Bovine Embryonic Fibroblasts Research Articles

Lumpy Skin Disease Virus Infection Activates Autophagy and Endoplasmic Reticulum Stress-Related Cell Apoptosis in Primary Bovine Embryonic Fibroblast Cells.

Poxviruses have been associated with humans for centuries. From smallpox to mpox to lumpy skin disease virus (LSDV), members of the poxvirus family have continued to threaten the lives of humans and domestic animals. A complete understanding of poxvirus-mediated cellular processes will aid in the response to challenges from the viruses. In this study, we demonstrate that LSDV infection results in an abnormal ultrastructure of the endoplasmic reticulum (ER) lumen in primary bovine embryonic fibroblast (BEF) cells, and we further show that an ER imbalance occurs in LSDV-infected BEF cells. Additionally, we believe that ER stress-related apoptosis plays a role in the late apoptosis of BEF cells infected with LSDV, primarily through the activation of the CCAAT/enhancer binding protein homologous protein (CHOP)-Caspase-12 signal. In addition to cell apoptosis, a further investigation showed that LSDV could also activate autophagy in BEF cells, providing additional insight into the exact causes of LSDV-induced BEF cell death. Our findings suggest that LSDV-induced BEF cell apoptosis and autophagy may provide new avenues for laboratory diagnosis of lumpy skin disease progression and exploration of BEF cell processes.

Naïve-like conversion of bovine induced pluripotent stem cells from Sertoli cells

Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naïve-like conversion, based on a naïve medium (NM). The results showed successful conversion of the primed bovine iPSCs to naïve-like state within 3–4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naïve-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naïve-like iPSCs. We further analyzed the naïve markers and differentiation potential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naïve-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naïve-like bovine iPSCs, and improve the conversion efficiency.

In vitro characterization of anodized magnesium alloy as a potential biodegradable material for biomedical applications

Pure metals and their alloys are used to partially or completely restore bone fractures. Biodegradable metals emerged as promising candidates for fracture fixation devices since they are able to self-degrade in the body environment. The main challenge of the devices is to reach an adequate degradation rate relative to the bone healing and also to present safe degradation by-products. Magnesium alloys are very much studied because of their promising properties, but the main limitation for their application in biomedical devices is the hydrogen evolution that results from their corrosion in aqueous media. This work assesses the effects of low potential anodizing process of AZ91 alloys in basic media on surface topography, electrochemical response, hydrogen evolution and cell attachment compared with the non-treated alloys. A comparative approach to determine the electrochemical parameters for assessing the degradation rate is also discussed. Results show that the electrochemical treatment of anodizing at low voltage in 5 mol/L KOH solution generates magnesium oxide/hydroxide on the surface which could act as a barrier to prevent fast degradation, and consequently to reduce hydrogen release. In turn, this treatment improved the adhesion of bovine embryonic fibroblasts (BEFs) and MCT3T3 pre-osteoblastic cells to the surface, showing that it could be a good candidate to be used in temporary implants.

Efficient Myogenic/Adipogenic Transdifferentiation of Bovine Fibroblasts in a 3D Bioprinting System for Steak-Type Cultured Meat Production.

The interest in cultured meat is increasing because of the problems with conventional livestock industry. Recently, many studies related to cultured meat have been conducted, but producing large-sized cultured meat remains a challenge. It is aimed to introduce 3D bioprinting for producing large cell aggregates for cultured meat production. A hydrogel scaffold is produced at the centimeter scale using a bioink consisting of photocrosslinkable materials for digital light processing-based (DLP) printing, which has high printing accuracy and can produce geometrically complex structures. The light exposure time for hydrogel photopolymerization by DLP bioprinting is optimized based on photorheometry and cell viability assays. Naturally immortalized bovine embryonic fibroblast cells transformed with MyoD and PPARγ2 instead of primary cells are used as the latter have difficulties in maintaining stemness and are associated with animal ethics issues. The cells are mixed into the hydrogel for printing. Myogenesis and adipogenesis are induced simply by changing the medium after printing. Scaffolds are obtained successfully with living cells and large microchannels. The cooked cultured meat maintains its size and shape upon cutting. The overall dimensions are 3.43cm×5.53cm×0.96cm. This study provides proof-of-concept for producing 3D cultured meat using bioinks.

Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C.

Feeder cells play important roles in In-vitro culture of stem cells. However, the preparation protocol of feeder cells produced by bovine embryonic fibroblast cells (bEFs) is still lack. In this study, the preparation of bEF-feeder by Mitomycin C was optimized with different concentrations and treatment time. The cell viability of bEFs was detected by CCK8 and 5-Ethynyl-2'-deoxyuridine. The growth of bESCs in each bEFs-feeder group was assessed by alkaline phosphatase staining and CCK8. Quantitative real time PCR was used to detect the mRNA expression of pluripotency-related genes of bESCs. Results showed that the proliferation of bEFs was significantly repressed while bEFs were treated with 14 ug/mL or 16 ug/mL Mitomycin C for 3h, and the cell viability within 2-4days after treatment was consistent with the 1st day. The numbers of bESCs clones in bEF-feeder treated with 14μg/mL Mitomycin C for 3h or 16μg/mL Mitomycin C for 3h were significantly higher than that in bEF-feeder treated with 8μg/mL Mitomycin C for 8h or bEFs treated with 6μg/mL Mitomycin C for 9h. The mRNA expression of pluripotency-related genes in bESCs cultured by bEF-feeder were higher than the MEF-feeder, the clone morphology of bESCs cultured in bEF-feeder was rounder and sharper than the MEF-feeder. In conclusion, the bEF-feeder prepared with 14μg/mL Mitomycin C for 3h or 16μg/mL Mitomycin C for 3h could effectively maintains the growth of bESCs, and bEF-feeder is more suitable for bESCs culture than the MEF-feeder.

Identification of candidate proteins regulated by long-term caloric restriction and feed efficiency in longissimus dorsi muscle in Korean native steer.

We aimed to investigate candidate proteins related to long-term caloric restriction and feed efficiency in bovine longissimus dorsi muscle (LM). A total of 31 Korean native steers were randomly distributed to ad libitum (n = 16) or caloric restriction group (n = 15) to conduct two feeding trials for 13 mon. In the first trial (10–18 mon of age), steers were fed with 100% ad libitum (NEg = 0.63 Mcal/kg) or caloric restriction (80% of the previous day’s feed intake of ad libitum group). In the second trial (18–23 mon of age), the energy value of 100% ad libitum diet was 1.13 Mcal/kg NEg and those in caloric restriction group diet was 0.72 Mcal/kg NEg. At the endpoint of this experiment, in each group, 6 animals were selected with high (n = 3) or low feed efficiency (n = 3) to collect muscle tissue samples (6 animals/group). From muscle tissues of 23 mo of age, we excavated 9 and 12 differentially expressed (two-fold or more) proteins in a nutritional group and feed efficiency group using two-dimensional electrophoresis, respectively. Of these proteins, heat shock protein beta-6 was up-regulated in both the caloric restriction and the low feed efficiency group. In bovine embryonic fibroblasts, the mRNA expression of heat shock protein beta-6 increased after adipogenic differentiation, however, decreased after myogenic differentiation. Our data provide that heat shock protein beta-6 may be an adipogenic protein involved in the mechanism of caloric restriction and feed efficiency in the LM of the steer.

Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply.

Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B12) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine–folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle’s minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine–folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest (p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism.

82 Methyl Donor Supplementation Alters Cytosine Methylation and Biological Processes of Cells Cultured in Divergent Glucose Media Reflecting Improvements in Mitochondrial Respiration and Cell Growth Rate

Abstract We hypothesized that supplementation of one-carbon metabolites (OCM: methionine, folate, choline, and vitamin B12) to bovine embryonic tracheal fibroblasts in divergent glucose media would alter cytosine methylation, and alterations in cytosine methylation will reflect biological processes matching previously improved mitochondrial respiration, cell proliferation, and cell growth rate data. Cells were cultured with 1g/L glucose (Low) or 4.5g/L glucose (High). Control medium (CON) contained basal concentrations of folate (0.001g/L), choline (0.001g/L), vitamin B12 (4µg/L), and methionine (0.015g/L). The OCM were supplemented at 2.5 and 5 times (2.5X and 5X, respectively) the CON media, except methionine was limited to 2X across all supplemented treatments. Cells were passaged three times in their treatment media before DNA extraction. Reduced representation bisulfite sequencing was adopted to analyze and compare the genomic methylation patterns within and across treatments using edgeR. Biological processes (BP) were retrieved based on the nearest genes of differentially methylated cytosines (P &amp;lt; 0.01) for each comparison between treatments. In both Low and High treatments, greater OCM increased the proportion of hypomethylated vs. hypermethylated cytosines. Functional analyses pointed out positive regulation of BP related to energy metabolism, except for the contrasts within the High group. Among the BP, we can highlight positive regulation of: GTPase activity, catalytic activity, molecular function, protein modification processes, phosphorylation, protein phosphorylation, cellular protein metabolic processes, MAPK cascade, and metabolic processes. These data support previously reported results from this experiment that showed increased mitochondrial respiration, cell proliferation, and growth rates with increasing OCM levels. We interpret these data to imply that when energy and OCM requirements are met for growth and basal methylation levels, DNA methylation levels decrease which may allow for greater transcription. Thus, OCM can be utilized for other functions such as polyamine synthesis, nucleotide synthesis, energetic metabolites, and phosphatidylcholine synthesis. USDA is an equal opportunity provider and employer.

390 Micronutrients, One-Carbon Metabolism, and Epigenetics: Potential Developmental and Production Outcomes

Abstract One-carbon metabolism is the network of biochemical pathways in which methyl groups are transferred from one compound to another for methylation processes. Expansion of the core pathway connects one-carbon metabolism to polyamine synthesis, nucleotide synthesis, redox metabolism, and the citric acid cycle. One-carbon metabolites (OCM) are methyl donors and cofactors which play key roles in the one-carbon metabolism pathway and include B-vitamins (choline, vitamin B12, vitamin B6, riboflavin, and folate), minerals (cobalt and sulfur) and amino acids (methionine, serine, and glycine). One-carbon metabolites are fundamental methyl donors for epigenetic modifications. Immediately post-fertilization, the embryonic genome undergoes epigenetic remodeling, and is the time when cell division is greatest (cell divisions/total cell number). At this time OCM supplementation may have its greatest impact on programming of offspring development, growth and postnatal performance, due to established metabolic roles in epigenetics (methyl transfer), growth (polyamine and nucleotide synthesis), and energetics. Limited data are available, however, which directly investigates the developmental effects of OCM supplementation in ruminants. In dairy cows, OCM supplementation in late gestation increased calf birth weight, nutrient sensing pathway activation, and offspring performance through the pre-weaning period. Methyl deficient diets during the pre-conception period in ewes altered the offspring hepatic methylome at 90 days of gestation, as well as the body composition and insulin tolerance of ram lambs at 22-mo of age. Bovine embryonic fibroblasts cultured in vitro with increasing OCM had greater growth rates and mitochondrial respiration parameters. Additional research into the area of one-carbon metabolism and the roles that OCM supplementation may play on postnatal function will provide new knowledge that could lead to altered management practices and increased efficiency of beef cattle. USDA is an equal opportunity provider and employer.

Targeted histone demethylation improves somatic cell reprogramming into cloned blastocysts but not postimplantation bovine concepti†.

Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

PSXIV-3 Expression of genes within the methionine-folate cycle are altered by glucose and one-carbon metabolite supplementation in bovine embryonic fibroblasts

Abstract We hypothesized that supplementation with one-carbon metabolites (OCM; methionine, folate, choline, and vitamin B12) in divergent glucose media would alter expression of genes in the methionine-folate cycle of DNA methylation in bovine embryonic tracheal fibroblasts. Cells were cultured in EMEM medium containing 1 g/L (Low) or 4.5 g/L (High) of glucose with no added OCM (CON), or 2.5, 5, or 10 times folate, choline, and B12 (2.5X, 5X, or 10X, respectively) in the CON media. Methionine was limited to 2X CON. The data were analyzed as a completely randomized design with a 2 × 4 factorial arrangement of treatments. Cells were passaged three times in treated media before RNA extraction. Gene expression analysis was conducted on Methionine Adenosyltransferase 2A and 2B (MAT2A and MAT2B) DNA Methyltransferase 1, 3A, and 3B (DNMT1, DNMT3A, and DNMT3B), S-Adenosylhomocysteine Hydrolase (AHCY), and Methionine Synthase (MTR). There was no glucose × OCM interaction for DNMT1, DNMT3A, DNMT3B, AHCY, or MTR (P &amp;gt; 0.17). Expression of MAT2B was greater (P &amp;lt; 0.01) in 2.5X, 5X, and 10X OCM in High media compared with all other treatments. Expression of DNMT1 was greater in CON, 5X, and 10X OCM in Low media compared with all other treatments. Expression of MTR was greater (P = 0.03) in CON and 10X OCM treatments compared with 2.5X and 5X levels. Finally, MAT2A and AHCY expression was affected by glucose level, with MAT2A being greater (P = 0.02) in Low compared with High, and AHCY being greater (P &amp;lt; 0.01) in High compared with Low media. These data suggest that glucose and OCM supplementation differentially affect the expression of genes in the methionine-folate cycle of DNA methylation. Additionally, these data demonstrate that additional work elucidating the changes in protein abundance of these enzymes, concentrations of methionine-folate cycle intermediates (specifically S-adenosylmethionine:S-adenosylhomocysteine), as well as changes in DNA methylation are needed.

One-carbon metabolite supplementation improves growth of bovine embryonic fibroblasts cultured in divergent glucose media.

One-carbon metabolite supplementation improves growth of bovine embryonic fibroblasts cultured in divergent glucose media.

Effect of glutamine on heat-shock protein beta 1 (HSPB1) expression during myogenic differentiation in bovine embryonic fibroblast cells.

The objective of this study was to examine the effects of glutamine on heat-shock protein beta 1 (HSPB1) expression in bovine embryonic fibroblast cells during myogenesis. First, to elucidate the role of glutamine on HSPB1 expression during myogenesis, we treated with glutamine in myogenic lineage determinant (MyoD) over-expressed bovine embryonic fibroblast cells (BEFS-MyoD cells). Second, knockdown of HSPB1 using small interference RNA was performed to evaluate whether muscle development by glutamine is dependent on HSPB1 in BEFS-MyoD cells. As a result, glutamine promoted the mRNA level of HSPB1, Myogenin, Desmin, and mTOR as well as myotube formation, and protein synthesis (p<0.05). The inhibition of HSPB1 expression during myogenesis has shown to repress the expression of myogenic marker genes (MyoD, Myogenin, Desmin) (p<0.01), formation of myotubes and protein synthesis (p<0.05). According to the results, it is concluded that glutamine regulates HSPB1 expression during myogenesis.

Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts.

Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG1/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKN1C was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2, PEG3, and PEG10, were obtained. These results illustrate the epigenetic characteristic of bovine parthenogenetic embryos and contribute to the identification of novel imprinted genes in cattle.

BEFS-PPARγ2 cells incubated with trans-11 C18:1 exhibit more beneficial fatty acid synthesis

Responses of the PPARγ2 (an adipogenic lineage determinant) over-expressed immortalized bovine embryonic fibroblast cell line (BEFS-PPARγ2 cells) to trans-11 C18:1 (TVA) incubation were evaluated. BEFS-PPARγ2 cells were well differentiated. Stearoyl-CoA desaturase 1, an endoplasmic reticulum enzyme that can catalyze conversion of TVA into cis-9, trans-11 CLA, exhibited a significantly (p<0.05) higher mRNA expression after 4 h, compared with controls. Enzyme activity peaked, and was significantly (p<0.05) higher after 6 h, compared with controls. With extension of the incubation time, TVA absorption into cells was significantly (p<0.05) higher with significantly (p<0.05) more conversion into cis-9, trans-11 CLA, compared with shorter incubation times. TVA incubated cells exhibited significantly (p<0.05) higher levels of arachidonic, eicosapentaenoic, and docosahexaenoic acids than controls. BEFS-PPARγ2 cells can be used as a model system and TVA incubation is a good approach for research into cis-9, trans-11 CLA-fortified beef production.

Application of a novel population of multipotent stem cells derived from skin fibroblasts as donor cells in bovine SCNT.

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

187 INCREASED EFFICIENCY OF DERIVING BOVINE STEM CELL-LIKE COLONIES USING VALPROIC ACID AND SMALL-MOLECULE COCKTAILS

Nonviral reprogramming of bovine embryonic and adult fibroblasts was undertaken using electroporation of a polycistronic plasmid carrying human reprogramming factors Oct4, Sox2, Lin28, and Nanog (Addgene Plasmid #20922). Because of difficulties encountered in reliably and reproducibly reprogramming bovine cells to a pluripotent state, several small-molecule combinations have been tested to support reprogramming. Previously, a combination of three molecules, (sodium butyrate, PD0325901 and SB431542; NaB-PD-SB) in induced pluripotent stem (iPS) medium [Minimum Essential Medium Alpha, 20% FBS, 1x insulin-transferrin-selenium (ITS), 2 mM Glutamax (Gibco, Grand Island, NY, USA), 100 μM nonessential amino acids (NEAA), 50 U mol–1 penicillin, 50 mg mL–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL–1 human leukaemia inhibitory factor, and 10 ng mL–1 basic fibroblast growth factor] was found to accelerate the reprogramming process of bovine iPS cells, with colonies observed at 12 days post-electroporation, as opposed to 21 days without the addition of the small-molecule cocktail. The addition of 1 mM valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, in combination with the other small-molecule cocktail (NaB-PD-SB), was found to improve the reprogramming efficiency of bovine adult and embryonic fibroblasts, with colonies observed at Day 10 post-electroporation, and the average number of colonies present per 10-cm dish, rising from an average of 5 colonies observed in the NaB-PD-SB cocktail to 27 with the addition of VPA. Colonies grown in the presence of VPA had a consistent morphology, forming compact domed colonies consisting of small round cells with well-defined borders. It was observed that colonies growing in the presence of VPA tended to have a better defined border and grew larger in size than those grown in the presence of NaB-PD-SB alone. Colonies began to differentiate after 21 days and were no longer alkaline phosphatase positive after this time. Cells were either harvested for mRNA extraction or differentiated into embryoid bodies (EB). Expression of pluripotency genes, Oct4, Sox2, Klf4, Nanog, and Alkaline Phosphatase were significantly increased in the presence of VPA compared to nonreprogrammed somatic bovine fibroblasts, with expression profiles similar to those grown in the NaB-PD-SB cocktail. Embryoid bodies were analysed for gene expression of different germ layer markers, FOXA2 (endoderm), Nestin and TUBB3 (ectoderm), and Desmin (mesoderm) using quantitative RT–PCR. For both EBs derived from the NaB-PD-SB cocktail and those with VPA, at least one marker from each germ layer was present, demonstrating the potential potency of these cells. At least a 10-fold increase in expression of these was observed in comparison to somatic fibroblast cells. It is apparent that the addition of small molecules can assist in the reprogramming of bovine iPS cells, and addition of VPA to the cocktail results in more consistent putative bovine iPS colonies. Further work is needed to identify the causes for early differentiation of colonies in order to obtain fully reprogrammed pluripotent stem cells.

SGO1 Maintains Bovine Meiotic and Mitotic Centromeric Cohesions of Sister Chromatids and Directly Affects Embryo Development

Shugoshin (SGO) is a critical factor that enforces cohesion from segregation of paired sister chromatids during mitosis and meiosis. It has been studied mainly in invertebrates. Knowledge of SGO(s) in a mammalian system has only been reported in the mouse and Hela cells. In this study, the functions of SGO1 in bovine oocytes during meiotic maturation, early embryonic development and somatic cell mitosis were investigated. The results showed that SGO1 was expressed from germinal vesicle (GV) to the metaphase II stage. SGO1 accumulated on condensed and scattered chromosomes from pre-metaphase I to metaphase II. The over-expression of SGO1 did not interfere with the process of homologous chromosome separation, although once separated they were unable to move to the opposing spindle poles. This often resulted in the formation of oocytes with 60 replicated chromosomes. Depletion of SGO1 in GV oocytes affected chromosomal separation resulting in abnormal chromosome alignment at a significantly higher proportion than in control oocytes. Knockdown of SGO1 expression significantly decreased the embryonic developmental rate and quality. To further confirm the function(s) of SGO1 during mitosis, bovine embryonic fibroblast cells were transfected with SGO1 siRNAs. SGO1 depletion induced the premature dissociation of chromosomal cohesion at the centromere and along the chromosome arm giving rise to abnormal appearing mitotic patterns. The results of this study infer that SGO1 is involved in the centromeric cohesion of sister chromatids and chromosomal movement towards the spindle poles. Depletion of SGO1 causes arrestment of cell division in meiosis and mitosis.

Growth inhibition of mouse embryonic stem (ES) cells on the feeders from domestic animals

Mouse embryonic stem cells (mESCs) can be propagated in vitro on the feeders of mouse embryonic fibroblasts. In this study, we found growth inhibition of mESCs cultured on embryonic fibroblast feeders derived from different livestock animals. Under the same condition, mESCs derived from mouse embryonic fibroblast feeders were seen on the mass-like colonies and round or oval images, and more significant growth in the total number of colonies ( p <0.05) and viable cells in the colonies ( p <0.01) than that from goat embryonic fibroblast feeders, and viable cells in the colonies ( p <0.05) than that from porcine embryonic fibroblast feeders. The feeders from bovine embryonic fibroblasts also reduced viable cells in the colonies, but were not significantly different in the total number of colonies and viable cells in the colonies with mouse embryonic fibroblast feeders. mESCs on the different embryonic fibroblast feeders were expressed as stem cell-specific markers Oct 4 and stage-specific embryonic antigen 1 (SSEA 1). Here, our results indicate that the feeders from goat, porcine and bovine embryonic fibroblasts inhibit the proliferation of mESCs. Key words : Domestic animals, feeders, mouse embryonic stem cells (mESCs), growth.