Fetal Bovine Serum Treatment Research Articles

Omics insights into spermatozoa activation induced by Fetal bovine serum in viviparous black rockfish (Sebastes schlegelii)

Black rockfish (Sebastes schlegelii) is an economically important marine species with the characteristics of viviparity. The spermatozoa were transferred into the ovary by mating and stored for several months until fertilization. Little is known about spermatozoa activation and its mechanism in black rockfish. In this study, the suitable medium for spermatozoa activation in vitro was explored, and the underlying mechanism was studied by omics analysis. Fetal bovine serum (FBS) could significantly enhance spermatozoa motility in vitro. Omics analysis showed 559 differentially expressed genes (DEGs) and 1311 differentially methylated genes (DMGs) were identified after FBS treatment. Transcriptome analysis revealed that FBS-induced spermatozoa motility activation is associated with spermatozoa capacitation regulated by the cAMP-SRC-PKA, cGMP-PKG and phospholipase D signaling pathway. Spermatozoa capacitation-related gene hsp90aa1 and chemotaxis-related gene cxcr4 were two of the important DMGs. Methylome analysis further revealed that FBS-induced epigenetic modifications are involved in spermatozoa capacitation and chemotaxis. 36 overlaps were identified between DMGs and DEGs, of which five genes were demonstrated to play a role in spermatozoa physiology, required for flagellum stability and spermatozoa motility. The results could provide new clues for understanding spermatozoa activation's molecular mechanism and help establish activation and/or immobilizing media for improving either artificial fertilization or cryopreservation in black rockfish.

MicroRNA-1246 regulates proliferation, invasion, and differentiation in human vascular smooth muscle cells by targeting cystic fibrosis transmembrane conductance regulator (CFTR).

MicroRNA (miRNA) plays a key role in the proliferation and invasion of vascular smooth muscle cells (VSMCs). However, the role and underlying mechanism of miRNAs in VSMCs are not fully understood. Therefore, this study was designed to investigate the role and mechanism of microRNA-1246 (miR-1246) in VSMCs. VSMCs were cultured, and the proliferation of VSMCs was stimulated by platelet-derived growth factor (PDGF-BB) or 15% fetal bovine serum (FBS). The quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of miR-1246 and cystic fibrosis transmembrane conductance regulator (CFTR) in VSMCs. The CCK-8 assay and transwell assay were used to detect the proliferation and invasion of VSMCs. Target gene prediction and screening and luciferase reporter assays were used to verify downstream target genes of miR-1246. Western blotting was used to detect the protein expression levels of PCNA, α-SMA, SM-MHC, Collagen-1, and Cyclin D1 in VSMCs. PDGF-BB and FBS treatment induced VSMCs proliferation and the upregulation of miR-1246 expression. Overexpression of miR-1246 promoted VSMCs proliferation, invasion, and differentiation towards synthetic phenotype, while knockdown of miR-1246 had opposite effects. In addition, CFTR was found to be a direct target for miR-1246, and miR-1246 inhibited the expression of CFTR. Moreover, overexpression of CFTR inhibited VSMC proliferation and synthetic differentiation, while overexpression of miR-1246 partly abolished the effects of CFTR overexpression on VSMCs proliferation and differentiation. Our data suggest that MiR-1246 promotes VSMC proliferation, invasion, and differentiation to synthetic phenotype by regulating CFTR. MiR-1246 may be a potential therapeutic target for atherosclerosis.

Protective effects of docosahexaenoic acid against non-alcoholic hepatic steatosis through activating of JAK2/STAT3 signaling pathway

Non-alcoholic fatty liver disease is the most common cause of hepatic dysfunction. In the present study, human normal hepatocyte L02 cells were treated with 50% fetal bovine serum to induce the formation of hepatic steatosis in vitro, and then the cells were treated with docosahexaenoic acid to investigate its protective effect on Non-alcoholic fatty liver disease. Our results showed that 50% of fetal bovine serum significantly induced intracellular lipid accumulation and hepatocyte fatty degeneration within 48 h. The expression level of adipose formation-related genes was significantly up-regulated, such as PPARγ, C/EBPα and SREBP-1; meanwhile, the content of cellular total lipid, total cholesterol and triglycerides were significantly increased after 50% fetal bovine serum treatment. Interestingly, docosahexaenoic acid treatment could inhibit FBS-induced intracellular lipid accumulation in L02 cells and the expression of lipogenic genes. Moreover, docosahexaenoic acid treatment could reduce hepatic steatosis-induced oxidative stress and endoplasmic reticulum stress response, and these responses were shown by the modification of antioxidant enzyme activities and GRP78, CHOP expression. In addition, the results showed that docosahexaenoic acid can activate the JAK2/STAT3 signaling pathway in fatty liver L02 cell; inhibition of JAK2/STAT3 signaling pathway by WP1066 abolished the beneficial effects of docosahexaenoic acid on hepatic steatosis accompanied with the increased expression of lipogenic genes and endoplasmic reticulum stress response. Above all, the present study showed that docosahexaenoic acid can alleviate non-alcoholic hepatic steatosis by activating JAK2/STAT3 signaling pathway.

P015 Removal of selected non-specific reactivity in single antigen bead assays via FBS pre-treatment

Aim Non-specific reactivity is a known complication of single antigen bead (SAB), solid phase assays. Several approaches have been tried to reduce these reactions but are mostly related to Class I (eg: iBeads, acid treatment). No such treatments exist for Class II. Presently, the current lot of Class II SABs (LS2A01, Lot#: 011) has shown strong non-specific reactivity with some DRB1∗04 and DRB1∗16 bearing beads in patients who are otherwise non-sensitized; MFI values up to 4000. Previously, we reported that pre-treatment of patient serum with fetal bovine serum (FBS) effectively reduced non-specific binding to HLA Class I and Class II FlowPRA beads. In this study we sought to determine if FBS treatment would be effective in reducing/eliminating this non-specific class II reactivity. Methods Sera from 20 non-sensitized patients, each exhibiting the DR4/DR16 pattern, were pre-treated with FBS. Briefly, 5ul of FBS was added to 95ul of patient serum; incubated for 30 min at 37 °C; centrifuged and then used in the SAB assay. As controls, we treated sera from patients with well documented HLA antibody including DR4/DR16 (N = 10) and patients without HLA antibody (N = 10). Results Our results showed that in 100% (20/20) of the effected patients the DR4/DR16 reactivity was eliminated or significantly reduced. (Fig.). Importantly, patients with bona fide HLA antibody had no change in MFI values and additional reactivity was not observed in patients without HLA antibody following FBS treatment. Download : Download high-res image (339KB) Download : Download full-size image Conclusions In this study, we have demonstrated that the non-specific DR4/DR16 reactivity pattern was eliminated by pre-treatment with FBS. We speculate that this pattern is a result of reactivity against bovine peptides (possibly derived from culture media) present in the grove of the DR4/DR16 alleles. Pre-treating sera with FBS provides an exogenous source of soluble target proteins that interact with the antibodies thereby preventing their subsequent interaction with the beads.

Human Fibroblast–Like Cultures in the Presence of Platelet-Rich Plasma as a Single Growth Factor Source

The purpose of this study was to compare the proliferation, morphology, and antigenic expression of human fibroblast-like cells between primary cultures treated with platelet-rich plasma (PRP) or fetal bovine serum (FBS) as the growth factor source. Cells from human gingival tissue samples obtained from healthy volunteers during oral surgery were studied. Isolated cells were cultured in media supplemented with 10% PRP or FBS. Platelet-rich plasma was prepared from the venous blood of each patient. The authors studied short- and long-term cell cultures in the presence of PRP or FBS as the sole growth factor source in order to determine (a) cell growth rate, by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay; (b) cell morphology, by electronic microscopy; and (c) antigenic expression, by flow cytometry and confocal microscopy. In short-term cultures, the cell growth rate was higher with PRP versus FBS treatment. No differences in morphology or expression of vimentin, fibronectin, or α-actin antigens were observed between PRP and FBS cultures. In long-term cultures, PRP and FBS did not significantly differ in cell growth rate but differed in morphology and in the expression of vimentin, fibronectin, and α-actin. The PRP enhances cell proliferation over the short term and induces cell differentiation of fibroblast-like cells to myofibroblast-like cells over the long term, suggesting that fibroblast differentiation to myofibroblasts may underlie the action mechanism of PRP in soft tissue regeneration.

63 THE EFFECT OF A MODIFIED CRYOPRESERVATION METHOD ON THE VIABILITY OF FROZEN–THAWED PRIMORDIAL GERM CELL ON THE KOREAN NATIVE CHICKEN (Ogye)

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus, conservation of genetic material in chickens was attempted by preserving primordial germ cells (PGC) in LN2. This study established a method for preserving chicken PGC that enables long-term storage in LN2 for preservation of species. The purpose of this study was to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on the viability of cryopreserved PGC in the Korean native chicken (Ogye). Primordial germ cells were separated from a germinal gonad using a fine glass micropipette under a microscope and were suspended in a freezing medium containing freezing and protecting agents [e.g. dimethyl sulfoxide (DMSO) and ethylene glycol (EG)]. The PGC were then purified using the magnetic-activated cell sorting (MACS) method. The viability of the PGC in both groups was determined by the trypan blue exclusion method. The values of the 0, 5, 10, and 15% DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36%, respectively. The viability of PGC after freeze-thawing was significantly higher for the 10% EG plus FBS treatment than for the 10% EG plus CS treatment (P < 0.05; 64.36 v. 50.66%). This study established a method for preserving chicken PGC that enables systematic storage and labelling of cryopreserved PGC in LN2 at a germplasm repository and ease of entry into a database. In the future, the importance of this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germ line chimeras.

ROCK inhibitor, Y-27632, reduces FBS-induced structural alteration in organ-cultured mesenteric artery

Abstract Background Chronic treatment with fetal bovine serum (FBS) causes gradual vasoconstriction, vascular wall thickening, and contractility reduction in organ-cultured vascular tissues. We have previously demonstrated that Rho-associated kinase (ROCK) inhibitors prevent the functional alterations of small arteries in response to the FBS treatment. Here, we tested a further hypothesis that the chronic inhibition of ROCK has a protective effect on FBS-induced structural alterations. Methods To verify the new hypothesis, the rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor and then western blot, immunohistochemistry, apoptosis assay, and electron microscopy were performed using organ-cultured arterial rings. Results Chronic treatment with Y-27632 maintained the arterial diameter by preventing FBS-induced gradual arterial constriction during organ culture. Y-27632 also reduced the apoptosis and the loss of contractile myosin and actin filaments of smooth muscle cells. In addition, Y-27632 protected the morphological integrity between the endothelial cell layer and smooth muscle cell layer by preventing endothelial cell detachment and platelet endothelial cell adhesion molecule (PECAM) expression decrement. Conclusions Chronic ROCK inhibition provides protective effects against FBS-stimulated structural in addition to functional alterations of vascular smooth muscle cells and endothelial cells. These results strongly suggest that the RhoA/ROCK signaling is crucial for maintaining the structural and functional phenotypes of vasculature, and hence, chronic ROCK inhibition may provide protective effects on excessive growth factor-related vascular diseases including hypertension and atherosclerosis.

한국재래닭 (오계) 원시생식세포에 있어 동결방법의 개선이 융해 후 생존율에 미치는 영향

This study was conducted to establish methods for preserving chicken primordial germ cells (PGCs) for long-term storage in liquid nitrogen and for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on the viability of cryopreserved PGCs from Korean Native Chicken (Ogye). PGCs separated from a germinal gonad of an early embryo at day 5.5-6 (stage 28) were suspended in a freezing medium containing freezing and protective agents (dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The values from 0, 5, 10, and 15 % DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36 %, respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% EG + FCS treatment (p

Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids.

Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was 15.1±0.2×104 as the least for the low density group, and 29.3±2.8×104 as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids.

An in vitro comparison study: The effects of fetal bovine serum concentration on retinal progenitor cell multipotentiality

Retinal progenitor cells (RPCs) are an excellent resource for retinal replacement therapy, because they show enormous potential to differentiate into retinal-specific cell types. While the differentiating influence of serum has long been appreciated, the effects of serum concentration on RPC differentiation into specified retinal neural cells have not been investigated. Using cultured murine RPCs, this study compared the effects of different levels of fetal bovine serum (FBS) (1%, 5%, 10% and 20%) on RPC differentiation in vitro. RPC multipotentiality was assessed by using quantitative polymerase chain reaction (qPCR) to determine the relative expression levels of 10 genes involved in retinal development. In addition, analyses of cell morphology and retinal development-related protein expression were performed using microscopy and immunocytochemistry. The data revealed that 1% FBS-induced cultures preferentially generated rhodopsin- and PKC-α-positive cells. Calbindin and AP2α expression levels were greater in 5% FBS-induced cultures. Brn3a was expressed at similar levels in 1%, 5% and 10% FBS treatment conditions but diminished in 20% FBS conditions. Twenty percent FBS induced more glial fibrillary acid protein (GFAP)-immunoreactive cells corresponding to glia populations. These findings suggest that the concentration of FBS plays an important role in RPC differentiation in vitro. Treatment with low levels of FBS favors differentiation of rhodopsin-positive photoreceptors, interneurons and retinal ganglion cells (RGCs), while high FBS concentrations preferentially induce differentiation of glia cells. These results are expected to facilitate research in the treatment of neurodegenerative retinal diseases.

Establishment and Characterization of Gene Expression of Two New Ovine Mammary Cell Lines: Preliminary Results.

Some post-translational modifications are necessary for the production of biopharmaceutical proteins, with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression is an essential work. The main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as beta lactoglobulin, alpha casein, beta casein and kappa casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed beta lactoglobulin and beta casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0.001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed beta lactoglobulin, beta casein and kappa casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of beta lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P<0.05). However, the medium did not have effect on growth speed of LD (P>0.05) and no difference was observed at the NDL treated with LOS (P>0.05). The NDL was positive for staining with vimentin and cytokeratin. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of beta lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. Research supported by FAPESP (2008/56138-2) and CNPq. (poster)

A role for SOX2 in the generation of microtubule-associated protein 2-positive cells from microglia

We recently demonstrated that, as a type of multipotential stem cells, microglia give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells. In this study, we investigated the role of SOX2, a high-mobility group DNA binding domain transcription factor, in the generation of microglia-derived MAP2-positive and GFAP-positive cells. Western blot analysis demonstrated that expression of SOX2 was upregulated by treatment with 70% fetal bovine serum treatment. Immunocytochemical analyses demonstrated that SOX2 expression was evident in the nuclei of microglia-derived MAP2-positive and GFAP-positive cells, whereas it was not present in the nuclei of microglia. These assays also showed that Sox2 siRNA inhibited the generation of MAP2-positive and GFAP-positive cells from microglia. Interestingly, this activity was also inhibited by Smad4 siRNA, which reduces SOX2 expression. These results indicate that SOX2 upregulation is involved in the generation of microglia-derived MAP2-positive and GFAP-positive cells through SMAD4.

Role of Nuclear Factor-κB and Protein Kinase C Signaling in the Expression of the Kinin B1Receptor in Human Vascular Smooth Muscle Cells

Kinin B1 receptor expression was characterized in human umbilical artery smooth muscle cells to further elucidate the function and specificity of three previously proposed pathways [nuclear factor-kappaB (NF-kappaB), protein kinase C, and agonist autoregulation] that regulate this inducible G protein-coupled receptor. Radioligand binding assays, real-time reverser transcription/polymerase chain reaction with an optional actinomycin D treatment period, and NF-kappaB immunofluorescence were primarily employed in these primary cell cultures. Various stimulatory compounds that increase receptor mRNA stability only (human and bovine sera, cycloheximide) or that stimulate NF-kappaB nuclear translocation and both mRNA concentration and stability [interleukin (IL)-1beta, phorbol 12-myristate 13-acetate (PMA)] all increased the density of binding sites for the tritiated B1 receptor agonist [3H]Lys-des-Arg9-bradykinin (without change in receptor affinity) in cell-based assays. Small interfering RNA assays indicated that NF-kappaB p65 is necessary for the effective expression of the cell surface B1 receptor under basal or IL-1beta, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1beta-, FBS-, or PMA-induced stabilization of B1 receptor mRNA was inhibited by the addition of the protein kinase C inhibitor 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF-109203x), which also diminished the Bmax under FBS or PMA treatment. Lys-des-Arg9-bradykinin had little effect on NF-kappaB activation, the Bmax, or receptor mRNA abundance or stability. Both NF-kappaB and protein kinase C signaling are required for the effective expression of the kinin B1 receptor in human umbilical artery smooth muscle cells.

Fetal bovine serum suppresses apoptosis in the small intestine after total ischemia and reperfusion in mice

Background/Purpose Total ischemia/reperfusion (I/R) of the small intestine induces cellular apoptosis. In this study, the authors investigated the effects of fetal bovine serum (FBS) on apoptosis and related proapoptotic and antiapoptotic factors in the I/R-injured small intestine of mice. Methods The mice underwent total I/R of the small intestine and were treated with oral gavages of normal saline or FBS for 3 days, concluding with total ischemia of the small intestine. Samples of the I/R-injured small intestine, representing various predefined time-points post-I/R (immediately before and after 1 hour of total ischemia and at 1, 6, and 24 hours after initiation of reperfusion), were subjected to terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining to study the cellular apoptosis, and Western blot analysis to measure expression of p53, bcl-2 and bax, with caspase-activity assay used to determine the activity of caspase 3. Results For the saline-treated mice, increased cellular apoptosis, suppressed expression of p53 and bcl-2 (with decreased bcl-2 to bax ratio) and increased caspase-3 activity were found for the I/R-injured small intestine. By contrast, FBS treatment suppressed cellular apoptosis, producing up-regulation of p53 and bcl-2 (with increased bcl-2 to bax ratio to 5.4-fold of the saline-treated group) and inhibiting the activity of caspase 3 (P < .05). Conclusions The results of our study suggest that I/R-induced apoptosis of the mouse small intestine may be related to p53 and bcl-2 suppression (with decreased bcl-2 to bax ratio) and activation of caspase 3 and that such apoptosis seems to be suppressed by FBS treatment.

P‐II‐04 Early Phase Regulation of Human Mesenchymal Stem Cells and the Unique Profile of the Proliferation by Bone Morphogenetic Protein‐2

Human mesenchymal stem cells (hMSCs) obtained from a single donor of the iliac crest, was further investigated for the cell proliferation, cell cycle profiles, gene expressions and the ultrastructures by electron microscopy. The hMSCs significantly increased the cell number by day 2 after by treatment of bone morphogenetic protein (BMP)‐2 alone, or basic fibroblast growth factor (bFGF) alone or combination of both in the serum‐free condition (P &lt; 0.01). The hMSCs demonstrated the remarkable proliferation cell nuclear antigen notably at day 1 and pituitary tumor transforming gene all through the experiment, suggesting the cell cycle progression by BMP‐2 treatment as well as the strong cellular nuclear BrdU expression by immunocytochemistry. The fluorescence‐activated cell sorter also demonstrated the similar pattern of the cell cycle progression between BMP‐2 treatment in the serum‐free and 10 % fetal bovine serum treatment. The BMP‐2 treated hMSCs demonstrated the heterochromatin in the nucleus, suggesting the cell differentiation and the well‐developed granular endoplasmic reticulums, indicating the protein production. The hMSCs successfully proliferate, the cell cycle is progressed, and the cell ultrastructure morphology suggest the remarkable nuclear and of granular endoplasmic reticulum induction by BMP‐2 treatment in the serum‐free condition.

Bone morphogenetic protein-2 regulates proliferation of human mesenchymal stem cells.

Human mesenchymal stem cells obtained from the iliac crest of a single donor were investigated for cell proliferation, cell cycle profile, gene expression, and ultrastructural changes using electron microscopy. The human mesenchymal stem cells significantly increased their cell number by day 2 after treatment with bone morphogenetic protein-2 alone, or basic fibroblast growth factor alone or combinations of both proteins under serum-free conditions (p < 0.01). The human mesenchymal stem cells showed marked expression of cell nuclear antigen, notably at day 1, and pituitary tumor transforming gene throughout the experiment, suggesting cell cycle progression by bone morphogenetic protein-2 treatment. In addition, strong cellular nuclear bromodeoxyuridine incorporation was seen by immunocytochemistry. Fluorescence-activated cell sorting also showed a similar pattern of cell cycle progression with bone morphogenetic protein-2 treatment in serum-free medium and 10% fetal bovine serum treatment. The bone morphogenetic protein-2-treated human mesenchymal stem cells showed heterochromatin in the nucleus, suggesting cell differentiation, and well-developed granular endoplasmic reticulum, indicative of protein production. Overall, the human mesenchymal stem cells successfully proliferated with appropriate cell cycle progression and the cell ultrastructural morphology suggested marked nuclear and granular endoplasmic reticulum induction by bone morphogenetic protein-2 treatment in serum-free medium.

Regulation of oxytocin receptor expression in cultured human myometrial cells by fetal bovine serum and lysophospholipids.

Oxytocin receptor (OTR) expression in human myometrium increases over 150-fold from the beginning of pregnancy to the end. In the present studies, we examined potential mechanisms of OTR up-regulation, using myometrial cells in primary culture from women in late gestation. OTR ligand-binding sites and steady-state mRNA levels were down regulated by serum starvation, and up-regulated by restoration of fetal bovine serum (FBS). Transcriptional activity of the OTR gene was the same with or without FBS treatment, but FBS increased OTR mRNA half-life about 5-fold. Lysophospholipids (lysophosphatidic acid and sphingosine 1-phosphate), which are present in serum, had similar effects as FBS. Lysophospholipid receptor mRNAs of the endothelial differentiation gene (Edg) family (Edgs 1, 3, 4, and 5) were demonstrated in myometrial cells by RT-PCR. These G protein-coupled receptors have been shown to be coupled to G(i/o) and to mediate activation of phosphoinositol 3-phosphate kinase. Indeed, the effects of the lysophospholipids and FBS were completely blocked by pertussis toxin, a G(i/o) inhibitor. Likewise, inhibition of G(i/o) signaling by elevation of intracellular cAMP or inhibition of phosphoinositol 3-phosphate kinase blocked FBS effects on OTR mRNA stability. We do not presently understand the mechanisms of OTR up-regulation in human myometrium in vivo, but the present studies might lead to the description of mRNA-stabilizing factors whose activity can be quantified in tissue samples during pregnancy to elucidate the process of OTR up-regulation.

Acute hypoxia induces elevation of ornithine decarboxylase activity in neonatal rat brain slices.

Recent studies in vivo have demonstrated that ornithine decarboxylase (ODC) activity in the fetal rat brain is elevated 4-5-fold by acute maternal hypoxia. This hypoxic-associated increase is seen in the rat brain in both the newborn and the adult. Because of the intimate involvement of ODC in transcription and translation, as well as in growth and development, it is imperative that the manner in which hypoxia affects the regulation of this enzyme be better understood. In order to achieve this, a brain preparation in vitro was required to eliminate the confounding effects of the dam on the fetal and newborn brain ODC activity in vivo. Therefore, brain slices from 3-4-day-old (P-3) newborn rats were utilized to test the hypothesis that ODC activity increases in response to hypoxia in vitro. Cerebral slices from the P-3 rat pups were allowed to equilibrate and recover in artificial cerebrospinal fluid (ACSF) continuously bubbled with a mixture of 95% O2 and 5% CO2 for 1 h before beginning hypoxic exposures. Higher basal ODC activities were obtained by treating the slices with 0.03% fetal bovine serum (FBS) and 0.003% bovine serum albumin (BSA), rather than with ACSF alone. Hypoxia was induced in the slices by replacing the gas with 40%, 21%, 10%, or 5% O2, all with 5% CO2 and balance N2. With FBS and BSA treatment, ODC activity was maintained at about 0.15-0.11 nM CO2 mg-1 protein h-1 throughout the experiment, which was 2-3-fold higher than that without FBS and BSA. ODC activity increased significantly and peaked between 1 h and 2 h after initiation of hypoxia. For instance, with 21% O2, ODC activity increased approximately 1.5-fold at 1 h and approximately 2-fold at 2 h. These studies demonstrate that: (1) the hypoxic-induced increases observed in vivo in the fetal and newborn rat brain ODC activity can be approximated in a newborn rat brain slice preparation in vitro; (2) newborn rat brain slice preparations may provide an alternative to methods in vivo or cell culture methods for studying the regulation of acute hypoxic-induced enzymes; and (3) high, stable baseline ODC activities in brain slices suggest that the cells in the slice are capable of active metabolism if FBS and BSA are available to mimic conditions in vivo.

Adipocyte differentiation selectively represses the serum inducibility of c-jun and junB by reversible transcription-dependent mechanisms.

Nonterminally differentiated 3T3 T adipocytes are resistant to growth stimulation by 10% (vol/vol) fetal bovine serum even though they can be induced to proliferate with extremely high serum concentrations. We now report that in adipocytes 10% fetal bovine serum also fails to typically induce c-jun or junB. Rather, after 10% fetal bovine serum treatment, c-jun and junB expression is markedly repressed after a brief initial slight induction. Gel mobility shift studies confirm that AP-1 DNA binding activity is inhibited in adipocytes. Repression in c-jun and junB inducibility in adipocytes results from transcriptional mechanisms, can be reversed by treatment with protein synthesis inhibitors or higher serum concentrations, and does not affect c-fos or c-myc expression. These data suggest that adipocyte differentiation selectively and transcriptionally represses the inducibility of c-jun and junB so as to decrease the cell's ability to proliferate in response to 10% fetal bovine serum.

Effects of serum type on growth and permeability properties of cultured endothelial cells

Serum is frequently added to defined basal media as a source of certain nutrients and macromolecular growth factors essential for cell growth. The many different sera commercially available may not be equally suitable for all cell types. The effects of four sera, fetal bovine serum (FBS), calf bovine serum (CS), equine serum (ES-1), and plasma-derived equine serum (ES-2), on growth and permeability properties of cultured porcine endothelial cells were determined. The rate of DNA synthesis, measured as [ 3H]thymidine incorporation, reached a peak at around 24 h, regardless of serum type, and was most marked with ES-1- or ES-2-treated cells. However, when estimated by total DNA, FBS, CS, or ES-1 treatment resulted in greater cell proliferation than ES-2. Based on protein synthetic rate and total cell protein, both FBS and CS appeared to be most growth supporting. At 72 h after cell plating, albumin passage across cultured endothelial monolayers was elevated in ES-1- and ES-2-treated cells compared with FBS- or CS-treated cells. “Leaky” cell monolayers were most marked with ES-1-treated cells. Cells grown in ES-2- and particularly in ES-1-enriched media were larger and more spindle-shaped compared with the typical cobblestone appearance of cells cultured in media enriched with either FBS or CS. These data suggest that CS, but not ES-1 or ES-2, is an excellent substitute for FBS to support desirable growth properties of macrovascular endothelial cells in culture.