Gut mucosa dissociation protocols influence cell type proportions and single-cell gene expression levels.

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of the cellular landscape of organs. Most single-cell protocols require fresh material, which limits sample size per experiment, and consequently, introduces batch effects. This is especially true for samples acquired through complex medical procedures, such as intestinal mucosal biopsies. Moreover, the tissue dissociation procedure required for obtaining single cells is a major source of noise; different dissociation procedures applied to different compartments of the tissue induce artificial gene expression differences between cell subsets. To overcome these challenges, we have developed a one-step dissociation protocol and demonstrated its use on cryopreserved gut mucosal biopsies. Using flow cytometry and scRNA-seq analysis, we compared this one-step dissociation protocol with the current gold standard, two-step collagenase digestion, and an adaptation of a recently published alternative, three-step cold-active Bacillus licheniformus protease digestion. Both cell viability and cell type composition were comparable between the one-step and two-step collagenase dissociation, with the former being more time-efficient. The cold protease digestion resulted in equal cell viability, but better preserves the epithelial cell types. Consequently, to analyze the rarer cell types, such as glial cells, larger total biopsy cell numbers are required as input material. The multi-step protocols affected cell types spanning multiple compartments differently. In summary, we show that cryopreserved gut mucosal biopsies can be used to overcome the logistical challenges and batch effects in large scRNA-seq studies. Furthermore, we demonstrate that using cryopreserved biopsies digested using a one-step collagenase protocol enables large-scale scRNA-seq, FACS, organoid generation and intraepithelial lymphocyte expansion.

Survival and cellular heterogeneity of epithelium in cultured mouse and rat precision-cut intestinal slices.

Precision-cut intestinal slices (PCIS) are used to study intestinal (patho)physiology, drug efficacy, toxicity, transport and metabolism ex vivo. One of the factors that limit the use of PCIS is a relatively short life-span. Moreover, culture-induced changes in cellular composition of PCIS remain largely uncharacterized. In this study, we demonstrated the epithelial cell heterogeneity in mouse and rat PCIS and its alterations during culture. In addition, we evaluated whether the presence of niche growth factors impacts the survival of PCIS epithelial cells. We showed that freshly prepared PCIS retained the main epithelial cell types, namely absorptive enterocytes, goblet cells, enteroendocrine cells, stem cells, transit-amplifying cells and Paneth cells. Once placed in culture, PCIS displayed progressive epithelial damage, and loss of these epithelial cell types. Cells comprising the intestinal stem cell niche were especially sensitive to the damage, and the addition of niche growth factors beneficially affected the survival of stem cells and transit-amplifying cells in PCIS during culture. In conclusion, this study provides new insights into the dynamic changes in cellular composition of epithelium in cultured PCIS, paving the way to future toxicological and pharmacological studies in an informed and reliable ex vivo setting.

Understanding human gut diseases at single-cell resolution.

Our understanding of gut functioning and pathophysiology has grown considerably in the past decades, and advancing technologies enable us to deepen this understanding. Single-cell RNA sequencing (scRNA-seq) has opened a new realm of cellular diversity and transcriptional variation in the human gut at a high, single-cell resolution. ScRNA-seq has pushed the science of the digestive system forward by characterizing the function of distinct cell types within complex intestinal cellular environments, by illuminating the heterogeneity within specific cell populations and by identifying novel cell types in the human gut that could contribute to a variety of intestinal diseases. In this review, we highlight recent discoveries made with scRNA-seq that significantly advance our understanding of the human gut both in health and across the spectrum of gut diseases, including inflammatory bowel disease, colorectal carcinoma and celiac disease.

Predictive Value of Precision-Cut Kidney Slices as an Ex Vivo Screening Platform for Therapeutics in Human Renal Fibrosis.

Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGFß or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation; (2) expression of markers of TGFß and PDGF signaling; and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis.

Investigating fibrosis and inflammation in an ex vivo NASH murine model.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, characterized by excess fat accumulation (steatosis). Nonalcoholic steatohepatitis (NASH) develops in 15-20% of NAFLD patients and frequently progresses to liver fibrosis and cirrhosis. We aimed to develop an ex vivo model of inflammation and fibrosis in steatotic murine precision-cut liver slices (PCLS). NASH was induced in C57Bl/6 mice on an amylin and choline-deficient l-amino acid-defined (CDAA) diet. PCLS were prepared from steatohepatitic (sPCLS) and control (cPCLS) livers and cultured for 48 h with LPS, TGFß1, or elafibranor. Additionally, C57Bl/6 mice were placed on CDAA diet for 12 wk to receive elafibranor or vehicle from weeks 7 to 12. Effects were assessed by transcriptome analysis and procollagen Iα1 protein production. The diets induced features of human NASH. Upon culture, all PCLS showed an increased gene expression of fibrosis- and inflammation-related markers but decreased lipid metabolism markers. LPS and TGFß1 affected sPCLS more pronouncedly than cPCLS. TGFß1 increased procollagen Iα1 solely in cPCLS. Elafibranor ameliorated fibrosis and inflammation in vivo but not ex vivo, where it only increased the expression of genes modulated by PPARα. sPCLS culture induced inflammation-, fibrosis-, and lipid metabolism-related transcripts, explained by spontaneous activation. sPCLS remained responsive to proinflammatory and profibrotic stimuli on gene expression. We consider that PCLS represent a useful tool to reproducibly study NASH progression. sPCLS can be used to evaluate potential treatments for NASH, as demonstrated in our elafibranor study, and serves as a model to bridge results from rodent studies to the human system.NEW & NOTEWORTHY This study showed that nonalcoholic steatohepatitis can be studied ex vivo in precision-cut liver slices obtained from murine diet-induced fatty livers. Liver slices develop a spontaneous inflammatory and fibrogenic response during culture that can be augmented with specific modulators. Additionally, the model can be used to test the efficacy of pharmaceutical compounds (as shown in this investigation with elafibranor) and could be a tool for preclinical assessment of potential therapies.

Macromolecular Crowding as a Tool to Screen Anti-fibrotic Drugs: The Scar-in-a-Jar System Revisited.

An unsolved therapeutic problem in fibrosis is the overproduction of collagen. In order to screen the effect of anti-fibrotic drugs on collagen deposition, the Scar-in-a-Jar approach has been introduced about a decade ago. With macromolecular crowding a rapid deposition of collagen is seen, resulting in a substantial decrease in culture time, but the system has never been tested in an adequate way. We therefore have compared six different macromolecular crowders [Ficoll PM 70 (Fc70), Ficoll PM 400 (Fc400), a mixture of Ficoll 70 and 400 (Fc70/400), polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 500 (DxS500), and carrageenan (CR)] under profibrotic conditions (addition of TGFß1) with primary human adult dermal fibroblasts in the presence of 0.5 and 10% FBS. We found that (1) collagen deposition and myofibroblast formation was superior with 0.5% FBS, (2) DxS500 and CR results in an aberrant collagen deposition pattern, (3) ND670 does not increase collagen deposition, and (4) CR, DxS500, and Fc40/700 affected important phenotypical properties of the cells when cultured under pro-fibrotic conditions, whereas PVP40 and PVP360 did less or not. Because of viscosity problems with PVP360, we conclude that PVP40 is the most optimal crowder for the screening of anti-fibrotic drugs. Finally, the effect of various concentrations of Imatinib, Galunisertib, Omipalisib or Nintedanib on collagen deposition and myofibroblast formation was tested with PVP40 as the crowder.

Exploring organ-specific features of fibrogenesis using murine precision-cut tissue slices.

Fibrosis is the hallmark of pathologic tissue remodelling in most chronic diseases. Despite advances in our understanding of the mechanisms of fibrosis, it remains uncured. Fibrogenic processes share conserved core cellular and molecular pathways across organs. In this study, we aimed to elucidate shared and organ-specific features of fibrosis using murine precision-cut tissue slices (PCTS) prepared from small intestine, liver and kidneys. PCTS displayed substantial differences in their baseline gene expression profiles: 70% of the extracellular matrix (ECM)-related genes were differentially expressed across the organs. Culture for 48 h induced significant changes in ECM regulation and triggered the onset of fibrogenesis in all PCTS in organ-specific manner. TGFß signalling was activated during 48 h culture in all PCTS. However, the degree of its involvement varied: both canonical and non-canonical TGFß pathways were activated in liver and kidney slices, while only canonical, Smad-dependent, cascade was involved in intestinal slices. The treatment with galunisertib blocked the TGFßRI/SMAD2 signalling in all PCTS, but attenuated culture-induced dysregulation of ECM homeostasis and mitigated the onset of fibrogenesis with organ-specificity. In conclusion, regardless the many common features in pathophysiology of organ fibrosis, PCTS displayed diversity in culture-induced responses and in response to the treatment with TGFßRI kinase inhibitor galunisertib, even though it targets a core fibrosis pathway. A clear understanding of the common and organ-specific features of fibrosis is the basis for developing novel antifibrotic therapies.

Inhibition of tyrosine kinase receptor signaling attenuates fibrogenesis in an ex vivo model of human renal fibrosis.

Poor translation from animal studies to human clinical trials is one of the main hurdles in the development of new drugs. Here, we used precision-cut kidney slices (PCKS) as a translational model to study renal fibrosis and to investigate whether inhibition of tyrosine kinase receptors, with the selective inhibitor nintedanib, can halt fibrosis in murine and human PCKS. We used renal tissue of murine and human origins to obtain PCKS. Control slices and slices treated with nintedanib were studied to assess viability, activation of tyrosine kinase receptors, cell proliferation, collagen type I accumulation, and gene and protein regulation. During culture, PCKS spontaneously develop a fibrotic response that resembles in vivo fibrogenesis. Nintedanib blocked culture-induced phosphorylation of platelet-derived growth factor receptor and vascular endothelial growth factor receptor. Furthermore, nintedanib inhibited cell proliferation and reduced collagen type I accumulation and expression of fibrosis-related genes in healthy murine and human PCKS. Modulation of extracellular matrix homeostasis was achieved already at 0.1 µM, whereas high concentrations (1 and 5 µM) elicited possible nonselective effects. In PCKS from human diseased renal tissue, nintedanib showed limited capacity to reverse established fibrosis. In conclusion, nintedanib attenuated the onset of fibrosis in both murine and human PCKS by inhibiting the phosphorylation of tyrosine kinase receptors; however, the reversal of established fibrosis was not achieved.

Transcriptomic characterization of culture-associated changes in murine and human precision-cut tissue slices.

Our knowledge of complex pathological mechanisms underlying organ fibrosis is predominantly derived from animal studies. However, relevance of animal models for human disease is limited; therefore, an ex vivo model of human precision-cut tissue slices (PCTS) might become an indispensable tool in fibrosis research and drug development by bridging the animal-human translational gap. This study, presented as two parts, provides comprehensive characterization of the dynamic transcriptional changes in PCTS during culture by RNA sequencing. Part I investigates the differences in culture-induced responses in murine and human PCTS derived from healthy liver, kidney and gut. Part II delineates the molecular processes in cultured human PCTS generated from diseased liver, kidney and ileum. We demonstrated that culture was associated with extensive transcriptional changes and impacted PCTS in a universal way across the organs and two species by triggering an inflammatory response and fibrosis-related extracellular matrix (ECM) remodelling. All PCTS shared mRNA upregulation of IL-11 and ECM-degrading enzymes MMP3 and MMP10. Slice preparation and culturing activated numerous pathways across all PCTS, especially those involved in inflammation (IL-6, IL-8 and HMGB1 signalling) and tissue remodelling (osteoarthritis pathway and integrin signalling). Despite the converging effects of culture, PCTS display species-, organ- and pathology-specific differences in the regulation of genes and canonical pathways. The underlying pathology in human diseased PCTS endures and influences biological processes like cytokine release. Our study reinforces the use of PCTS as an ex vivo fibrosis model and supports future studies towards its validation as a preclinical tool for drug development.

PI3K inhibition reduces murine and human liver fibrogenesis in precision-cut liver slices.

BACKGROUND: Liver fibrosis results from continuous inflammation and injury. Despite its high prevalence worldwide, no approved antifibrotic therapies exist. Omipalisib is a selective inhibitor of the PI3K/mTOR pathway that controls nutrient metabolism, growth and proliferation. It has shown antifibrotic properties in vitro. While clinical trials for idiopathic pulmonary fibrosis have been initiated, an in-depth preclinical evaluation is lacking. We evaluated omipalisib's effects on fibrogenesis using the ex vivo model of murine and human precision-cut tissue slices (PCTS). METHODS: Murine and human liver and jejunum PCTS were incubated with omipalisib up to 10 µM for 48 h. PI3K pathway activation was assessed through protein kinase B (Akt) phosphorylation and antifibrotic efficacy was determined via a spectrum of fibrosis markers at the transcriptional and translational level. RESULTS: During incubation of PCTS the PI3K pathway was activated and incubation with omipalisib prevented Akt phosphorylation (IC50 = 20 and 1.5 nM for mouse and human, respectively). Viability of mouse and human liver PCTS was compromised only at the high concentration of 10 and 1-5 µM, respectively. However, viability of jejunum PCTS decreased with 0.1 (mouse) and 0.01 µM (human). Spontaneously increased fibrosis related genes and proteins were significantly and similarly suppressed in mouse and in human liver PCTS. CONCLUSIONS: Omipalisib has antifibrotic properties in ex vivo mouse and human liver PCTS, but higher concentrations showed toxicity in jejunum PCTS. While the PI3K/mTOR pathway appears to be a promising target for the treatment of liver fibrosis, PCTS revealed likely side effects in the intestine at higher doses.

Growth factors of stem cell niche extend the life-span of precision-cut intestinal slices in culture: A proof-of-concept study.

Precision-cut intestinal slices (PCIS) is an ex vivo culture technique that found its applications in toxicology, drug transport and drug metabolism testing, as well as in fibrosis research. The main limiting factor of PCIS as experimental model is the relatively short viability of tissue slices. Here, we describe a strategy for extending the life-span of PCIS during culture using medium that is routinely used for growing intestinal organoids. Mouse and rat PCIS cultured in standard medium progressively showed low ATP/protein content and severe tissue degradation, indicating loss of tissue viability. In turn, organoid medium, containing epithelial growth factor (EGF), Noggin and R-spondin, maintained significantly higher ATP/protein levels and better preserved intestinal architecture of mouse PCIS at 96 h. In contrast, organoid medium that additionally contained Wnt, had a clear positive effect on the ATP content of rat PCIS during 24 h of culture, but not on slice histomorphology. Our proof-of-concept study provides early evidence that employing organoid medium for PCIS culture improved tissue viability during extended incubation. Enabling lasting PCIS cultures will greatly widen their range of applications in predicting long-term intestinal toxicity of xenobiotics and elucidating their mechanism of action, among others.

Evaluating the antifibrotic potency of galunisertib in a human ex vivo model of liver fibrosis.

BACKGROUND AND PURPOSE: Liver fibrosis is a major cause of liver-related mortality and, so far, no effective antifibrotic drug is available. Galunisertib, a TGF-ß receptor type I kinase inhibitor, is a potential candidate for the treatment of liver fibrosis. Here, we evaluated the potency of galunisertib in a human ex vivo model of liver fibrosis. EXPERIMENTAL APPROACH: Antifibrotic potency and associated mechanisms were studied ex vivo, using both healthy and cirrhotic human precision-cut liver slices. Fibrosis-related parameters, both transcriptional and translational level, were assessed after treatment with galunisertib. KEY RESULTS: Galunisertib showed a prominent antifibrotic potency. Phosphorylation of SMAD2 was inhibited, while that of SMAD1 remained unchanged. In healthy and cirrhotic human livers, spontaneous transcription of numerous genes encoding collagens, including collagen type I, α 1, collagen maturation, non-collageneous extracellular matrix (ECM) components, ECM remodelling and selected ECM receptors was significantly decreased. The reduction of fibrosis-related transcription was paralleled by a significant inhibition of procollagen I C-peptide released by both healthy and cirrhotic human liver slices. Moreover, galunisertib showed similar antifibrotic potency in human and rat lives. CONCLUSIONS AND IMPLICATIONS: Galunisertib is a drug that deserves to be further investigated for the treatment of liver fibrosis. Inhibition of SMAD2 phosphorylation is probably a central mechanism of action. In addition, blocking the production and maturation of collagens and promoting their degradation are related to the antifibrotic action of galunisertib.

Meta-Analysis on Randomized Controlled Trials of Vaccines with QS-21 or ISCOMATRIX Adjuvant: Safety and Tolerability.

BACKGROUND AND OBJECTIVES: QS-21 shows in vitro hemolytic effect and causes side effects in vivo. New saponin adjuvant formulations with better toxicity profiles are needed. This study aims to evaluate the safety and tolerability of QS-21 and the improved saponin adjuvants (ISCOM, ISCOMATRIX and Matrix-M™) from vaccine trials. METHODS: A systematic literature search was conducted from MEDLINE, EMBASE, Cochrane library and Clinicaltrials.gov. We selected for the meta-analysis randomized controlled trials (RCTs) of vaccines adjuvanted with QS-21, ISCOM, ISCOMATRIX or Matrix-M™, which included a placebo control group and reported safety outcomes. Pooled risk ratios (RRs) and their 95% confidence intervals (CIs) were calculated using a random-effects model. Jadad scale was used to assess the study quality. RESULTS: Nine RCTs were eligible for the meta-analysis: six trials on QS-21-adjuvanted vaccines and three trials on ISCOMATRIX-adjuvanted, with 907 patients in total. There were no studies on ISCOM or Matrix-M™ adjuvanted vaccines matching the inclusion criteria. Meta-analysis identified an increased risk for diarrhea in patients receiving QS21-adjuvanted vaccines (RR 2.55, 95% CI 1.04-6.24). No increase in the incidence of the reported systemic AEs was observed for ISCOMATRIX-adjuvanted vaccines. QS-21- and ISCOMATRIX-adjuvanted vaccines caused a significantly higher incidence of injection site pain (RR 4.11, 95% CI 1.10-15.35 and RR 2.55, 95% CI 1.41-4.59, respectively). ISCOMATRIX-adjuvanted vaccines also increased the incidence of injection site swelling (RR 3.43, 95% CI 1.08-10.97). CONCLUSIONS: Our findings suggest that vaccines adjuvanted with either QS-21 or ISCOMATRIX posed no specific safety concern. Furthermore, our results indicate that the use of ISCOMATRIX enables a better systemic tolerability profile when compared to the use of QS-21. However, no better local tolerance was observed for ISCOMATRIX-adjuvanted vaccines in immunized non-healthy subjects. This meta-analysis is limited by the relatively small number of individuals recruited in the included trials, especially in the control groups.

New Luminescent Polynuclear Metal Complexes with Anticancer Properties: Toward Structure-Activity Relationships.

A series of new heterodinuclear luminescent complexes with two different organic ligands have been synthesized and characterized. A luminescent Ru(II)(polypyridine) moiety and a metal-based anticancer fragment (AuCl, (p-cymene)RuCl2, (p-cymene)OsCl2, (Cp*)RhCl2, or Au-thioglucose) are the two general features of these complexes. All of the bimetallic compounds have been evaluated for their antiproliferative properties in vitro in human cancer cell lines. Only the complexes containing an Au(I) fragment exhibit antiproliferative activity in the range of cisplatin or higher. The photophysical and electrochemical properties of the bimetallic species have been investigated, and fluorescence microscopy experiments have been performed successfully. The most promising bimetallic cytotoxic complexes (i.e., with the Au-thioglucose scaffold) have shown to be easily taken up by cancer cells at 37 °C in the cytoplasm or in specific organelles. Interestingly, experiments repeated at 4 °C showed no uptake of the bimetallic species inside cells, which confirms involvement of active transport processes. To evaluate the role of glucose transporters in the cell uptake of the gold complexes, inhibition of the GluT-1 (glucose transporter isoform with high level of expression in cancer cells) was achieved, showing only scarce influence on the compounds' uptake. Finally, the observed absence of interactions with nucleic acid model structures suggests that the gold compounds may have different intracellular targets with respect to cisplatin.

Assessment of the developmental toxicity of nanoparticles in an ex vivo 3D model, the murine limb bud culture system.

The rapid growth of nanotechnological products for biomedical applications has exacerbated the need for suitable biological tests to evaluate the potential toxic effects of nanomaterials. The possible consequences of exposure during embryo and fetal development are of particular concern. The limb bud culture is an ex vivo 3D model in which growth, cell differentiation, and tissue organization occur and both molecular and functional endpoints can be quantitatively assessed. We employed this model to assess biochemical and morphological changes induced during organogenesis by two classes of nanostructured materials: quantum dot nanocrystals and organic polyglycerol sulfate dendrimers (dPGS). We show that quantum dots carrying mercaptopropionic acid (QD-MPA) on the surface, commonly used in biological studies, inhibit the development of limb buds from CD1 wildtype and Col2a1; Col10a1; Col1a1 triple transgenic fluorescent reporter mice, as revealed by changes in several morphological and biochemical markers. QD-MPA interfere with chondrogenesis and osteogenesis and disrupt the expression of COL10A1 and COL1A1, key markers of differentiation. In contrast, equivalent (3-100 nM) concentrations of dPGS do not adversely affect limb development. Neither QD-MPA nor dPGS-Cy5 alters the expression of several markers of cell proliferation or apoptosis. Collectively, these results suggest that murine limb buds in culture constitute a convenient, inexpensive and reliable developmental model for the assessment of the nanotoxicological effects of nanocrystals and polymers. In these 3D cultures, any effect that is observed can be directly ascribed to the nanostructures per se or a degradation component released from the complex nanostructure.

New heteronuclear gold(I)-platinum(II) complexes with cytotoxic properties: are two metals better than one?

A series of mono- and heterodinuclear gold(I) and platinum(II) complexes with a new bipyridylamine-phosphine ligand have been synthesized and characterized. The X-ray structures of the ligand precursor 4-iodo-N,N-di(pyridin-2-yl)benzamide, and of one gold derivative are reported. All the complexes display antiproliferative properties in vitro in human cancer cells in the range of cisplatin or higher, which appear to correlate with compounds' uptake. Interestingly, studies of the interactions of the compounds with models of DNA indicate different mechanisms of actions with respect to cisplatin. The biological activity study of these complexes provides useful information about the interest of designing multimetallic complexes for enhanced cytotoxic properties.