The potential and limitations of intrahepatic cholangiocyte organoids to study inborn errors of metabolism

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process

Effect of dietary soybean oil inclusion on liver-related transcription factors in a pig model for metabolic diseases.

Dietary fatty acids (FA) are components of the lipids, which contribute to membrane structure, energy input, and biological functions related to cellular signaling and transcriptome regulation. However, the consumers still associate dietary FA with fat deposition and increased occurrence of metabolic diseases such as obesity and atherosclerosis. Previous studies already demonstrated that some fatty acids are linked with inflammatory response, preventing metabolic diseases. To better understand the role of dietary FA on metabolic diseases, for the first time, a study to identify key transcription factors (TF) involved in lipid metabolism and inflammatory response by transcriptome analysis from liver samples of animal models was performed. The key TF were identified by functional enrichment analysis from the list of differentially expressed genes identified in liver samples between 35 pigs fed with 1.5% or 3.0% soybean oil. The functional enrichment analysis detected TF linked to lipid homeostasis and inflammatory response, such as RXRA, EGFR, and SREBP2 precursor. These findings demonstrated that key TF related to lipid metabolism could be modulated by dietary inclusion of soybean oil. It could contribute to nutrigenomics research field that aims to elucidate dietary interventions in animal and human health, as well as to drive food technology and science

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Modern Day Heracles: Patient-derived Liver Organoids to Model Rare Pediatric Liver Diseases

Rare pediatric liver disorders can be very destructive to the lives of patients and their families. Often, therapeutic options are limited to symptomatic care and precise disease mechanisms remain elusive. The relatively low incidence of each individual disease and the often widespread geographic distribution of patients complicate research and treatment development. In this thesis, we have investigated the potential of patient-derived liver organoids to study such rare pediatric liver diseases. We characterized intrahepatic cholangiocyte organoids (ICOs) from several monogenic liver disorders and showed that organoids express affected proteins of diseases such as cystic fibrosis, Wilson disease and methylmalonic acidemia. We uncovered that ICOs possess a hybrid phenotype, combining hepatocyte and cholangiocyte characteristics. This led us to investigate whether patient-derived liver organoids would be interesting to study the rare perinatal disease biliary atresia (BA), in which the hepatobiliary tree is occluded and becomes fibrotic. We found that organoids from different hepatobiliary regions (intrahepatic, extrahepatic and gallbladder) of BA patients can be cultured and biobanked. Further characterization showed that BA patient organoids display BA specific growth behavior and increased sensitivity to viral infections. While we showed that various hepatocyte and cholangiocyte functions can be studied in liver organoids, our data also indicated that several hepatocyte functions are currently limited in this in vitro system. Therefore, we devised a novel culture strategy to increase hepatic maturation in liver organoids. We found that hepatic functions, such as drug metabolism, improve when liver organoid cells are cultured on hollow fiber membranes coated with extracellular matrix proteins of the hepatic niche. Moreover, we demonstrated that hepatic transepithelial transporter defects such as progressive familial intrahepatic cholestasis type 3 can be studied in patient-derived liver organoids. In summary, we have demonstrated that patient-derived liver organoids are a useful tool to study various rare liver and metabolic disorders affecting the hepatobiliary tract. Several diseases such as BA, cystic fibrosis, Wilson disease, progressive familial intrahepatic cholestasis type 3 and methylmalonic acidemia can currently be studied with ICOs. Although ICOs were previously praised as a new break-through hepatocyte model, we have shown that organoids from all hepatobiliary regions are at least equally useful for researching cholangiopathies, such as BA. The development of new technologies, such as organ chips and co-culture strategies, are exciting not only to study the disease mechanisms of rare diseases but also to improve the hepatic maturity of ICOs. Applying the myriad of culture improvement strategies to ICOs will likely produce a fantastic patient-derived hepatocyte model. We have taken the first steps toward improving hepatic maturation of ICOs and in doing so have demonstrated that the ICO application range can be broadened to facilitate the study of (cholestatic) disease mechanisms. Similarly, we anticipate that these improvements in hepatic maturation of ICOs will aid in the development of safe therapeutics for (rare) liver diseases in a personalized manner in the future

Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same

Simple Summary Regenerative medicine using platelet-based blood products or adult stem cells offers the prospect of better clinical outcomes with many diseases. In veterinary medicine, most progress has been made with the development and therapeutic use of these regenerative therapeutics in horses, but the clinical need is given in dogs as well. Our aim was to transfer previous advances in the development of horse regenerative therapeutics, specifically the use of platelet lysate for feeding stem cell cultures, to the dog. Here, we describe the scalable production of canine platelet lysate, which could be used in regenerative biological therapies. We also evaluated the canine platelet lysate for its suitability in feeding canine stem cell cultures in comparison to equine platelet lysate used for equine stem cell cultures. Platelet lysate production from canine blood was successful, but the platelet lysate did not support stem cell culture in dogs in the same beneficial way observed with the equine platelet lysate and stem cells. In conclusion, canine platelet lysate can be produced in large scales as described here, but further research is needed to improve the cultivation of canine stem cells. Abstract Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications

Three-dimensional finite element analysis of occlusal splint and implant connection on stress distribution in implant-supported fixed dental prosthesis and peri-implantal bone

Made available in DSpace on 2019-09-12T16:53:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2017The aim of this study was to investigate the influence of occlusal splint therapy on the stress distribution of implant-supported fixed dental prosthesis (FDP) and peri-implantal bone structures using three-dimensional (3D) finite element analysis (FEA). The system consisted of two implants (positioned on region of second premolar and second molar) as retainers of fixed porcelain-fused-to-metal 3-unit FDP (first molar as pontic element). Two implant connections systems (external and internal hexagon) were tested. Static axial loads simulating functional (100 N) and overloaded (300 N) chewing were applied on the occlusal surface of the FDP covered or not with the occlusal splint. Maximum von Mises stress and bioperformance were accessed for the implants and bone tissues (cortical and cancellous) models. Analytic results indicated that independently of the load condition, implant region, and connection type, the presence of the occlusal splint decreased the stress developed in the implants. Both bone tissues showed increased levels of von Mises stress, and the bioperformance index was far from their maximum yield strength. The internal hexagon implants presented lower stress on premolar and molar regions than external hexagon implants under functional load and overload. The presence of the occlusal splint device over implant-supported FDP may be clinically useful for forwarding stresses towards the bone structure to maintain implants for long term. (C) 2017 Elsevier B.V. All rights reserved.[Marsico, Vivian dos Santos; de Assis Claro, Cristiane Aparecida; Amaral, Marina; Vitti, Rafael Pino; Claro Neves, Ana Christina; da Silva Concilio, Lais Regiane] Universidade de Taubaté (Unitau), Dent Sch, Dept Prosthodont[Lehmann, Roberto Brunow] Fluminense Fed Univ, Dept Elect Engn, Volta Redonda, RJ, Brazi