Effect of inoculum density on human-induced pluripotent stem cell expansion in 3D bioreactors.

OBJECTIVE: For optimized expansion of human-induced pluripotent stem cells (hiPSCs) with regards to clinical applications, we investigated the influence of the inoculum density on the expansion procedure in 3D hollow-fibre bioreactors. MATERIALS AND METHODS: Analytical-scale bioreactors with a cell compartment volume of 3 mL or a large-scale bioreactor with a cell compartment volume of 17 mL were used and inoculated with either 10 × 106 or 50 × 106 hiPSCs. Cells were cultured in bioreactors over 15 days; daily measurements of biochemical parameters were performed. At the end of the experiment, the CellTiter-Blue® Assay was used for culture activity evaluation and cell quantification. Also, cell compartment sections were removed for gene expression and immunohistochemistry analysis. RESULTS: The results revealed significantly higher values for cell metabolism, cell activity and cell yields when using the higher inoculation number, but also a more distinct differentiation. As large inoculation numbers require cost and time-extensive pre-expansion, low inoculation numbers may be used preferably for long-term expansion of hiPSCs. Expansion of hiPSCs in the large-scale bioreactor led to a successful production of 5.4 × 109 hiPSCs, thereby achieving sufficient cell amounts for clinical applications. CONCLUSIONS: In conclusion, the results show a significant effect of the inoculum density on cell expansion, differentiation and production of hiPSCs, emphasizing the importance of the inoculum density for downstream applications of hiPSCs. Furthermore, the bioreactor technology was successfully applied for controlled and scalable production of hiPSCs for clinical use.

Online measurement of oxygen enables continuous noninvasive evaluation of human-induced pluripotent stem cell (hiPSC) culture in a perfused 3D hollow-fiber bioreactor.

For clinical and/or pharmaceutical use of human-induced pluripotent stem cells (hiPSCs), large cell quantities of high quality are demanded. Therefore, we combined the expansion of hiPSCs in closed, perfusion-based 3D bioreactors with noninvasive online monitoring of oxygen as culture control mechanism. Bioreactors with a cell compartment volume of 3 or 17 ml were inoculated with either 10 × 106 or 50 × 106 cells, and cells were expanded over 15 days with online oxygen and offline glucose and lactate measurements being performed. The CellTiter-Blue® Assay was performed at the end of the bioreactor experiments for indirect cell quantification. Model simulations enabled an estimation of cell numbers based on kinetic equations and experimental data during the 15-day bioreactor cultures. Calculated oxygen uptake rates (OUR), glucose consumption rates (GCR), and lactate production rates (LPR) revealed a highly significant correlation (p < 0.0001). Oxygen consumption, which was measured at the beginning and the end of the experiment, showed a strong culture growth in line with the OUR and GCR data. Furthermore, the yield coefficient of lactate from glucose and the OUR to GCR ratio revealed a shift from nonoxidative to oxidative metabolism. The presented results indicate that oxygen is equally as applicable as parameter for hiPSC expansion as glucose while providing an accurate real-time impression of hiPSC culture development. Additionally, oxygen measurements inform about the metabolic state of the cells. Thus, the use of oxygen online monitoring for culture control facilitates the translation of hiPSC use to the clinical setting.

Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches.

The proportions of body fat and fat-free mass are determining factors of adiposity-associated diseases. Work in Caenorhabditis elegans has revealed evolutionarily conserved pathways of fat metabolism. Nevertheless, analysis of body composition and fat distribution in the nematodes has only been partially unraveled because of methodological difficulties. We characterized metabolic C. elegans mutants by using novel and feasible BODIPY 493/503-based fat staining and flow cytometry approaches. Fixative as well as vital BODIPY staining procedures visualize major fat stores, preserve native lipid droplet morphology, and allow quantification of fat content per body volume of individual worms. Colocalization studies using coherent anti-Stokes Raman scattering microscopy, Raman microspectroscopy, and imaging of lysosome-related organelles as well as biochemical measurement confirm our approaches. We found that the fat-to-volume ratio of dietary restriction, TGF-ß, and germline mutants are specific for each strain. In contrast, the proportion of fat-free mass is constant between the mutants, although their volumes differ by a factor of 3. Our approaches enable sensitive, accurate, and high-throughput assessment of adiposity in large C. elegans populations at a single-worm level.

Association analysis between the prostaglandin E synthase 2 R298H polymorphism and body mass index in 8079 participants of the KORA study cohort.

CONTEXT: The H-allele of the R298H polymorphism in the prostaglandin E synthase 2 (PTGES2) gene was associated with lower risk of diabetes type 2. AIM: To explore the association between the PTGES2 R298H SNP and body mass index (BMI). METHODS: We analyzed the R298H SNP (rs13283456) and three haplotype single-nucleotide polymorphisms (rs884115, rs10987883, and rs4837240) covering a 20 kb gene region in population-based surveys of the Kooperative Gesundheitsforschung in der Region Augsburg study cohort with 8079 participants. RESULTS: A statistically significant difference in BMI between the heterozygous PTGES2 R298H genotype and the homozygous R/R genotype was found in males but not in females. Males with the R/H genotype showed a decrease in BMI of -0.30 BMI units (95% CI: -0.55, -0.04, p = 0.02) in comparison to R/R males. A haplotype comprising the minor allele of PTGES2 R298H showed a significant decrease of -0.23 BMI units in males (-0.45, -0.02; p = 0.04) but not in females. Other haplotypes and haplotype single-nucleotide polymorphisms were not significantly associated with BMI. CONCLUSION: We found a marginal but significant influence of the PTGES2 298H SNP on BMI in a large population-based study.

Association between functional FABP2 promoter haplotypes and body mass index: analyses of 8072 participants of the KORA cohort study.

Studies in relatively small cohorts provide preliminary evidence that functional fatty acid binding protein 2 (FABP2) promoter haplotypes are associated with type 2 diabetes and BMI. Here, we studied the influence of the haplotypes on BMI by using 8072 male and female participants of the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) cohort. By linear regression analysis, we found in males a reduction of -0.39 BMI units (95% CI: -0.73, -0.05, p = 0.024) in homozygous FABP2 promoter haplotype B carriers. Carriers of haplotype B showed a significant decrease in BMI of -0.19 BMI units (95% CI: -0.35, -0.02, p = 0.027). In accordance, a significant reduction in BMI of the minor haplotype carriers in the BMI point categories of 25-30 (BMI units: -0.10, 95% CI: -0.18, -0.01, p = 0.03) and < 30 (BMI units: -0.37, 95% CI: -0.67, -0.07, p = 0.02) were found. In summary, the minor FABP2 promoter haplotype B contributes to a reduced BMI in men. This provides evidence that functional FABP2 contributes to multifactorialy regulated body weight.

MTTP variants and body mass index, waist circumference and serum cholesterol level: Association analyses in 7582 participants of the KORA study cohort.

The microsomal triglyceride transfer protein (MTTP) is a key regulator in the assembly and secretion of chylomicrons and very low density lipoprotein (VLDL) in the intestine and in liver. Associations between MTTP variants and traits of the metabolic syndrome are carried out in relatively small cohorts and are not consistent. We analysed MTTP polymorphisms in 7582 participants of the KORA study cohort. Seven htSNPs covering a 52kb region of the MTTP locus and two cSNPs (I128T, H297Q) were selected. A MTTP haplotype containing the minor allele of H297Q showed a significant decrease of -0.636 (95% CI: -1.226, -0.046; p=0.035) BMI units in females but not in males. In comparison to homozygous H-carriers for the major allele of the MTTP H297Q polymorphism, homozygous Q297Q carriers showed a significant decrease in BMI of -0.425B MI units (95% CI: -0.74, -0.12; p=0.007), in waist circumference of -0.990 cm (95% CI: 1.74, -0.24; p=0.01) and in total cholesterol of -0.039 mmol/l (95% CI: -0.07, 0; p=0.03). Heterozygous Q-carriers displayed a reduction in BMI of -0.183 BMI unit (95% CI: -0.33, -0.04; p=0.012), in waist circumference of -0.45 cm (95% CI: 0.8, -0.1; p=0.01) and in total cholesterol of -0.103 mmol/l (95% CI: -0.18, -0.03; p=0.01). Gender stratified statistics revealed a significant reduction of -0.657 BMI units (95% CI: -1.14, -0.18; p=0.007), -1.437 cm waist circumference (95% CI: -2.55, -0.32; p=0.01) and -0.052 mmol/l total cholesterol (95% CI: -0.1, -0.01; p=0.03) for females homozygous for the Q297Q polymorphism. Females carrying the Q-allele showed a decrease of -0.259 BMI unit (95% CI: -0.48, -0.04; p=0.023), -0.662 cm waist circumference (95% CI: -1.18, -0.14; p=0.01) and -0.111 mmol/l total cholesterol (95% CI: -0.21, -0.01; p=0.03). Our association analysis in a large population based study cohort provides evidence that the minor allele of the MTTP H297Q polymorphism is associated with lower BMI, waist circumference and total cholesterol in females but not in males.

Nutrition concepts for elite distance runners based on macronutrient and energy expenditure.

CONTEXT: Elite distance runners (EDR) must optimize their nutrition to maintain their demanding training schedules. OBJECTIVE: To develop a nutrition concept for EDR based on energy and macronutrient expenditures. DESIGN: This theoretical study provides calculations for macronutrient and energy expenditures of EDR. Anthropometric and metabolic characteristics of EDR were assumed based on average real EDR. SETTING: University of Kiel. PATIENTS OR OTHER PARTICIPANTS: Three prototypic types of male EDR described in the literature as type I (TI; body mass = 72 kg, respiratory quotient = 0.9 at rest, fast-twitch muscle fibers = 60% to 70%), type II (TII; body mass = 67 kg, respiratory quotient = 0.82 at rest, fast-twitch muscle fibers = 50%), and type III (TIII; body mass = 60 kg, respiratory quotient = 0.75 at rest, fast-twitch muscle fibers = 30% to 40%). MAIN OUTCOME MEASURE(S): We calculated the macronutrient and energy expenditures of the 3 types of EDR according to body mass, respiratory quotient, and percentage of fast-twitch muscle fibers. RESULTS: We found that the average energy expenditure was 3750 kcal . d(-1) for TI runners, 3463 kcal . d(-1) for TII runners, and 3079 kcal . d(-1) for TIII runners. The carbohydrate (CHO) expenditure reached an average value of 10.0 g . kg(-1) . d(-1) for TI runners, 8.0 g . kg(-1) . d(-1) for TII runners, and 4.7 g . kg(-1) . d(-1) for TIII runners. When the EDR accomplished running sessions at a pace >or=100% of maximum oxygen consumption, all types of runners had a CHO demand of about 10 g . kg(-1) . d(-1). The TI and TII runners need a CHO intake of 8 to 10 g . kg(-1) . d(-1). For the TIII runners, a CHO intake >6 g . kg(-1) . d(-1) is necessary during anaerobic training sessions. CONCLUSIONS: Nutrition concepts must be differentiated for EDR according to metabolic and anthropometric characteristics of the runners and their special training emphases.

Analysis of the transcriptional regulation of the FABP2 promoter haplotypes by PPARgamma/RXRalpha and Oct-1.

Variants of the human intestinal fatty acid binding protein 2 gene (FABP2) are associated with traits of the metabolic syndrome. Relevant FABP2 promoter polymorphisms c.-80_-79insT, c.-136_-132delAGTAG, c.-168_-166delAAGinsT, c.-260G>A, c.-471G>A, and c.-778G>T result in two haplotypes A and B. Activation of haplotypes by rosiglitazone stimulated PPARgamma/RXRalpha leads to 2-fold higher activity of haplotype B than A. As shown by chimeric FABP2 promoter constructs, the higher responsiveness of FABP2 haplotype B is mainly but not solely determined by polymorphism c.-471G>A. As shown by EMSA and promoter-reporter assays, Oct-1 interacts with the -471 region of FABP2 promoters, induces the activities of both FABP2 promoter haplotypes and abolishes the different activities of haplotypes induced by rosiglitazone activated PPARgamma/RXRalpha. In conclusion, our findings suggest a functional role of PPARgamma/RXRalpha and Oct-1 in the regulation of the FABP2 gene.

Transcriptional regulation of the fatty acid binding protein 2 (FABP2) gene by the hepatic nuclear factor 1 alpha (HNF-1alpha).

The human fatty acid binding protein (FABP2) is involved in intestinal absorption and intracellular trafficking of long-chain fatty acids. Here we investigate transcriptional regulation of FABP2 by the endodermal hepatic nuclear factor 1 alpha (HNF-1alpha). In electromobility shift and supershift assays we show the presence of two adjacent HNF-1alpha binding sites within the FABP2 promoter regions -185 to -165 and -169 to -149. HNF-1alpha activates an FABP2 promoter luciferase construct by 3.5 and 20-fold in Caco-2 and Hela cells, respectively. Mutational analysis of HNF-1alpha elements resulted in about 50% reduction of basal and HNF-1alpha induced activity of FABP2 promoter constructs, predominantly caused by deletion of the -185 to -165 site. Thus, our data suggest a major role of HNF-1alpha in control of FABP2 expression in intestine via a functional HNF-1alpha recognition element within FABP2 promoter region -185 to -165.

Type 2 diabetes-associated fatty acid binding protein 2 promoter haplotypes are differentially regulated by GATA factors.

The human intestinal fatty acid binding protein 2 (FABP2) mediates fat absorption by binding and intracellular trafficking of long-chain free fatty acids. Studies with knockout mice and association analysis of polymorphisms revealed that FABP2 is a susceptibility gene for type 2 diabetes (noninsulin dependent diabetes mellitus [NIDDM]) and related traits. Relevant FABP2 promoter polymorphisms c.-80_-79insT (rs5861422), c.-136_-132delAGTAG (rs5861423), c.-168_-166delAAGinsT (rs1973598), c.-260G>A (rs6857641), c.-471G>A (rs2282688), and c.-778G>T (rs10034579) result in two haplotypes A and B, whereby B possesses two- to three-fold lower transcriptional activity than A. We show in luciferase reporter gene assays by a series of chimeric FABP2 promoter constructs in intestinal Caco-2 cells that polymorphism c.-80_-79insT essentially determines different activities of the FABP2 promoter. In accordance, in electrophoretic mobility shift assays (EMSAs), transcriptional factors GATA-5 and -6 bind with higher binding affinities to the FABP2 promoter region containing the -80A allele compared to B. As functional consequence, haplotype A is twice as much more activated by GATA factors than haplotype B in liver Huh7 cells. Additionally, a construct bearing the -80B allele in the background of haplotype A reversed the activity from A to B. Thus, the GATA mediated differential activation of FABP2 haplotypes depends on polymorphism c.-80_-79insT. This provides the molecular basis for the variant specific transcriptional regulation of the diabetes type 2-associated FABP2 gene.

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4alpha.

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4alpha (HNF-4alpha), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4alpha binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4alpha by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4alpha, that are both candidate genes for diabetes type 2, may be a powerful approach.