Assessment of limiting dietary amino acids in broiler chickens offered reduced crude protein diets.

As lowering crude protein (CP) in poultry diets continues to minimize amino acid excess, it is important to understand the limiting order of amino acids and the impact of their deficiencies. Therefore, a pair of experiments were conducted to observe the effects of individual amino acid deletions on growth performance, carcass traits, and nutrient utilization. Both experiments involved 3 control diets based on wheat and soybean meal, including a 210.0 g/kg CP industry control (IC), 186.7 g/kg CP positive control (PC) supplemented with feed-grade amino acids to match the IC amino acid profile, 186.7 g/kg CP negative control (NC) with reducing N corrected apparent metabolizable energy (AMEN) by 0.5 MJ/kg and removing feed-grade amino acids beyond L-Lys-HCl, DL-Met, and L-Thr from PC. Ten deletion diets where the following supplemented amino acids were individually removed from the PC: Val, Ile, Leu, Trp, Arg, His, Phe + Tyr, glycine equivalence (Glyequi), Pro, and Energy (0.5 MJ/kg reduction in AMEN of the PC). All diets were formulated to contain similar concentrations of digestible Lys, total sulfur amino acid (TSAA) and Thr. Experimental diets were offered to broiler chickens from 15 to 22 d post-hatch in a cage study (Exp. 1) to gain digestibility and nutrient utilization data; whereas they were offered from 15 to 35 d post-hatch in a floor-pen study (Exp. 2) to gain performance and carcass yield data. The removal of supplemented Val, Arg, and Ile resulted in reduction on broiler performance (P < 0.05), and the removal of Val, Arg, Ile, and Glyequi negatively influenced carcass traits (P < 0.05). Results from both experiments indicate that Val and Arg are co-limiting in wheat-soybean meal diets, but that Ile and Glyequi may potentially limit breast and thigh development.

Graded methionine dietary inclusions influence growth performance and apparent ileal amino acid digestibility coefficients and disappearance rates in broiler chickens.

Graded quantities of 1.38, 2.76 and 4.14 g/kg L-methionine were included in a control diet formulated to contain 3.07 g/kg digestible methionine. Each of the 4 dietary treatments was offered to 6 replicate cages (initially 8 birds per cage) from 1 to 21 d post-hatch. The parameters assessed included growth performance, nutrient utilisation (apparent metabolisable energy [AME], AME:GE ratios, N retention, N-corrected apparent metabolisable energy [AMEn]), apparent digestibility coefficients and disappearance rates of amino acids in the distal ileum. They also included free amino concentrations in systemic plasma (brachial vein) at 20 d post-hatch and in hepatic tissue at 14 and 21 d post-hatch. Graded L-methionine inclusions quadratically influenced weight gain (r = 0.688; P = 0.001) and FCR (r = 0.780; P < 0.001). It may be deduced from the quadratic regressions that 3.43 g/kg L-methionine supported maximum weight gain of 1,036 g/kg and 3.50 g/kg L-methionine minimum FCR of 1.193, from 1 to 21 d post-hatch. The control diet contained specified levels of 3.07 g/kg digestible methionine and 13.0 g/kg digestible lysine. Thus, an inclusion of 3.465 g/kg L-methionine corresponded to a total of 6.535 g/kg methionine or a methionine-to-lysine ratio of 50.3, which is higher than standard recommendations. The implications of this and other outcomes of the present study are reported and discussed.

Implications of excreta uric acid concentrations in broilers offered reduced-crude protein diets and dietary glycine requirements for uric acid synthesis.

In a previous experiment, male Ross 308 broiler chickens were offered dietary treatments with 3 levels of crude protein (222, 193, 165 g/kg) and 3 feed grains (ground maize, ground wheat, whole wheat) from 7 to 35 d post-hatch. Maize-based diets supported superior growth performance in comparison to wheat-based diets. Uric acid concentrations in excreta were retrospectively determined and related to total nitrogen (N) excreta concentrations. Uric acid concentrations ranged from 28.5 to 69.4 mg/g and proportions of uric acid-N to total excreta-N ranged from 27.4% to 42.6% in broiler chickens offered the 3 × 3 factorial array of dietary treatments. Proportions of uric acid-N to total N in excreta in birds offered the 165 g/kg CP, maize-based diet were significantly lower by 10.6 percentage units (27.4% versus 38.0%; P = 0.00057) than their wheat-based counterparts. Total excreta analysed had been collected from 35 to 37 d post-hatch when feed intakes and excreta outputs were monitored. There were linear relationships between proportions of uric acid-N to total N in excreta in birds offered the three 165 g/kg CP diets with weight gain (r = -0.587; P = 0.010), feed intake (r = -0.526; P = 0.025) and feed conversion ratios (r = 0.635; P = 0.005). The possibility that increasing uric acid-N proportions in excreta is indicative of excessive ammonia accumulations compromising growth performance is discussed. The mean proportion of dietary glycine involved in uric acid excretion was 49.2% across all dietary treatments but ranged from 25.0% to 80.9%. Thus, the appropriate amount of dietary glycine is variable and largely dependent on the volume of uric acid synthesised and excreted.

Protein digestive dynamics of meat and bone meals in broiler chickens.

This study determined the variations in protein digestibilities and digestion rates in broiler chickens offered diets containing 7 different meat and bone meals (MBM). A total of 252 male Ross 308 broiler chickens were offered 7 atypical diets largely based on maize and MBM from 24 to 28 d post-hatch. Each experimental diet was offered to 6 replicates with 6 birds per replicate cage. Excreta were collected in their entirety from 25 to 27 d post-hatch and on 28 d post-hatch. Digesta samples were collected from the proximal jejunum, distal jejunum, proximal ileum and distal ileum. Apparent digestibilities of protein were determined in each segment and apparent digestibilities of amino acids were measured in the distal ileum. There were significant differences in apparent protein digestibility coefficients in the proximal jejunum (P = 0.006), where broiler chickens offered the high ash beef meal (diet 7) generated the lowest protein digestibility in the proximal jejunum (0.318). Similarly, there were significant differences in apparent digestibility coefficients in the distal jejunum (P < 0.001) and distal ileum (P < 0.001) but not in the proximal ileum. More pronounced differences were found in the disappearance rate of protein and there were significant differences in all 4 segments of the small intestine (P < 0.001). Broiler chickens offered the high ash beef meal had the lowest protein disappearance rate (P < 0.001). No difference was observed in the predicted protein digestion rate (P = 0.486) but chickens offered the high ash beef meal had the lowest potential digestible protein (0.662, P = 0.034) whereas the highest potential digestible protein (0.739) was detected in diet 5 (containing a beef meal). This study contributed to the establishment of a preliminary database to include digestion rates of starch and protein into practical diet formulation.

Capping dietary starch:protein ratios in moderately reduced crude protein, wheat-based diets showed promise but further reductions generated inferior growth performance in broiler chickens.

The hypothesis that capping dietary starch:protein ratios would enhance the performance of broiler chickens offered reduced-crude protein (CP) diets was tested in this experiment. A total of 432 off-sex, male Ross 308 chicks were allocated to 7 dietary treatments from 7 to 35 d post-hatch. The experimental design consisted of a 3 × 2 factorial array of treatments with the seventh treatment serving as a positive control. Three levels of dietary CP (197.5, 180.0 and 162.5 g/kg) with either uncapped or capped dietary starch:protein ratios constituted the factorial array of treatments, whilst the positive control diet contained 215.0 g/kg CP. The positive control diet had an analysed dietary starch:protein ratio of 1.50 as opposed to a ratio of 1.68 in the uncapped 197.5 g/kg CP diet and 1.41 in the corresponding capped diet and the capped 197.5 g/kg CP diet displayed promise. The growth performance this diet matched the positive control but outperformed the uncapped 197.5 g/kg CP diet by 10.4% (2,161 vs. 1,958; P = 0.009) in weight gain, by 3.10% (3,492 vs. 3,387; P = 0.019) in feed intake on the basis of pair-wise comparisons and numerically improved FCR by 4.04% (1.616 vs. 1.684). However, the growth performance of birds offered the 180.0 and 162.5 g/kg CP dietary treatments was remarkably inferior, irrespective of dietary starch:protein ratios. This inferior growth performance was associated with poor feathering and even feather-pecking and significant linear relationships between feather scores and parameters of growth performance were observed. The amino acid profile of feathers was determined where cysteine, glutamic acid, glycine, proline and serine were dominant in a crude protein content of 931 g/kg. Presumably, the feathering issues observed were manifestations of amino acid inadequacies or imbalances in the more reduced-CP diets and consideration is given to the implications of these outcomes.

Reducing agent and exogenous protease additions, individually and in combination, to wheat- and sorghum-based diets interactively influence parameters of nutrient utilisation and digestive dynamics in broiler chickens.

The objective of the study was to investigate the possibility that tandem inclusions of a reducing agent and a protease may advantage chicken-meat production and to ascertain if the established benefits of including sodium metabisulphite in sorghum-based diets extend to wheat-based diets. The study comprised a 2 × 2 × 2 factorial array of treatments in which either nutritionally iso-nitrogenous and iso-energetic wheat- or sorghum-based diets, without and with sodium metabisulphite (2.75 g/kg), without and with protease (1,000 units/kg) were offered to broiler chickens from 7 to 28 days post-hatch. The effects of dietary treatments on growth performance, nutrient utilisation, protein (N) and starch digestibility coefficients and digestive dynamics were determined. A preliminary investigation into the effects of two treatments on concentrations of free amino acids and glucose in the portal circulation was conducted. There was significant feed grain by sodium metabisulphite interactions (P = 0.03 to 0.005) for parameters of nutrient utilisation (AME, ME:GE ratios, N retention, AMEn). For example, sodium metabisulphite inclusions in sorghum-based diets enhanced AME by 0.18 MJ (12.47 versus 12.29 MJ/kg) but depressed AME by 0.43 MJ (11.88 versus 12.31 MJ/kg) in wheat-based diets. There was a linear relationship between starch:protein disappearance rate ratios in the distal ileum with weight gain (r = -0.484; P = 0.0012) indicating that condensed ratios (or absorption of more protein relative to starch) advantaged growth performance. Concentrations of free amino acids in the portal circulation or the post-enteral availability of certain amino acids, including the branched-chain amino acids, methionine, phenylalanine and threonine, were significantly correlated to FCR. For example, threonine concentrations were negatively correlated to FCR (r = -0.773; P = 0.005). Finally, tandem inclusions of sodium metabisulphite and protease in sorghum-based diets may hold merit but it appears that the established 'energy sparing' effects of sodium metabisulphite inclusions in sorghum-based diets are not duplicated in wheat-based diets.

Effect of N-acetylcysteine infusion on exercise-induced modulation of insulin sensitivity and signaling pathways in human skeletal muscle.

-Reactive oxygen species (ROS) produced in skeletal muscle may play a role in potentiating the beneficial responses to exercise; however, the effects of exercise-induced ROS on insulin action and protein signaling in humans has not been fully elucidated. Seven healthy, recreationally active participants volunteered for this double-blind, randomized, repeated-measures crossover study. Exercise was undertaken with infusion of saline (CON) or the antioxidant N-acetylcysteine (NAC) to attenuate ROS. Participants performed two 1-h cycling exercise sessions 7-14 days apart, 55 min at 65% V̇o2peak plus 5 min at 85%V̇o2peak, followed 3 h later by a 2-h hyperinsulinemic euglycemic clamp (40 mIU·min(-1)·m(2)) to determine insulin sensitivity. Four muscle biopsies were taken on each trial day, at baseline before NAC infusion (BASE), after exercise (EX), after 3-h recovery (REC), and post-insulin clamp (PI). Exercise, ROS, and insulin action on protein phosphorylation were evaluated with immunoblotting. NAC tended to decrease postexercise markers of the ROS/protein carbonylation ratio by -13.5% (P = 0.08) and increase the GSH/GSSG ratio twofold vs. CON (P < 0.05). Insulin sensitivity was reduced (-5.9%, P < 0.05) by NAC compared with CON without decreased phosphorylation of Akt or AS160. Whereas p-mTOR was not significantly decreased by NAC after EX or REC, phosphorylation of the downstream protein synthesis target kinase p70S6K was blunted by 48% at PI with NAC compared with CON (P < 0.05). We conclude that NAC infusion attenuated muscle ROS and postexercise insulin sensitivity independent of Akt signaling. ROS also played a role in normal p70S6K phosphorylation in response to insulin stimulation in human skeletal muscle.

Proteomics of Huntington's disease-affected human embryonic stem cells reveals an evolving pathology involving mitochondrial dysfunction and metabolic disturbances.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the Huntingtin gene, where excessive (≥ 36) CAG repeats encode for glutamine expansion in the huntingtin protein. Research using mouse models and human pathological material has indicated dysfunctions in a myriad of systems, including mitochondrial and ubiquitin/proteasome complexes, cytoskeletal transport, signaling, and transcriptional regulation. Here, we examined the earliest biochemical and pathways involved in HD pathology. We conducted a proteomics study combined with immunocytochemical analysis of undifferentiated HD-affected and unaffected human embryonic stem cells (hESC). Analysis of 1883 identifications derived from membrane and cytosolic enriched fractions revealed mitochondria as the primary dysfunctional organ in HD-affected pluripotent cells in the absence of significant differences in huntingtin protein. Furthermore, on the basis of analysis of 645 proteins found in neurodifferentiated hESC, we show a shift to transcriptional dysregulation and cytoskeletal abnormalities as the primary pathologies in HD-affected cells differentiating along neural lineages in vitro. We also show this is concomitant with an up-regulation in expression of huntingtin protein in HD-affected cells. This study demonstrates the utility of a model that recapitulates HD pathology and offers insights into disease initiation, etiology, progression, and potential therapeutic intervention.

N-acetylcysteine alters substrate metabolism during high-intensity cycle exercise in well-trained humans.

We investigated the effects of N-acetylcysteine (NAC) on metabolism during fixed work rate high-intensity interval exercise (HIIE) and self-paced 10-min time-trial (TT10) performance. Nine well-trained male cyclists (V̇O2peak, 69.4 ± 5.8 mL · kg(-1) · min(-1); peak power output (PPO), 385 ± 43 W; mean ± SD) participated in a double-blind, repeated-measures, randomised crossover trial. Two trials (NAC supplementation and placebo) were performed 7 days apart consisting of 6 × 5 min HIIE bouts at 82% PPO (316 ± 40 W) separated by 1 min at 100 W, and then after 2 min of recovery at 100 W, TT10 was performed. Expired gases, venous blood, and electromyographic (EMG) data were collected. NAC did not influence blood glutathione but decreased lipid peroxidation compared with the placebo (P < 0.05). Fat oxidation was elevated with NAC compared with the placebo during HIIE bouts 5 and 6 (9.9 ± 8.9 vs. 3.9 ± 4.8 µmol · kg(-1) · min(-1); P < 0.05), as was blood glucose throughout HIIE (4.3 ± 0.6 vs. 3.8 ± 0.6 mmol · L(-1); P < 0.05). Blood lactate was lower with NAC after TT10 (3.3 ± 1.3 vs. 4.2 ± 1.3 mmol · L(-1); P < 0.05). Median EMG frequency of the vastus lateralis was lower with NAC during HIIE (79 ± 10 vs. 85 ± 10 Hz; P < 0.05), but not TT10 (82 ± 11 Hz). Finally, NAC decreased mean power output 4.9% ± 6.6% (effect size = -0.3 ± 0.4, mean ± 90% CI) during TT10 (305 ± 57 W vs. 319 ± 45 W). These data suggest that NAC alters substrate metabolism and muscle fibre type recruitment during HIIE, which is detrimental to time-trial performance.

Overexpression of αvß6 integrin alters the colorectal cancer cell proteome in favor of elevated proliferation and a switching in cellular adhesion that increases invasion.

Many proteins enhance cancer progression toward life-threatening metastases. These include linking proteins called integrins that mediate cell adhesion to the extracellular matrix (ECM), consequently altering both function and phenotype. Specific neoexpression of the ß6 integrin subunit correlates with the epithelial-to-mesenchymal transition, metastasis, and poor overall patient survival. While ß6 is implicated in these processes, exactly how it affects signaling and/or proteolytic pathways in metastasis remains unclear. A membrane-enriched peptide immobilized pH gradient isoelectric focusing (IPG-IEF) shotgun proteomics study was undertaken in which subclones of the SW480 colorectal cancer cell line transfected with a vector inducing unregulated ß6 integrin overexpression were compared with the "empty" mock vector control cell line. ß6 overexpression induced a significant change in 708 proteins and was found to be localized across most intracellular locations, some involving cellular processes and pathways underpinning cancer progression. Proteomics data have been deposited to the ProteomeXchange with identifier PXD000230. ß6 expression increased cell proliferation 4-fold while decreasing cell adhesion to many integrin ECM substrates. ß6 expression also enhanced cell invasion and promoted the expression/repression of many established cancer-related pathways.

Improved membrane proteomics coverage of human embryonic stem cells by peptide IPG-IEF.

Protein biomarkers are fundamental tools for the characterization of stem cells and for tracking their differentiation and maturation down developmental lineages. Technology development allowing increased coverage of difficult cellular proteomes should allow for the discovery of new and novel membrane protein biomarkers for use by the stem cell research community. The amphipathic and highly hydrophobic nature and relative low abundance of many membrane proteins present significant analytical challenges. These difficulties are amplified when the source material (tissue or cells) is only available in limited quantities (e.g., embryonic stem cells). Recent advances in enrichment for purer membrane fractions, the enzymatic and chemical digestion of membrane proteins in the presence of solvents or chaotropes, and the use of "shotgun" proteomics methodologies have gradually resulted in increased membrane proteome coverage with numbers of predicted integral membrane proteins now in excess of 1000 being routinely reported. We have recently demonstrated the advantages of using peptide isoelectric focusing in the first dimension on immobilized pH gradients (peptide IPG-IEF) followed by reversed phase chromatography and tandem MS to increase membrane proteome coverage. This study looked at achieving a similar level of membrane proteome coverage using modifications to reported methodologies while restricting the number of characterized human embryonic stem cells to 10(7) cells. Two-thousand two-hundred and ninety-two (2292) nonredundant proteins were identified with two or more high accuracy peptide matches from 260 mug of a human embryonic stem cell membrane enriched fraction with a false discovery rate of 0.32%. Gene Ontology (GO) mapping predicted 1279 (44.9%) of this list to be membrane proteins of which 395 proteins were predicted to be derived from the plasma membrane compartment. The TMHMM algorithm predicted 904 integral membrane proteins with up to 16 transmembrane helices. Collectively, we assert that the substantial membrane proteome coverage achieved using these procedures will enable rapid advances in the identification and quantitation of novel membrane proteins as markers of differentiation status and/or genetic mutation from relatively low numbers of cultured embryonic stem cells.

Inherited surfactant deficiency caused by uniparental disomy of rare mutations in the surfactant protein-B and ATP binding cassette, subfamily a, member 3 genes.

OBJECTIVE: To characterize inheritance of homozygous, rare, recessive loss-of-function mutations in surfactant protein-B (SFTPB) or ATP binding cassette, subfamily A, member 3 (ABCA3) genes in newborns with lethal respiratory failure. STUDY DESIGN: We resequenced genes from parents whose infants were homozygous for mutations in SFTPB or ABCA3. For infants with only 1 heterozygous parent, we performed microsatellite analysis for chromosomes 2 (SFTPB) and 16 (ABCA3). RESULTS: We identified 1 infant homozygous for the g.1549C > GAA mutation (121ins2) in SFTPB for whom only the mother was heterozygous and 3 infants homozygous for mutations in ABCA3 (p.K914R, p.P147L, and c.806_7insGCT) for whom only the fathers were heterozygous. For the SP-B-deficient infant, microsatellite markers confirmed maternal heterodisomy with segmental isodisomy. Microsatellite analysis confirmed paternal isodisomy for the 3 ABCA3-deficient infants. Two ABCA3-deficient infants underwent lung transplantation at 3 and 5 months of age, respectively, and 2 infants died. None exhibited any nonpulmonary phenotype. CONCLUSIONS: Uniparental disomy should be suspected in infants with rare homozygous mutations in SFTPB or ABCA3. Confirmation of parental carrier status is important to provide recurrence risk and to monitor expression of other phenotypes that may emerge through reduction to homozygosity of recessive alleles.

Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media.

The unique pluripotential characteristic of human embryonic stem cells heralds their use in fields such as medicine, biotechnology, biopharmaceuticals, and developmental biology. However, the current availability of sufficient quantities of embryonic stem cells for such applications is limited, and generating sufficient numbers for downstream therapeutic applications is a key concern. In the absence of feeder layers or their conditioned media, human embryonic stem cells readily differentiate to form embryoid bodies, indicating that trophic factors secreted by the feeder layers are required for long-term proliferation and maintenance of pluripotency. Adding further complexity to the elucidation of the factors required for the maintenance of pluripotency is the variability of different fibroblast feeder layers (of mouse or human origin) to effectively support human embryonic stem cells. Currently, the deficiency of knowledge concerning the exact identity of factors within the pathways for self-renewal illustrates that a number of factors may be required to support pluripotent, undifferentiated growth of human embryonic stem cells. This study utilized a proteomic analysis (multidimensional chromatography coupled to tandem mass spectrometry) to isolate and identify proteins in the conditioned media of three mitotically inactivated fibroblast lines (human fetal, human neonatal, and mouse embryonic fibroblasts) used to support the undifferentiated growth of human embryonic stem cells. One-hundred seventy-five unique proteins were identified between the three cell lines using a

Transplantation of 3D scaffolds seeded with human embryonic stem cells: biological features of surrogate tissue and teratoma-forming potential.

AIM: To generate complex surrogate tissue by transplanting 3D scaffolds seeded with human embryonic stem cells (hESCs) between the liver lobules of severe combined immunodeficient (SCID) mice and to assess the teratoma-forming potential. MATERIALS & METHODS: 3D poly-(lactic-co-glycolic acid) (PLGA) scaffolds coated with laminin were seeded with hESCs and then transplanted between the liver lobules of SCID mice. After a period of in vivo differentiation, the scaffolds were retrieved and analyzed using reverse transcription polymerase chain reaction, immunofluorescent staining and scanning electron microscopy. RESULTS: A proportion of the hESCs within the scaffolds differentiated into cells that produced proteins characteristic of specific tissues, including endoderm and pancreatic markers glucogon-like peptide-1 receptor, islet amyloid polypeptide and Insulin. Markers of hepatic and neuronal lineages were also investigated. Major matrix proteins abundant in multiple tissue types, including collagen I, laminin and collagen IV, were found to be profuse within the scaffold pores. Transplantation of the seeded scaffolds between liver lobules also resulted in extensive vascularization both from host blood vessel incursion and the differentiation of hESCs into endothelial progenitor cells. An investigation of teratoma-forming potential demonstrated that transplantation of 3D scaffolds seeded with hESCs will, under certain conditions, lead to the growth of teratomas. DISCUSSION: Transplantation of 3D scaffolds seeded with hESCs between liver lobules resulted in the development of surrogate tissue containing cells that produced proteins representing the pancreatic, hepatic and neuronal lineages, the assembly of an extracellular matrix structure and the formation of a vasculature. hESCs seeded within 3D scaffolds and transplanted into SCID mice were capable of forming teratomas. However, the formation and progression of teratoma growth is shown to be dependant on both the site of transplantation and the treatment of cells prior to transplantation.

Growth and differentiation of embryoid bodies derived from human embryonic stem cells: effect of glucose and basic fibroblast growth factor.

Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells.

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Wnt signaling and inhibition of bone morphogenetic proteins.