Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules.

Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.

A Novel Method to Isolate RNase MRP Using RNA Streptavidin Aptamer Tags.

Interactions between RNA-binding proteins and RNA molecules are at the center of multiple biological processes. Therefore, accurate characterization of the composition of ribonucleoprotein complexes (RNPs) is crucial. Ribonuclease (RNase) for mitochondrial RNA processing (MRP) and RNase P are highly similar RNPs that play distinct roles at the cellular level; as a consequence, the specific isolation of either of these complexes is essential to study their biochemical function. Since their protein components are nearly identical, purification of these endoribonucleases using protein-centric methods is not feasible. Here, we describe a procedure employing an optimized high-affinity streptavidin-binding RNA aptamer, termed S1m, to purify RNase MRP free of RNase P. This report details all steps from the RNA tagging to the characterization of the purified material. We show that using the S1m tag allows efficient isolation of active RNase MRP.

Molecular and Functional Characterization of Human Intestinal Organoids and Monolayers for Modeling Epithelial Barrier.

BACKGROUND: Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier. METHODS: Patient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib. RESULTS: In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib. CONCLUSIONS: We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.

A novel experimental approach for the selective isolation and characterization of human RNase MRP.

RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of RNase MRP is desired to be able to study its biochemical function, composition and activity in both healthy and disease situations. Due to the high similarity with RNase P, a method to specifically isolate the RNase MRP complex is currently lacking. By fusing a streptavidin-binding RNA aptamer, the S1m-aptamer, to the RNase MRP RNA we have been able to compare the relative expression levels of wildtype and mutant MRP RNAs. Moreover, we were able to isolate active RNase MRP complexes. We observed that mutant MRP RNAs are expressed at lower levels and have lower catalytic activity compared to the wildtype RNA. The observation that a single nucleotide substitution at position 40 in the P3 domain but not in other domains of RNase MRP RNA severely reduced the binding of the Rpp25 protein subunit confirmed that the P3 region harbours the main binding site for this protein. Altogether, this study shows that the RNA aptamer tagging approach can be used to identify RNase MRP substrates, but also to study the effect of mutations on MRP RNA expression levels and RNase MRP composition and endoribonuclease activity.

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport, antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

LINE-1 Hypermethylation in Serum Cell-Free DNA of Relapsing Remitting Multiple Sclerosis Patients.

Concentrations of cell-free DNA (cfDNA) circulating in blood and its epigenetic variation, such as DNA methylation, may provide useful diagnostic or prognostic information. Long interspersed nuclear element-1 (LINE-1) constitutes approximately 20% of the human genome and its 5'UTR region is CpG rich. Due to its wide distribution, the methylation level of the 5'UTR of LINE-1 can serve as a surrogate marker of global genomic DNA methylation. The aim of the current study was to investigate whether the methylation status of LINE-1 elements in serum cell-free DNA differs between relapsing remitting multiple sclerosis (RRMS) patients and healthy control subjects (CTR). Serum DNA samples of 6 patients and 6 controls were subjected to bisulfite sequencing. The results showed that the methylation level varies among distinct CpG sites in the 5'UTR of LINE-1 repeats and revealed differences in the methylation state of specific sites in this element between patients and controls. The latter differences were largely due to CpG sites in the L1PA2 subfamily, which were more frequently methylated in the RRMS patients than in the CTR group, whereas such differences were not observed in the L1HS subfamily. These data were verified by quantitative PCR using material from 18 patients and 18 control subjects. The results confirmed that the methylation level of a subset of the CpG sites within the LINE-1 promoter is elevated in DNA from RRMS patients in comparison with CTR. The present data suggest that the methylation status of CpG sites of LINE repeats could be a basis for development of diagnostic or prognostic tests.

RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences.

The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels.

Multiplex peptide-based B cell epitope mapping.

B cell epitope mapping is widely applied to determine antibody-binding sites. Several methods exist to map B cell epitopes and here we describe three methods that are characterized by the simultaneous analysis of multiple peptides. In the first approach a microarray of overlapping synthetic peptides derived from an antigenic protein is used and the binding of the antibodies is analyzed by fluorescently labeled secondary antibodies. This method is particularly suited for the identification of linear epitopes of an established target protein. In the second approach the binding of antibodies to a random synthetic peptide library immobilized on microbeads is determined by enzyme-conjugated secondary antibodies and the selection of antibody-bound beads by a light microscope. This method can be applied when information on the identity of the antigenic protein is lacking. In the third method an antigen is proteolytically digested and antibody binding to the resulting peptides is analyzed by surface plasmon resonance imaging (iSPR). The latter method can be applied when the purified antigenic protein is available.