Single Cell Gene Expression Analysis in a 3D Microtissue Liver Model Reveals Cell Type-Specific Responses to Pro-Fibrotic TGF-ß1 Stimulation.

3D cell culture systems are widely used to study disease mechanisms and therapeutic interventions. Multicellular liver microtissues (MTs) comprising HepaRG, hTERT-HSC and THP-1 maintain multicellular interactions and physiological properties required to mimic liver fibrosis. However, the inherent complexity of multicellular 3D-systems often hinders the discrimination of cell type specific responses. Here, we aimed at applying single cell sequencing (scRNA-seq) to discern the molecular responses of cells involved in the development of fibrosis elicited by TGF-ß1. To obtain single cell suspensions from the MTs, an enzymatic dissociation method was optimized. Isolated cells showed good viability, could be re-plated and cultured in 2D, and expressed specific markers determined by scRNA-seq, qRT-PCR, ELISA and immunostaining. The three cell populations were successfully clustered using supervised and unsupervised methods based on scRNA-seq data. TGF-ß1 led to a fibrotic phenotype in the MTs, detected as decreased albumin and increased αSMA expression. Cell-type specific responses to the treatment were identified for each of the three cell types. They included HepaRG damage characterized by a decrease in cellular metabolism, prototypical inflammatory responses in THP-1s and extracellular matrix remodeling in hTERT-HSCs. Furthermore, we identified novel cell-specific putative fibrosis markers in hTERT-HSC (COL15A1), and THP-1 (ALOX5AP and LAPTM5).

Implementation of a Human Renal Proximal Tubule on a Chip for Nephrotoxicity and Drug Interaction Studies.

Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-ß-d-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation).

Sphingomyelin and phosphatidylcholine contrarily affect the induction of apoptosis in intestinal epithelial cells.

SCOPE: The major alimentary sources for the plasma membrane lipid sphingomyelin (SM) are dairy products, eggs, and meat. We recently reported that the SM metabolite ceramide induces cathepsin D mediated apoptosis in murine intestinal epithelial cells (IECs) and increases inflammation in acute colitis. We investigated the impact of SM and phosphatidylcholine on apoptosis in human IECs and point out BH3-interacting death agonist (BID) as link between cathepsin D and apoptosis. METHODS AND RESULTS: HT-29 and isolated human IECs were stimulated with SM or phosphatidylcholine. SM treatment resulted in increased apoptosis. Phosphatidylcholine showed contrary effects. Western revealed higher amounts of cathepsin D and BID activation upon lipid stimulation. Western blotting revealed BID activation through SM in both an induced and a spontaneous mouse model of colitis. CONCLUSION: Dietary phospholipids may induce or abolish apoptosis in IECs and seem to play a role in the pathogenesis of inflammatory bowel diseases. This nutritional factor might be considered when evaluating the pathogenesis of inflammatory bowel diseases. Effects of SMase- and SM treatment on inflammation in dextran sulfate sodium induced animal models of colitis and in vitro experiments are discussed as controversial. Variable sources of SM, feeding techniques, and mouse strains might play a role.

A new heterotopic transplant animal model of intestinal fibrosis.

BACKGROUND: Severe mucosal tissue damage requiring efficient wound healing is a main feature of inflammatory bowel disease but excessive tissue repair promotes fibrosis. The clinical investigation of fibrosis is confined to the limited amount of biological material available from patients. This makes the establishment of a new animal model, a highly desirable goal. We investigated whether intestinal fibrosis occurs after heterotopic transplantation of small bowel resections in rats. METHODS: Donor (Brown Norway or Lewis rats) small bowel resections were transplanted subcutaneously into the neck of recipients (Lewis rats). Grafts were explanted 2, 7, 14, and 21 days after transplantation. RESULTS: Heterotopic intestinal transplants remained viable for 21 days. Rapid loss of crypt structures at day 2 after intestinal transplantation was followed by lymphocyte infiltration and obliteration of the intestinal lumen by fibrous tissue at day 21. Loss of the intestinal epithelium was confirmed by the lack of cytokeratin staining in immunohistochemistry. Collagen expression was increased with time after transplantation as confirmed by real-time PCRs, Elastica van Gieson, and Sirius Red staining. Lumen obliteration was connected with increased expression of potent mediators of fibrosis such as α5ß6 integrin, interleukin (IL)-13, and transforming growth factor ß. Myofibroblast phenotype was demonstrated by the presence of both α-smooth muscle actin and vimentin in the obliterated lumen. CONCLUSIONS: We established a method for heterotopic transplantation of small bowel resections. A variety of histologic and molecular features of fibrosis were observed in the heterotopic intestinal grafts which suggests, that this new in vivo model will be instrumental in studying pathogenesis and treatment of intestinal fibrosis.