, Denmark.

The processes involved in renewal of the epithelium that lines the mouse stomach remain unclear. Apart from the cells in the isthmus, several other populations located deeper in the gastric glands have been suggested to contribute to the maintenance of the gastric epithelium. Here, we reveal that Lrig1 is expressed in the basal layer of the forestomach and the lower part of glands in the corpus and pylorus. In the glandular epithelium of the stomach, Lrig1 marks a heterogeneous population comprising mainly non-proliferative cells. Yet, fate-mapping experiments using a knock-in mouse line expressing Cre specifically in Lrig1 cells demonstrate that these cells are able to contribute to the long-term maintenance of the gastric epithelium. Moreover, when cultured in vitro, cells expressing high level of Lrig1 have much higher organoid forming potential than the corresponding cellular populations expressing lower levels of Lrig1. Taken together, these observations show that Lrig1 is expressed primarily by differentiated cells, but that these cells can be recruited to contribute to the maintenance of the gastric epithelium. This confirms previous observations that cells located in the lower segments of gastric glands can participate in tissue replenishment.
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http://dx.doi.org/10.1038/s41598-018-33578-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189208PMC
October 2018

COX-2-PGE Signaling Impairs Intestinal Epithelial Regeneration and Associates with TNF Inhibitor Responsiveness in Ulcerative Colitis.

EBioMedicine 2018 Oct 3;36:497-507. Epub 2018 Sep 3.

Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev DK-2730, Denmark. Electronic address:

Background: Inhibition of tumor necrosis factor-α (TNF) signaling is beneficial in the management of ulcerative colitis (UC), but up to one-third of patients do not have a clinical response of relevance to TNF inhibitors during induction therapy (i.e. primary non-responders [PNRs]). Through production of prostaglandins (PGs) and thromboxanes, cyclooxygenase-2 (COX-2) affects inflammation and epithelial regeneration and may in this way be implicated in treatment resistance to TNF inhibitors.

Methods: In this study, COX-2 expression was analyzed in human intestinal biopsies and patient-derived monocytes, and the downstream consequences of COX-2 activity was evaluated by assessing the influence of the down-stream effector, PGE, on intestinal epithelial stem cell self-renewal and differentiation using primary human intestinal organoids ("mini-guts").

Findings: We found that TNF stimulation induced COX-2 expression in monocytes isolated from responders (Rs), whereas COX-2 expression was constitutively high and non-inducible in monocytes from PNRs. Additionally, PGE in combination with proliferative signals transformed human intestinal epithelial cells to a proinflammatory state akin to flaring UC, whereas PGE in combination with differentiation signals supported robust mucin induction.

Interpretation: Our work indicates that COX-2-PGE signaling could be a novel target for the management of PNRs to TNF inhibitors. We additionally demonstrate that COX-2-PGE signaling has dual functions during tissue repair and normal lineage differentiation, explaining in part the lack of response to TNF inhibitors among PNRs. FUND: This work was funded by grants from the Novo Nordisk Foundation, the Lundbeck Foundation, the Vanderbilt Digestive Disease Research Center, NIH Grants, Aase and Ejnar Danielsen's Foundation and the A.P. Møller Foundation.
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http://dx.doi.org/10.1016/j.ebiom.2018.08.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197735PMC
October 2018

Inhibiting RHOA Signaling in Mice Increases Glucose Tolerance and Numbers of Enteroendocrine and Other Secretory Cells in the Intestine.

Gastroenterology 2018 10 20;155(4):1164-1176.e2. Epub 2018 Jun 20.

Section of Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic Research Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; Laboratory for Molecular Pharmacology, Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

Background & Aims: Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation.

Methods: We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for in vitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose.

Results: Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared with mice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucose tolerance compared with administration of saline.

Conclusions: In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of L cells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance.
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http://dx.doi.org/10.1053/j.gastro.2018.06.039DOI Listing
October 2018

Loss of PACS-2 delays regeneration in DSS-induced colitis but does not affect the model of colorectal cancer.

Oncotarget 2017 Dec 26;8(65):108303-108315. Epub 2017 Nov 26.

Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark.

PACS-2 is a multifunctional sorting protein that mediates cell homeostasis. We recently identified PACS-2 in a functional genome-wide siRNA screen for novel regulators of the metalloproteinase ADAM17, the main sheddase for ligands of the ErbB receptor family. Of note, we showed that mice have significantly reduced EGFR activity and proliferative index in the intestinal epithelium. As EGFR signaling is highly mitogenic for intestinal epithelial stem cells, and plays essential roles in intestinal epithelial regeneration and tumor development, we have now examined the role of PACS-2 in these processes. Specifically, we analyzed the role of 2-deficiency in a DSS-induced colitis model as well as in the genetic colon cancer model. We now report that loss of PACS-2 delays tissue regeneration after colonic injury with little effect on key inflammatory parameters. We did however not observe any apparent effects on tumor formation driven by excessive proliferative signaling downstream from APC-deficiency. Our findings reveal that the role of PACS-2 in regulating ADAM17-mediated shedding is not an obligate requirement for the epithelium to respond to the strong inflammatory or tumorigenic inducers in the models assessed here.
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http://dx.doi.org/10.18632/oncotarget.22661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752446PMC
December 2017

YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration.

Cell Stem Cell 2018 01 14;22(1):35-49.e7. Epub 2017 Dec 14.

BRIC - Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaloes Vej 5, 2200 Copenhagen N, Denmark; Novo Nordisk Foundation Center for Stem Cell Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark. Electronic address:

Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element.
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http://dx.doi.org/10.1016/j.stem.2017.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766831PMC
January 2018

Dietary Control of Skin Lipid Composition and Microbiome.

J Invest Dermatol 2018 05 14;138(5):1225-1228. Epub 2017 Dec 14.

BRIC-Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen N, Denmark; Novo Nordisk Foundation Center for Stem Cell Research, University of Copenhagen, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N, Denmark. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2017.12.005DOI Listing
May 2018

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia.

EMBO J 2017 10 12;36(20):3029-3045. Epub 2017 Sep 12.

MRC London Institute of Medical Sciences, Imperial College London, London, UK

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to loss-of-function disorders such as Hirschsprung disease.
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http://dx.doi.org/10.15252/embj.201696247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641678PMC
October 2017

A genetically inducible porcine model of intestinal cancer.

Mol Oncol 2017 11 10;11(11):1616-1629. Epub 2017 Oct 10.

Department of Molecular Medicine, Aarhus University Hospital, Denmark.

Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase-inducible oncogene cassette containing KRAS-G12D, cMYC, SV40LT - which inhibits p53 - and pRB and (b) a 4-hydroxytamoxifen (4-OHT)-inducible Flp recombinase activator cassette controlled by the intestinal epithelium-specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4-OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.
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http://dx.doi.org/10.1002/1878-0261.12136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664002PMC
November 2017

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

Nat Med 2017 Aug 3;23(8):954-963. Epub 2017 Jul 3.

Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, UK.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.
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http://dx.doi.org/10.1038/nm.4360DOI Listing
August 2017

Intestinal barrier integrity and inflammatory bowel disease: Stem cell-based approaches to regenerate the barrier.

J Tissue Eng Regen Med 2018 04 19;12(4):923-935. Epub 2017 Oct 19.

Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, Herlev, Denmark.

Disruption of normal barrier function is a fundamental factor in the pathogenesis of inflammatory bowel disease, which includes increased epithelial cell death, modified mucus configuration, altered expression and distribution of tight junction proteins, along with a decreased expression of antimicrobial peptides. Inflammatory bowel disease is associated with life-long morbidity for affected patients, and both the incidence and prevalence is increasing globally, resulting in substantial economic strain for society. Mucosal healing and re-establishment of barrier integrity are associated with clinical remission, as well as with an improved patient outcome. Hence, these factors are vital treatment goals, which conventionally are achieved by a range of medical treatments, although none are effective in all patients, resulting in several patients still requiring surgery at some point. Therefore, novel treatment strategies to accomplish mucosal healing and to re-establish normal barrier integrity in inflammatory bowel disease are warranted, and luminal stem cell-based approaches might have an intriguing potential. Transplantation of in vitro expanded intestinal epithelial stem cells derived either directly from mucosal biopsies or from directed differentiation of human pluripotent stem cells may constitute complementary treatment options for patients with mucosal damage, as intestinal epithelial stem cells are multipotent and may give rise to all epithelial cell types of the intestine. This review provides the reader with a comprehensive state-of-the-art overview of the intestinal barrier's role in healthy and diseased states, discussing the clinical application of stem cell-based approaches to accomplish mucosal healing in inflammatory bowel disease.
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http://dx.doi.org/10.1002/term.2506DOI Listing
April 2018

Isolation and In Vitro Characterization of Epidermal Stem Cells.

Methods Mol Biol 2017 ;1553:67-83

Biotech Research and Innovation Centre, Faculty of Health and Medical Sciences, University of Copenhagen, Ole Maaloes Vej 5, 2200, Copenhagen N, Denmark.

Colony-forming assays represent prospective methods, where cells isolated from enzymatically dissociated tissues or from tissue cultures are assessed for their proliferative capacity in vitro. Complex tissues such as the epithelial component of the skin (the epidermis) are characterized by a substantial cellular heterogeneity. Analysis of bulk populations of cells by colony-forming assays can consequently be convoluted by a number of factors that are not controlled for in population wide studies. It is therefore advantageous to refine in vitro growth assays by sub-fractionation of cells using flow cytometry. Using markers that define the spatial origin of epidermal cells, it is possible to interrogate the specific characteristics of subpopulations of cells based on their in vivo credentials. Here, we describe how to isolate, culture, and characterize keratinocytes from murine back and tail skin sorted by surface antigens associated with adult stem cell characteristics.
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http://dx.doi.org/10.1007/978-1-4939-6756-8_6DOI Listing
February 2018

Cell biology: Unconventional translation in cancer.

Nature 2017 01;541(7638):471-472

Biotech Research and Innovation Centre, University of Copenhagen, DK-2200 Copenhagen, Denmark.

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http://dx.doi.org/10.1038/nature21115DOI Listing
January 2017

Bimodal skin progenitors-a matter of place and time.

EMBO J 2016 12 14;35(24):2628-2630. Epub 2016 Nov 14.

BRIC - Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen N, Denmark.

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http://dx.doi.org/10.15252/embj.201695880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167342PMC
December 2016

Modeling human disease using organotypic cultures.

Curr Opin Cell Biol 2016 12 27;43:22-29. Epub 2016 Jul 27.

BRIC-Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark. Electronic address:

Reliable disease models are needed in order to improve quality of healthcare. This includes gaining better understanding of disease mechanisms, developing new therapeutic interventions and personalizing treatment. Up-to-date, the majority of our knowledge about disease states comes from in vivo animal models and in vitro cell culture systems. However, it has been exceedingly difficult to model disease at the tissue level. Since recently, the gap between cell line studies and in vivo modeling has been narrowing thanks to progress in biomaterials and stem cell research. Development of reliable 3D culture systems has enabled a rapid expansion of sophisticated in vitro models. Here we focus on some of the latest advances and future perspectives in 3D organoids for human disease modeling.
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http://dx.doi.org/10.1016/j.ceb.2016.07.003DOI Listing
December 2016

Stem cell heterogeneity revealed.

Nat Cell Biol 2016 05;18(6):587-9

BRIC-Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.

The skin forms a protective, water-impermeable barrier consisting of heavily crosslinked epithelial cells. However, the specific role of stem cells in sustaining this barrier remains a contentious issue. A detailed analysis of the interfollicular epidermis now proposes a model for how a composite of cells with different properties are involved in its maintenance.
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http://dx.doi.org/10.1038/ncb3368DOI Listing
May 2016

From Definitive Endoderm to Gut-a Process of Growth and Maturation.

Stem Cells Dev 2015 Sep 2;24(17):1972-83. Epub 2015 Jul 2.

BRIC - Biotech Research and Innovation Centre, University of Copenhagen , Copenhagen N, Denmark .

The intestine and colon carries out vital functions, and their lifelong maintenance is of the upmost importance. Research over the past decades has carefully addressed bowel function, how it is maintained and begun to unravel how disorders such as cancer and inflammatory bowel disease form. In contrast, very little is known about the molecular mechanisms that trigger tissue maturation during development. With this review, our aim is to carefully provide a critical appraisal of the literature to give a state-of-the-art view of intestinal development. Starting from definitive endoderm at gastrulation to the emergence of a structure with mature properties, the tissue undergoes complex morphogenetic processes that rely on both biophysical changes and secreted signaling molecules. We will also discuss how new and exciting developments using in vitro models are likely to provide new insights into this process and potential therapeutic strategies for gastrointestinal disorders.
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http://dx.doi.org/10.1089/scd.2015.0017DOI Listing
September 2015

Hippo signalling directs intestinal fate.

Nat Cell Biol 2015 Jan;17(1):5-6

Hippo signalling has been associated with many important tissue functions including the regulation of organ size. In the intestinal epithelium differing functions have been proposed for the effectors of Hippo signalling, YAP and TAZ1. These are now shown to have a dual role in the intestinal epithelium, regulating both stem cell proliferation and differentiation along a specific secretory lineage.
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http://dx.doi.org/10.1038/ncb3086DOI Listing
January 2015

Heterogeneity and plasticity of epidermal stem cells.

Development 2014 Jul;141(13):2559-67

BRIC - Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen N DK-2200, Denmark Wellcome Trust & Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK

The epidermis is an integral part of our largest organ, the skin, and protects us against the hostile environment. It is a highly dynamic tissue that, during normal steady-state conditions, undergoes constant turnover. Multiple stem cell populations residing in autonomously maintained compartments facilitate this task. In this Review, we discuss stem cell behaviour during normal tissue homeostasis, regeneration and disease within the pilosebaceous unit, an integral structure of the epidermis that is responsible for hair growth and lubrication of the epithelium. We provide an up-to-date view of the pilosebaceous unit, encompassing the heterogeneity and plasticity of multiple discrete stem cell populations that are strongly influenced by external cues to maintain their identity and function.
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http://dx.doi.org/10.1242/dev.104588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067958PMC
July 2014

Generation of multipotent foregut stem cells from human pluripotent stem cells.

Stem Cell Reports 2013 10;1(4):293-306. Epub 2013 Oct 10.

Wellcome Trust-Medical Research Council Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, West Forvie Site, Robinson Way, University of Cambridge, Cambridge CB2 0SZ, UK.

Human pluripotent stem cells (hPSCs) could provide an infinite source of clinically relevant cells with potential applications in regenerative medicine. However, hPSC lines vary in their capacity to generate specialized cells, and the development of universal protocols for the production of tissue-specific cells remains a major challenge. Here, we have addressed this limitation for the endodermal lineage by developing a defined culture system to expand and differentiate human foregut stem cells (hFSCs) derived from hPSCs. hFSCs can self-renew while maintaining their capacity to differentiate into pancreatic and hepatic cells. Furthermore, near-homogenous populations of hFSCs can be obtained from hPSC lines which are normally refractory to endodermal differentiation. Therefore, hFSCs provide a unique approach to bypass variability between pluripotent lines in order to obtain a sustainable source of multipotent endoderm stem cells for basic studies and to produce a diversity of endodermal derivatives with a clinical value.
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http://dx.doi.org/10.1016/j.stemcr.2013.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849417PMC
October 2015