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The application of cell-based therapeutics requires development of refined and scalable culture processes. Stirred tank bioreactors facilitate growth of human mesenchymal stromal cells (hMSC) while meeting these needs. A process for expansion of hMSCs in a 50 L bioreactor was developed. Parameters evaluated include agitation rate, pH and dissolved oxygen (DO) control set-points, and media formulation. The pH and DO levels were determined empirically in 3 L bioreactors prior to implementation at the 50 L scale. The agitation operating range for microcarrier cultures in the 50 L bioreactor was calculated based on theoretical and empirical analyses of solid suspension and shear limitations. Additionally, small-scale experiments demonstrated that hMSC growth was improved in αMEM supplemented with human platelet lysate in comparison to DMEM supplemented with FBS. A 43-fold expansion of harvested hMSCs was achieved in 11 days in a 50 L bioreactor incorporating these process improvements. Cells expanded in the bioreactor exhibited the expected surface marker expression, trilineage differentiation potential, T cell growth inhibition and indoleamine 2,3-dioxygenase inducibility. The results highlight that identification of optimal pH, DO, agitation rates and culture medium for microcarrier-based bioreactor expansion of adherent cells is paramount to developing a platform to support cell-based therapies.

Several potent, cell permeable 4-aryl-dihydropyrimidinones have been identified as inhibitors of FATP4. Lipophilic ester substituents at the 5-position and substitution at the para-position (optimal groups being -NO(2) and CF(3)) of the 4-aryl group led to active compounds. In two cases racemates were resolved and the S enantiomers shown to have higher potencies.

Background & Aims: Fatty acid transport protein 5 (FATP5/Slc27a5) has been shown to be a multifunctional protein that in vitro increases both uptake of fluorescently labeled long-chain fatty acid (LCFA) analogues and bile acid/coenzyme A ligase activity on overexpression. The aim of this study was to further investigate the diverse roles of FATP5 in vivo. Methods: We studied FATP5 expression and localization in liver of C57BL/6 mice in detail. Furthermore, we created a FATP5 knockout mouse model and characterized changes in hepatic lipid metabolism (this report) and bile metabolism (the accompanying report by Hubbard et al). Results: FATP5 is exclusively expressed by the liver and localized to the basal plasma membrane of hepatocytes, congruent with a role in LCFA uptake from the circulation. Overexpression of FATP5 in mammalian cells increased the uptake of 14 C-oleate. Conversely, FATP5 deletion significantly reduced LCFA uptake by hepatocytes isolated from FATP5 knockout animals. Moreover, FATP5 deletion resulted in lower hepatic triglyceride and free fatty acid content despite increased expression of fatty acid synthetase and also caused a redistribution of lipids from liver to other LCFA-metabolizing tissues. Detailed analysis of the hepatic lipom of FATP5 knockout livers showed quantitative and qualitative alterations in line with a decreased uptake of dietary LCFAs and increased de novo synthesis. Conclusions: Our findings support the hypothesis that efficient hepatocellular uptake of LCFAs, and thus liver lipid homeostasis in general, is largely a protein-mediated process requiring FATP5. These new insights into the physiological role of FATP5 should lead to an improved understanding of liver function and disease.

Fatty acid transport protein-4 (FATP4) is the major FATP in the small intestine. We previously demonstrated, using in vitro antisense experiments, that FATP4 is required for fatty acid uptake into intestinal epithelial cells. To further examine the physiological role of FATP4, mice carrying a targeted deletion of FATP4 were generated. Deletion of one allele of FATP4 resulted in 48% reduction of FATP4 protein levels and a 40% reduction of fatty acid uptake by isolated enterocytes. However, loss of one FATP4 allele did not cause any detectable effects on fat absorption on either a normal or a high fat diet. Deletion of both FATP4 alleles resulted in embryonic lethality as crosses between heterozygous FATP4 parents resulted in no homozygous offspring; furthermore, no homozygous embryos were detected as early as day 9.5 of gestation. Early embryonic lethality has been observed with deletion of other genes involved in lipid absorption in the small intestine, namely microsomal triglyceride transfer protein and apolipoprotein B, and has been attributed to a requirement for fat absorption early in embryonic development across the visceral endoderm. In mice, the extraembryonic endoderm supplies nutrients to the embryo prior to development of a chorioallantoic placenta. In wild-type mice we found that FATP4 protein is highly expressed by the epithelial cells of the visceral endoderm and localized to the brush-border membrane of extraembryonic endodermal cells. This localization is consistent with a role for FATP4 in fat absorption in early embryogenesis and suggests a novel requirement for FATP4 function during development.

While intestinal transport systems for metabolites such as carbohydrates have been well characterized, the molecular mechanisms of fatty acid (FA) transport across the apical plasmalemma of enterocytes have remained largely unclear. Here, we show that FATP4, a member of a large family of FA transport proteins (FATPs), is expressed at high levels on the apical side of mature enterocytes in the small intestine. Further, overexpression of FATP4 in 293 cells facilitates uptake of long chain FAs with the same specificity as enterocytes, while reduction of FATP4 expression in primary enterocytes by antisense oligonucleotides inhibits FA uptake by 50%. This suggests that FATP4 is the principal fatty acid transporter in enterocytes and may constitute a novel target for antiobesity therapy.

Background & Aims: Fatty Acid Transport Protein 5 (FATP5) is a liver-specific member of the FATP/Slc27 family, which has been shown to exhibit both fatty acid transport and bile acid-CoA ligase activity in vitro. Here, we investigate its role in bile acid metabolism and body weight homeostasis in vivo by using a novel FATP5 knockout mouse model. Methods: Bile acid composition was analyzed by mass spectroscopy. Body weight, food intake, energy expenditure, and fat absorption were determined in animals fed either a low- or a high-fat diet. Results: Although total bile acid concentrations were unchanged in bile, liver, urine, and feces of FATP5 knockout mice, the majority of gallbladder bile acids was unconjugated, and only a small percentage was conjugated. Primary, but not secondary, bile acids were detected among the remaining conjugated forms in FATP5 deletion mice, suggesting a specific requirement for FATP5 in reconjugation of bile acids during the enterohepatic recirculation. Fat absorption in FATP5 deletion mice was largely normal, and only a small increase in fecal fat was observed on a high-fat diet. Despite normal fat absorption, FATP5 deletion mice failed to gain weight on a high-fat diet because of both decreased food intake and increased energy expenditure. Conclusions: Our findings reveal an important role for FATP5 in bile acid conjugation in vivo and an unexpected function in body weight homeostasis, which will require further analysis. FATP5 deletion mice provide a new model to study the intersection of bile acid metabolism, lipid metabolism, and body weight regulation.