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La edad es un factor de riesgo arrítmico que, en especies animales, está asociado a la remodelación del colágeno. En este trabajo caracterizamos la dinámica tisular del colágeno asociada a la edad en el ventrículo izquierdo humano y utilizamos estos resultados para simular su efecto sobre la generación de arritmias ventriculares.

La desregulación de microRNAs (miRNAs) se asocia con múltiples procesos incluidos el envejecimiento cardiaco y las enfermedades cardiovasculares (ECV). Por ello, se posicionan como prometedoras dianas terapéuticas. Para superar limitaciones en su uso como tal, se propone la creación de nanoestructuras de DNA (DNS) biocompatibles para crear terapias efectivas y seguras.

Para reducir el impacto del fracaso fármacológico debido a arritmias inesperadas, se apuesta por el uso temprano de modelos humanos. La edad, factor proarrítmico, está todavía desatendido en estas iniciativas. Aquí generamos modelos computacionales de electrofisiología cardiaca humana que consideran sus efectos para mejorar la estratificación del riesgo arrítmico farmacológico.

Pese a la relevancia de la edad como factor de riesgo de las enfermedades cardiovasculares, sus bases moleculares y funcionales están poco caracterizadas en humanos. Para mejorar esta comprensión hemos estudiado su dinámica transcripcional cronológica y biológica en ventrículo izquierdo humano y hemos desarrollado un modelo de envejecimiento celular cardiaco.

Simple Summary Prion diseases are neurodegenerative disorders affecting humans and animals. The development of in vitro cellular models from naturally susceptible species like humans or ruminants can potentially make a great contribution to the study of many aspects of these diseases, including the ability of prions to infect and replicate in cells and therapeutics. Our study shows for the first time how ovine mesenchymal stem cells derived from bone marrow and their neural-like progeny are able to react to scrapie prion infection in vitro and assesses the effects of this infection on cell viability and proliferation. Finally, we observe that the differentiation of ovine mesenchymal stem cells into neuron-like cells makes them more permissive to prion infection. Abstract Scrapie is a prion disease affecting sheep and goats and it is considered a prototype of transmissible spongiform encephalopathies (TSEs). Mesenchymal stem cells (MSCs) have been proposed as candidates for developing in vitro models of prion diseases. Murine MSCs are able to propagate prions after previous mouse-adaptation of prion strains and, although ovine MSCs express the cellular prion protein (PrPC), their susceptibility to prion infection has never been investigated. Here, we analyze the potential of ovine bone marrow-derived MSCs (oBM-MSCs), in growth and neurogenic conditions, to be infected by natural scrapie and propagate prion particles (PrPSc) in vitro, as well as the effect of this infection on cell viability and proliferation. Cultures were kept for 48–72 h in contact with homogenates of central nervous system (CNS) samples from scrapie or control sheep. In growth conditions, oBM-MSCs initially maintained detectable levels of PrPSc post-inoculation, as determined by Western blotting and ELISA. However, the PrPSc signal weakened and was lost over time. oBM-MSCs infected with scrapie displayed lower cell doubling and higher doubling times than those infected with control inocula. On the other hand, in neurogenic conditions, oBM-MSCs not only maintained detectable levels of PrPSc post-inoculation, as determined by ELISA, but this PrPSc signal also increased progressively over time. Finally, inoculation with CNS extracts seems to induce the proliferation of oBM-MSCs in both growth and neurogenic conditions. Our results suggest that oBM-MSCs respond to prion infection by decreasing their proliferation capacity and thus might not be permissive to prion replication, whereas ovine MSC-derived neuron-like cells seem to maintain and replicate PrPSc.

INTRODUCTION: Murine microglia expressing the Alzheimer's disease-linked TREM2R47H mutation display variable decrease in phagocytosis, while impaired phagocytosis is reported following loss of TREM2. However, no data exist on TREM2+/R47H human microglia. Therefore, we created human pluripotent stem cell (hPSC) monocytes and transdifferentiated microglia-like cells (tMGs) to examine the effect of the TREM2+/R47H mutation and loss of TREM2 on phagocytosis. METHODS: We generated isogenic TREM2+/R47H, TREM2+/-, and TREM2-/- hPSCs using CRISPR/Cas9. Following differentiation to monocytes and tMGs, we studied the uptake of Escherichia coli fragments and analyzed amyloid plaque clearance from cryosections of APP/PS1+/- mouse brains. RESULTS: We demonstrated that tMGs resemble cultured human microglia. TREM2+/- and TREM2-/- hPSC monocytes and tMGs phagocytosed significantly less E. coli fragments and cleared less amyloid plaques than wild-type hPSC progeny, with no difference for TREM2+/R47H progeny. DISCUSSION: In vitro phagocytosis of hPSC monocytes and tMGs was not affected by the TREM2+/R47H mutation but was significantly impaired in TREM2+/- and TREM2-/- progeny. ispartof: ALZHEIMERS & DEMENTIA vol:15 issue:3 pages:453-464 ispartof: location:United States status: published

Cardiac tissue slices preserve the heterogeneous structure and multicellularity of the myocardium and allow its functional characterization. However, access to human ventricular samples is scarce. We aim to demonstrate that slices from small transmural core biopsies collected from living donors during routine cardiac surgery preserve structural and functional properties of larger myocardial specimens, allowing accurate electrophysiological characterization. In pigs, we compared left ventricular transmural core biopsies with transmural tissue blocks from the same ventricular region. In humans, we analyzed transmural biopsies and papillary muscles from living donors. All tissues were vibratomesliced. By histological analysis of the transmural biopsies, we showed that tissue architecture and cellular organization were preserved. Enzymatic and vital staining methods verifed viability. Optically mapped transmembrane potentials confrmed that action potential duration and morphology were similar in pig biopsies and tissue blocks. Action potential morphology and duration in human biopsies and papillary muscles agreed with published ranges. In both pigs and humans, responses to increasing pacing frequencies and β-adrenergic stimulation were similar in transmural biopsies and larger tissues. We show that it is possible to successfully collect and characterize tissue slices from human myocardial biopsies routinely extracted from living donors, whose behavior mimics that of larger myocardial preparations both structurally and electrophysiologically. Fil: Oliván Viguera, Aida. Universidad de Zaragoza; España Fil: Pérez Zabalza, María. Universidad de Zaragoza; España Fil: García Mendívil, Laura. Universidad de Zaragoza; España Fil: Mountris, Konstantinos A.. Universidad de Zaragoza; España Fil: Orós Rodrigo, Sofía. Universidad de Zaragoza; España Fil: Ramos Marquès, Estel. Universidad de Zaragoza; España Fil: Vallejo Gil, José María. University Hospital Miguel Servet; España Fil: Fresneda Roldán, Pedro Carlos. University Hospital Miguel Servet; España Fil: Fañanás Mastral, Javier. University Hospital Miguel Servet; España Fil: Vázquez Sancho, Manuel. University Hospital Miguel Servet; España Fil: Matamala Adell, Marta. University Hospital Miguel Servet; España Fil: Sorribas Berjón, Fernando. University Hospital Miguel Servet; España Fil: Bellido Morales, Javier André. University Hospital Miguel Servet; España Fil: Mancebón Sierra, Francisco Javier. University Hospital Miguel Servet; España Fil: Vaca Núñez, Alexánder Sebastián. University Hospital Miguel Servet; España Fil: Ballester Cuenca, Carlos. University Hospital Miguel Servet; España Fil: Marigil, Miguel Ángel. Hospital San Jorge; España Fil: Pastor, Cristina. Aragón Institute of Health Sciences; España Fil: Ordovás, Laura. Aragón Agency for Research and Development; España. Universidad de Zaragoza; España Fil: Köhler, Ralf. Aragón Institute of Health Sciences; España. Aragón Agency for Research and Development; España Fil: Diez, Emiliano Raúl. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Cátedra de Fisiología Humana Normal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Pueyo, Esther. Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina; España. Universidad de Zaragoza; España

Cardiomyocytes&rsquo, geometry and connexin 43 (CX43) amount and distribution are structural features that play a pivotal role in electrical conduction. Their quantitative assessment is of high interest in the study of arrhythmias, but it is usually hampered by the lack of automatic tools. In this work, we propose a software algorithm (Myocyte Automatic Retrieval and Tissue Analyzer, MARTA) to automatically detect myocytes from fluorescent microscopy images of cardiac tissue, measure their morphological features and evaluate the expression of CX43 and its degree of lateralization. The proposed software is based on the generation of cell masks, contouring of individual cells, enclosing of cells in minimum area rectangles and splitting of these rectangles into end-to-end and middle compartments to estimate CX43 lateral-to-total ratio. Application to human ventricular tissue images shows that mean differences between automatic and manual methods in terms of cardiomyocyte length and width are below 4 &mu, m. The percentage of lateral CX43 also agrees between automatic and manual evaluation, with the interquartile range approximately covering from 3% to 30% in both cases. MARTA is not limited by fiber orientation and has an optimized speed by using contour filtering, which makes it run hundreds of times faster than a trained expert. Developed for CX43 studies in the left ventricle, MARTA is a flexible tool applicable to morphometric and lateralization studies of other markers in any heart chamber or even skeletal muscle. This open-access software is available online.

INTRODUCTION: Tauopathies are neurodegenerative diseases characterized by TAU protein-related pathology, including frontotemporal dementia and Alzheimer's disease among others. Mutant TAU animal models are available, but none of them faithfully recapitulates human pathology and are not suitable for drug screening. METHODS: To create a new in vitro tauopathy model, we generated a footprint-free triple MAPT-mutant human induced pluripotent stem cell line (N279K, P301L, and E10+16 mutations) using clustered regularly interspaced short palindromic repeats-FokI and piggyBac transposase technology. RESULTS: Mutant neurons expressed pathogenic 4R and phosphorylated TAU, endogenously triggered TAU aggregation, and had increased electrophysiological activity. TAU-mutant cells presented deficiencies in neurite outgrowth, aberrant sequence of differentiation to cortical neurons, and a significant activation of stress response pathways. RNA sequencing confirmed stress activation, demonstrated a shift toward GABAergic identity, and an upregulation of neurodegenerative pathways. DISCUSSION: In summary, we generated a novel in vitro human induced pluripotent stem cell TAU-mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening. ispartof: ALZHEIMERS & DEMENTIA vol:14 issue:10 pages:1261-1280 ispartof: location:United States status: published

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)–mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs., Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.

Available online The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.scr.2020.101763 . The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal .

Summary Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes., Highlights • RMCE using positive/negative selection allows generation of transgenic hPSC lines in 15 days • The AAVS1 locus exerts transgene inhibition by inducing de novo DNA methylation • AAVS1-mediated silencing appears sequence and lineage-specific in vitro, yet is not present in vivo • AAVS1 does not meet the requirements to be considered a universal safe harbor locus in vitro, Verfaillie, Ordovás, and colleagues report an efficient and fast method to genetically engineer hPSC in the AAVS1 safe harbor locus free of random integration events. Their results demonstrate the suitability of the locus for several applications to study the hepatic lineage, but they uncover a so-far-unknown variable AAVS1-mediated transgene inhibition in undifferentiated and hepatocyte differentiating hESC. Therefore, AAVS1 cannot be considered a safe harbor locus.

Summary To understand how haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), we created induced pluripotent stem cells (iPSCs) from patients carrying the GRNIVS1+5G > C mutation (FTD-iPSCs). FTD-iPSCs were fated to cortical neurons, the cells most affected in FTD. Although generation of neuroprogenitors was unaffected, their further differentiation into CTIP2-, FOXP2-, or TBR1-TUJ1 double-positive cortical neurons, but not motorneurons, was significantly decreased in FTD-neural progeny. Zinc finger nuclease-mediated introduction of GRN cDNA into the AAVS1 locus corrected defects in cortical neurogenesis, demonstrating that PGRN haploinsufficiency causes inefficient cortical neuron generation. RNA sequencing analysis confirmed reversal of the altered gene expression profile following genetic correction. We identified the Wnt signaling pathway as one of the top defective pathways in FTD-iPSC-derived neurons, which was reversed following genetic correction. Differentiation of FTD-iPSCs in the presence of a WNT inhibitor mitigated defective corticogenesis. Therefore, we demonstrate that PGRN haploinsufficiency hampers corticogenesis in vitro., Graphical Abstract, Highlights • In vitro generation of cortical neurons from PGRN deficient FTD-iPSC is inefficient • Incorporation of GRN cDNA via ZFN technology rescues cortical neurons generation • WNT signaling may also play a major role in the defects observed, Verfaillie and colleagues describe the inefficient cortical neuron, but not motorneuron, generation, from FTD-patient-derived iPSCs carrying a mutation in the GRN gene. They show restoration of the defective phenotype following introduction of the GRN cDNA in FTD-iPSC using zinc finger nucleases and by inhibiting the WNT pathway.

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging.

Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process. ispartof: PLoS One vol:12 issue:11 pages:0186884- ispartof: location:United States status: published

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Knowledge about the 5′ regulatory region variation of candidate genes may help to improve breeding and preserve genetic diversity in ruminant species and reveal insights about their evolutionary history. Cis-regulatory regions of candidate genes involved in lipid metabolism and related to energy homeostasis were sequenced in 3 Bos taurus breeds and 1 Bos indicus breed to identify potentially functional polymorphism in non-coding regions. Searching for promoter variability in Fatty Acid Synthase (FASN), Stearoyl-CoADesaturase (SCD), Perilipin (PLIN), Glycerol-3 phosphate acyltransferase mitochondrial (GPAM) and Melanocortin-4 receptor (MC4R), 42 polymorphisms were found. Forty one of them were reported for the first time in B. taurus and B. indicus species. Through in silico analysis we noticed that many of them fall within putative binding sites for transcription factors closely involved in the regulation of the expression of adipogenic genes. A higher nucleotide diversity was found in B. indicus for FASN, SCD, PLIN and MC4R, whereas GPAM was the only more polymorphic promoter in B. taurus. The lower diversity in the B. taurus breeds may reflect their population history with a stronger selection pressure for some economic and productive traits. Alternatively, the ancestors of the indicine breed may have had a high genetic diversity. This research underlines the significance of local breeds for preserving genetic diversity for a sustainable agriculture. The domestication history of ruminant species and the critical role of the analyzed genes in lipid metabolic processes, make these putatively regulatory polymorphisms important targets in breeding and in genetic diversity studies.

Controlling meat traceability using SNPs is an effective method of ensuring food safety. We have analyzed several SNPs to create a panel for bovine genetic identification and traceability studies. One of these was the transversion g.329CT (Genbank accession no. AJ496781) on the cytochrome P450 17A1 gene, which has been included in previously published panels. Using minisequencing reactions, we have tested 701 samples belonging to eight Spanish cattle breeds. Surprisingly, an excess of heterozygotes was detected, implying an extreme departure from Hardy-Weinberg equilibrium (P0.001). By alignment analysis and sequencing, we detected that the g.329CT SNP is a false positive polymorphism, which allows us to explain the inflated heterozygotic value. We recommend that this ambiguous SNP, as well as other polymorphisms located in this region, should not be used in identification, traceability or disease association studies. Annotation of these false SNPs should improve association studies and avoid misinterpretations.

Summary Reasons for performing study: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. Objectives: To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. Methods: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. Results: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. Conclusions: Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. Potential relevance: To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.

We utilised DNA analysis to detect the presence of the digenean Phyllodistomum folium in three cyprinid species, Scardinius erythrophthalmus, Cyprinus carpio and Rutilus rutilus. DNA sequencing of the region containing the genes ITS1-5.8S-ITS2 revealed 100% sequence identity between DNA from the sporocysts found in zebra mussels and DNA from adults located in the urinary system of 29 cyprinid fish. A second genetically different (variation=1.6%) sequence was observed in two samples from R. rutilus. In our opinion, the existence of a complex of species reported as P. folium is supported by recent genetic studies, including our own results. The overall prevalence of P. folium in mussels from the Ebro River was 4.67% in 2006, although during the summer months the rates frequently exceeded 10%.

On the basis of QTL studies for milk-fat yield trait on BTA3, annexin 9 protein (ANXA9), fatty acid transport protein type 3 (SLC27A3) and diacylglycerol O-acyltransferase 1 (DGAT1) were selected as candidate genes. Three different single nucleotide polymorphisms (SNPs) of bovine ANXA9, SLC27A3 and DGAT1 genes have been tested in a selective genotyping design for milk-fat yield. Significant allele frequency differences were found for ANXA9 (p=0.02), in Holstein-Friesian animals with high and low breeding values for milk-fat yield. Regression analysis also showed a significant effect (p=0.0207) between estimated breeding values (EBVs) for fat milk content and ANXA9 polymorphism. So ANXA9 gene falls into a significant quantitative trait loci interval for milk-fat yield that was previously reported on bovine chromosome 3 in other dairy populations. Our results suggest that the ANXA9 gene polymorphism or a linked segregating QTL contributes to variation in milk-fat yield. © 2009 Elsevier Ltd.

The bovine transferrin gene (TF) is located at 125 cM on bovine chromosome 1 (BTA1); it codes for transferrin, a glycoprotein that is highly conserved in many species and that is responsible for iron transport. The TF gene has been located in several QTL regions, and some transferrin classes have been associated with fat and milk yields. We analyzed by means of allele-specific oligonucleotide real-time PCR the c.1455A>G SNP in exon 12 of the TF cDNA sequence (accession number U02564), which induces an Asp/Gly substitution at position 469 of the peptide. The c.1455A>G SNP was assayed in eight Spanish cattle breeds, as well as in two groups of Holstein-Friesian animals that had the highest and lowest estimated breeding values for milk fat yield. Analysis of the cSNP showed balanced frequencies in all breeds, with a mean of 0.44. Evaluation of a potential association between the cSNP and the groups of Holstein-Friesian animals selected for milk fat yield showed a significant association (P < 0.0006); the G allele was associated with high fat production. Significant differences in genotypic frequencies between the groups were also detected (P < 0.0028). These results lead us to suggest that the TF gene has an effect on milk fat yield.

The choice of housekeeping proteins or genes for internal standards should be made carefully, taking into account the cell and tissue type, the experimental conditions, and the healthy/disease state(s) under consideration. Furthermore, as the correlation between transcriptional and translational levels of commonly used housekeeping genes is often discussed, this study shed light on the transcriptional levels of beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the translational levels of beta-actin, GAPDH, and beta-tubulin in an amyotrophic lateral sclerosis mouse model.

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5′ flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

Fatty acid synthase (FASN) is a multifunctional protein that carries out the synthesis of fatty acids so it plays a central role in de novo lipogenesis in mammals. Previously, we defined the genetic structure and expression of the bovine FASN gene. Our mapping studies placed FASN on BTA19 (19q22) where several quantitative trait loci (QTL) affecting milk-fat content and related traits have been described. This study was conducted to identify polymorphisms in the bovine FASN gene and to study their association with milk-fat content. The bovine FASN gene was screened for polymorphisms in two cattle breeds. Sequence analysis revealed several single nucleotide polymorphisms (SNPs), and two of them were analysed: a G>C substitution in the untranslated exon 1 (g.763G>C), altering a potential Sp1 transcription factor-binding site, and an A>G substitution in exon 34 (g.16009A>G), which determines a non-conservative substitution of threonine by alanine. Allele-specific amplification of the SNPs in FASN revealed significant frequency differences for both polymorphisms in Holsteins with high and low breeding values for milk-fat content. The intragenic haplotypes comprising exon 1 (alleles G and C) and exon 34 (alleles A and G) polymorphisms were studied, and the existence of linkage disequilibrium between these SNPs was found (D(CG) = 0.048, P < 0.001). Our results suggest that the FASN gene polymorphisms contribute to variation in milk-fat content. We propose that the bovine FASN gene is a candidate gene for a milk-fat content QTL.

The Solute Carrier Family 27 Member 1 (SLC27A1) is an evolutionarily conserved protein involved in regulating the long chain fatty acid uptake into cells. It has been shown to be expressed in tissues undergoing rapid fatty acid metabolism such as heart, skeletal muscle and adipose tissues, but no expression is detected in liver. Here we report the molecular characterization of the bovine SLC27A1 gene and draw a comparison with orthologous genes of some monogastric species. The bovine SLC27A1 gene is organized in 13 exons and extends over more than 40 kb of genomic DNA. It codes for a protein of 646 amino acids with a predicted molecular weight of 71 kDa which has 92%, 88% and 88% similarity with the human, mouse and rat SLC27A1 proteins respectively. The bovine SLC27A1 RNA expression was high in heart, testis, nervous tissue and muscle and very low in liver. Surprisingly, adipose tissues showed very low RNA expression levels contrary to the results described for both human and mouse genes. On the other hand, discordances observed between the bovine SLC27A1 RNA and protein expression patterns suggest that complex regulation mechanisms may be involved in determining the final SLC27A1 protein levels in each tissue. Finally, we have identified an alternative transcript generated by exon skipping of exon 3 to 7 which could encode a cytosolic SLC27A1 isoform of ∼37 kDa.

GPAM maps in BTA26q22, where several QTLs affecting milk production, milk fat and protein content have been mapped. On the basis of the QTL location, the GPAM gene could be considered a good candidate gene for the mentioned traits. Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is the enzyme that catalyses the initial and committed step of glycerolipid synthesis and, therefore, it is a potential site for triacylglycerol synthesis regulation. In this study, the structure of the cDNA and the genomic DNA of the bovine GPAM gene were determined and the expression of its mRNA was studied. The cDNA of the gene was cloned by RT-PCR, 5′ and 3′ rapid amplification of cDNA ends. The GPAM mRNA sequence contains a 2,475-bp coding region and a 3,689-bp 3′ UTR. Its ORF encoded for an 825-amino acid protein and has an 89% homology with the coding regions of previously characterized mouse and human GPAM genes. The predicted amino acid sequence had an 89 and 93% similarity with mouse and human GPAM proteins, respectively. Using a 5′ RACE strategy, two different 5′ UTRs were cloned. Northern blot analysis confirmed the presence of two different transcripts. Adipose tissues and lung had the highest levels of GPAM mRNA expression, whereas it was barely detectable in liver. This expression pattern differs with those of non-ruminant animals where liver is one of the tissues with higher GPAM mRNA expression level.

Pancreatic endocrine progenitors obtained from human embryonic stem cells (hESCs) represent a promising source to develop cell-based therapies for diabetes. Although endocrine pancreas progenitor cells have been isolated from mouse pancreata on the basis of Ngn3 expression, human endocrine progenitors have not been isolated yet. As substantial differences exist between human and murine pancreas biology, we investigated whether it is possible to isolate pancreatic endocrine progenitors from differentiating hESC cultures by lineage tracing of NGN3. We targeted the 3' end of NGN3 using zinc finger nuclease-mediated homologous recombination to allow selection of NGN3eGFP(+) cells without disrupting the coding sequence of the gene. Isolated NGN3eGFP(+) cells express PDX1, NKX6.1, and chromogranin A and differentiate in vivo toward insulin, glucagon, and somatostatin single hormone-expressing cells but not to ductal or exocrine pancreatic cells or other endodermal, mesodermal, or ectodermal lineages. This confirms that NGN3(+) cells represent pancreatic endocrine progenitors in humans. In addition, this hESC reporter line constitutes a unique tool that may aid in gaining insight into the developmental mechanisms underlying fate choices in human pancreas and in developing cell-based therapies. ispartof: Stem Cells Translational Medicine vol:3 issue:4 pages:489-499 ispartof: location:England status: published

Translation of stem cell research to industrial and clinical settings mostly requires large quantities of cells, especially those involving large organs such as the liver. A scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiated cells. To increase the culture efficiency in bioreactor system, high surface to volume ratio needs to be achieved. We employed a microcarrier culture system for the expansion of undifferentiated human embryonic stem cells (hESCs) as well as for directed differentiation of these cells to hepatocyte-like cells. Cells in single cell suspension were attached to the bead surface in even distribution and were expanded to 1×10(6)cells/ml within 2 days of hESC culture with maintenance of the level of pluripotency markers. Directed differentiation into hepatocyte-like cells on microcarriers, both in static culture and stirred bioreactors, induced similar levels of hepatocyte-like cell differentiation as observed with cells cultured in conventional tissue culture plates. The cells expressed both immature and mature hepatocyte-lineage genes and proteins such as asialoglycoprotein receptor-1 (ASGPR-1) and albumin. Differentiated cells exhibited functional characteristics such as secretion of albumin and urea, and CYP3A4 activity could be detected. Microcarriers thus offer the potential for large-scale expansion and differentiation of hESCs induced hepatocyte-like cells in a more controllable bioreactor environment. ispartof: Journal of biotechnology vol:174 issue:1 pages:39-48 ispartof: location:Netherlands status: published

Human hepatocytes, suitable for treatment of patients with liver failure, for the creation of bioartificial (BAL) devices, or for studies for toxicity and metabolization studies in the pharmaceutical industry, are in short supply due to the lack of donor organs. Therefore, methods that allow ex vivo expansion of hepatocytes with mature function are being pursued. One cell source, believed to be a possible inexhaustible source of hepatocytes, is pluripotent stem cells (PSCs). However, directed differentiation of PSCs to cells with features of adult hepatocytes is not yet possible. Differentiated progeny remains mixed and PSC progeny does not have a number of the functional features of mature hepatocytes. In this review article, we will address tools being developed that allow for the identification of mature hepatocytes, in a non-invasive manner; to perform lineage tracing of PSC progeny; and novel culture systems being created for the in vitro differentiation of PSCs to hepatocyte like cells, and for the maintenance of primary liver derived hepatocytes or PSC-derived hepatic progeny in culture. As conventional two-dimensional (2D) static culture conditions poorly recapitulate the in vivo cellular environment, we will discuss bioreactor systems for liver tissue engineering, both macro-scale and micro-scale culture systems.

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10-20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development. ispartof: PLOS ONE vol:5 issue:8 ispartof: location:United States status: published

The solute carrier family 27 member 1 (SLC27A1) is an integral membrane protein involved in the transport of long-chain fatty acids across the plasma membrane. This protein has been implicated in diet-induced obesity and is thought to be important in the control of energy homeostasis. In previous reports, our group described the isolation and characterization of the bovineSLC27A1gene. The bovine gene is organized in 13 exons spanning over more than 40 kb of genomic DNA and maps in BTA 7 where several quantitative trait loci for fat related traits have been described. Because of its key role in lipid metabolism and its genomic localization, in the present work the search for variability in the bovineSLC27A1gene was carried out with the aim of evaluating its potential association with milk fat content in dairy cattle. By sequencing analysis of all exons and flanking regions 14 new single nucleotide polymorphisms (SNPs) were identified: 1 in the promoter, 7 in introns and 6 in exons. Allele frequencies of all the SNPs were calculated by minisequencing analysis in two groups of Holstein-Friesian animals with highest and lowest milk-fat content estimated breeding values as well as in animals of two Spanish cattle breeds, Asturiana de los Valles and Menorquina. In the conditions assayed, no significant differences between Holstein-Friesian groups were found for any of the SNPs, suggesting that theSLC27A1gene may have a poor or null effect on milk fat content. In Asturiana and Menorquina breeds all the positions were polymorphic with the exception of SNPs 1 and 8 in which C allele was fixed in both of them.

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