Autologous cell sources in therapeutic vasculogenesis: In vitro and in vivo comparison of endothelial colony-forming cells from peripheral blood and endothelial cells isolated from adipose tissue.

BACKGROUND AIMS: Autologous endothelial cells are promising alternative angiogenic cell sources in trials of therapeutic vasculogenesis, in the treatment of vascular diseases and in the field of tissue engineering. A population of endothelial cells (ECs) with long-term proliferative capability, endothelial colony-forming cells (ECFCs), can be isolated from human peripheral blood. ECFCs are considered an endothelial precursor population. They can be expanded in cell factories in sufficient numbers for clinical applications, but because the number of isolated primary ECs is low, the culture period required may be long. Another EC population that is easily available in the autologous setting and may be expanded in vitro through several population doublings are ECs from adipose tissue (AT-ECs). METHODS: Through extensive comparisons using whole-genome microarray analysis, morphology, phenotype and functional assays, we wanted to evaluate the potential of these EC populations for use in clinical neovascularization. RESULTS: Global gene expression profiling of ECFCs, AT-ECs and the classical EC population, human umbilical vein ECs, showed that the EC populations clustered as unique populations, but very close to each other. By cell surface phenotype and vasculogenic potential in vitro and in vivo, we also found the ECFCs to be extremely similar to AT-ECs. CONCLUSIONS: These properties, together with easy access in the autologous setting, suggest that both AT-ECs and ECFCs may be useful in trials of therapeutic neovascularization. However, AT-ECs may be a more practical alternative for obtaining large quantities of autologous ECs.

Simvastatin coating of TiO2 scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

Bone tissue engineering requires an osteoconductive scaffold, multipotent cells with regenerative capacity and bioactive molecules. In this study we investigated the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) on titanium dioxide (TiO2) scaffold coated with alginate hydrogel containing various concentrations of simvastatin (SIM). The mRNA expression of osteoblast-related genes such as collagen type I alpha 1 (COL1A1), alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP) and vascular endothelial growth factor A (VEGFA) was enhanced in hAD-MSCs cultured on scaffolds with SIM in comparison to scaffolds without SIM. Furthermore, the secretion of osteoprotegerin (OPG), vascular endothelial growth factor A (VEGFA), osteopontin (OPN) and osteocalcin (OC) to the cell culture medium was higher from hAD-MSCs cultured on scaffolds with SIM compared to scaffolds without SIM. The TiO2 scaffold coated with alginate hydrogel containing SIM promote osteogenic differentiation of hAD-MSCs in vitro, and demonstrate feasibility as scaffold for hAD-MSC based bone tissue engineering.

Fetal and placental development in early gestation of hyper-prolific sows.

Modern hyper-prolific sows produce large litters with a high within-litter variation in birth weight and an increased number of low-birth-weight piglets per litter with higher mortality rates and lower growth rates compared to heavier littermates. This study aimed to describe fetal development in hyper-prolific sows, to characterize differences between large and small fetuses, and to determine when within-litter variation in fetal weight can be detected. Forty-seven multiparous sows were blood-sampled and slaughtered at day 28, 33, 45, 50, and 56 of gestation. Number of fetuses were counted, fetal body and organ weights were measured, and the intrauterine positioning was recorded. Length, width, and area of each placenta was measured and the fetus weight/placental weight ratio was calculated. The umbilical cords of the smallest, medium and the largest fetus of each litter were sampled for histological analysis. In total measurements were obtained for 1161 fetuses. The results revealed no difference in fetal survival between the gestational days (P > 0.05). Intrauterine positioning near the cervix significantly reduced fetal weight at day 56 (P < 0.05). Total litter weight and average fetal weight increased with gestational age and individual fetal weight was negatively affected by litter size from day 33 and onwards (P < 0.05). The coefficient of variation for within-litter variation in fetal weight was higher at day 28 compared to the other gestational days (P < 0.05). Relative brain- and heart weights decreased from day 28-56 (P < 0.001). Small fetuses had relatively heavier brains and hearts at day 45, 50 and 56 (P < 0.001). Size of placenta, fetus weight/placental weight ratio and length of umbilical cord increased with gestational age (P < 0.001). There was a positive correlation between size of placenta and weight of individual fetus (P < 0.001), the weight of the fetus was positively correlated with umbilical cord length (P < 0.001) and the umbilical cross-sectional area was correlated to fetal weight at day 56 (P < 0.01). Individual fetal weight was positively correlated to the fetus weight/placental weight ratio (P < 0.001). In conclusion, fetal growth was affected by litter size, placental weight, and -area, and umbilical cord length. Lightweight fetuses were characterized by having placentas with lower weight and area and shorter umbilical cords. Lastly, within-litter variation in fetal weight was detectable at day 28, and the coefficient of variance remained stable from day 33-56.

Effect of increasing dietary methionine-to-lysine ratio during early gestation on fetal development and piglet birth weight.

It was hypothesized that increasing dietary methionine (Met) for sows in early gestation would have a positive effect on fetal and placental growth and development, thereby also increasing the birth weight of piglets. The objective of the study was to investigate the effect of increasing the total dietary methionine-to-lysine ratio (Met:Lys) from 0.29 (Control diet) to 0.41 (Met diet) from mating to day 50 of gestation. A total of 349 multiparous sows were allocated to either the Control or Met diet group. The sows' backfat thickness was measured pre-farrowing, post-farrowing, and at weaning in the previous cycle and on days 14, 50 and 112 of gestation in the current cycle. On day 50, three Control and six Met sows were slaughtered. In 116 litters, piglets were weighed and measured individually at farrowing. The dietary treatment did not affect the sows' backfat thickness before or during gestation (P > 0.05). The number of liveborn and stillborn piglets at farrowing were similar in both groups (P > 0.05) and no differences in average piglet birth weight, total litter weight at birth or within-litter variation in birth weight (P > 0.05) were observed. In conclusion, increasing the dietary Met:Lys ratio for sows in early gestation had no effect on piglet birth weight.

The effect of dietary omega-3 fatty acid supplementation on fetal growth, piglet birth weight and plasma fatty acid concentrations, using docosahexaenoic acid in early gestation in sows.

The objective of the study was to test the effect of the omega-3 fatty acid docosahexaenoic acid (DHA) on fetal and placental development as well as the birth weight of piglets. A total of 238 multiparous sows were allocated to either a control diet group or a DHA diet group with an omega-6 to omega-3 ratio of 9.8 and 2.4, respectively, from mating to day 43 of gestation. A blood sample was collected and back fat thickness was measured prior to mating, on days 14, 42 and 112 of gestation. On day 43 of gestation, 14 sows were slaughtered and measurements of fetuses and placentas were taken. Piglets in some litters were weighed individually at farrowing. Dietary treatment did not affect fetal characteristics and back fat thickness (P > 0.05). Dietary treatment increased the plasma concentrations of total omega-3 fatty acids in sows (P < 0.05). Sows fed the DHA diet had a shorter gestation length compared to the control sows (P < 0.05), but the number of born piglets was not affected (P > 0.05). The average piglet birth weight and the within-litter variation in birthweight were unaffected by dietary DHA (P > 0.05), however, sows fed DHA diet had fewer piglets under 800 g at birth compared to control sows (P < 0.05). In conclusion, addition of DHA decreased the dietary ratio of omega-6 to omega-3 fatty acids, increased plasma n-3 fatty acid concentrations in sows and decreased the number of piglets weighing under 800 g at birth.

Embryo-maternal communication: signalling before and during placentation in cattle and pig.

Communication during early pregnancy is essential for successful reproduction. In this review we address the beginning of the communication between mother and developing embryo; including morphological and transcriptional changes in the endometrium as well as epigenetic regulation mechanisms directing the placentation. An increasing knowledge of the embryo-maternal communication might not only help to improve the fertility of our farm animals but also our understanding of human health and reproduction.

Differential endometrial gene expression in pregnant and nonpregnant sows.

In an attempt to unveil molecular processes controlling the porcine placentation, we have investigated the pregnancy-induced gene expression in the endometrium using the Affymetrix GeneChip Porcine Genome Array. At Day 14 after insemination, at the time of initial placentation, samples were obtained from the endometrium of pregnant sows and sows inseminated with inactivated semen. Analysis of the microarray data revealed 263 genes to be significantly differentially expressed between the pregnant and nonpregnant sows. Most gene ontology terms significantly enriched at pregnancy had allocated more up-regulated genes than down-regulated genes. These terms included developmental process, transporter activity, calcium ion binding, apoptosis, cell motility, enzyme-linked receptor protein signaling pathway, positive regulation of cell proliferation, ion homeostasis, and hormone activity. Only the three terms oxidoreductase activity, lipid metabolic process, and organic acid metabolic process had an overrepresentation of down-regulated genes. A gene interaction network based on the genes identified in the gene ontology term developmental processes identified genes likely to be involved in the process of placentation. Pregnancy-specific localization of IL11RA to the surface epithelium of the endometrium suggests a role of interleukin 11 signaling in formation of the porcine epitheliochorial placenta. Furthermore, up-regulation of FGF9 mRNA in pregnant endometrium and localization of FGF9 to the apical cell domain of the glandular epithelium suggest the concept of endometrial FGF9 acting as an embryonic growth factor in the pig.

Alginate hydrogel enriched with enamel matrix derivative to target osteogenic cell differentiation in TiO2 scaffolds.

The purpose of bone tissue engineering is to employ scaffolds, cells, and growth factors to facilitate healing of bone defects. The aim of this study was to assess the viability and osteogenic differentiation of primary human osteoblasts and adipose tissue-derived mesenchymal stem cells from various donors on titanium dioxide (TiO2) scaffolds coated with an alginate hydrogel enriched with enamel matrix derivative. Cells were harvested for quantitative reverse transcription polymerase chain reaction on days 14 and 21, and medium was collected on days 2, 14, and 21 for protein analyses. Neither coating with alginate hydrogel nor alginate hydrogel enriched with enamel matrix derivative induced a cytotoxic response. Enamel matrix derivative-enriched alginate hydrogel significantly increased the expression of osteoblast markers COL1A1, TNFRSF11B, and BGLAP and secretion of osteopontin in human osteoblasts, whereas osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells seemed unaffected by enamel matrix derivative. The alginate hydrogel coating procedure may have potential for local delivery of enamel matrix derivative and other stimulatory factors for use in bone tissue engineering.

Analysis of the effects of five factors relevant to in vitro chondrogenesis of human mesenchymal stem cells using factorial design and high throughput mRNA-profiling.

The in vitro process of chondrogenic differentiation of mesenchymal stem cells for tissue engineering has been shown to require three-dimensional culture along with the addition of differentiation factors to the culture medium. In general, this leads to a phenotype lacking some of the cardinal features of native articular chondrocytes and their extracellular matrix. The factors used vary, but regularly include members of the transforming growth factor ß superfamily and dexamethasone, sometimes in conjunction with fibroblast growth factor 2 and insulin-like growth factor 1, however the use of soluble factors to induce chondrogenesis has largely been studied on a single factor basis. In the present study we combined a factorial quality-by-design experiment with high-throughput mRNA profiling of a customized chondrogenesis related gene set as a tool to study in vitro chondrogenesis of human bone marrow derived mesenchymal stem cells in alginate. 48 different conditions of transforming growth factor ß 1, 2 and 3, bone morphogenetic protein 2, 4 and 6, dexamethasone, insulin-like growth factor 1, fibroblast growth factor 2 and cell seeding density were included in the experiment. The analysis revealed that the best of the tested differentiation cocktails included transforming growth factor ß 1 and dexamethasone. Dexamethasone acted in synergy with transforming growth factor ß 1 by increasing many chondrogenic markers while directly downregulating expression of the pro-osteogenic gene osteocalcin. However, all factors beneficial to the expression of desirable hyaline cartilage markers also induced undesirable molecules, indicating that perfect chondrogenic differentiation is not achievable with the current differentiation protocols.

RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions.

Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA). While chromatin reprogramming has been widely studied in several species, only a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell stage) EGA and determine major metabolic changes that regulate totipotency. The period before EGA was dominated by transcripts responsible for cell cycle regulation, mitosis, RNA translation and processing (including ribosomal machinery), protein catabolism, and chromatin remodelling. Following EGA an increase in the abundance of transcripts involved in transcription, translation, DNA metabolism, histone and chromatin modification, as well as protein catabolism was detected. The further analysis of members of overlapping GO terms revealed that despite that comparable cellular processes are taking place before and after EGA (RNA splicing, protein catabolism), different metabolic pathways are involved. This strongly suggests that a complex metabolic switch accompanies EGA. In vitro conditions significantly altered RNA profiles before EGA, and the character of these changes indicates that they originate from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos showed striking overlap in functional annotation of transcripts during the EGA, suggesting conserved basic mechanisms regulating establishment of totipotency in mammalian development.

Enhanced in vitro osteoblast differentiation on TiO2 scaffold coated with alginate hydrogel containing simvastatin.

The aim of this study was to develop a three-dimensional porous bone graft material as vehicle for simvastatin delivery and to investigate its effect on primary human osteoblasts from three donors. Highly porous titanium dioxide (TiO2) scaffolds were submerged into simvastatin containing alginate solution. Microstructure of scaffolds, visualized by scanning electron microscopy and micro-computed tomography, revealed an evenly distributed alginate layer covering the surface of TiO2 scaffold struts. Progressive and sustained simvastatin release was observed for up to 19 days. No cytotoxic effects on osteoblasts were observed by scaffolds with simvastatin when compared to scaffolds without simvastatin. Expression of osteoblast markers (collagen type I alpha 1, alkaline phosphatase, bone morphogenetic protein 2, osteoprotegerin, vascular endothelial growth factor A and osteocalcin) was quantified using real-time reverse transcriptase-polymerase chain reaction. Secretion of osteoprotegerin, vascular endothelial growth factor A and osteocalcin was analysed by multiplex immunoassay (Luminex). The relative expression and secretion of osteocalcin was significantly increased by cells cultured on scaffolds with 10 µM simvastatin when compared to scaffolds without simvastatin after 21 days. In addition, secretion of vascular endothelial growth factor A was significantly enhanced from cells cultured on scaffolds with both 10 nM and 10 µM simvastatin when compared to scaffolds without simvastatin at day 21. In conclusion, the results indicate that simvastatin-coated TiO2 scaffolds can support a sustained release of simvastatin and induce osteoblast differentiation. The combination of the physical properties of TiO2 scaffolds with the osteogenic effect of simvastatin may represent a new strategy for bone regeneration in defects where immediate load is wanted or unavailable.

DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer.

DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome. Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts despite the occurrence of de novo methylation in early blastocysts. Comparable levels of DNA methylation were found in IV embryos and in 50% and 14% of SCNT early and late blastocysts, respectively. In conclusion, DNA methylation patterns were adversely affected by in vitro embryo production.