Bovine Calf Serum Research Articles

In vitro wear assessments of fixed and mobile UHMWPE total knee replacement

This work discusses the wear behaviour of two different ultra-high-molecular-weight-polyethylene tibial component designs. Mobile and fixed bearings were tested on a knee wear simulator for 5 million cycles using bovine calf serum as lubricant. We correlated the wear results with the chemical characterisation of the investigated materials: Fourier Transformed Infra Red Spectroscopy analyses, Differential Scanning Calorimetry and cross-link density measurements were used to assess the chemical features of this polyethylene.Mobile and fixed polyethylene inserts showed a different wear behaviour: the mobile designs components showed lower weight losses than the fixed components (109±6mg and 163±80mg, respectively). Significant statistical differences were observed in wear rate (P=0.035, Kolmogorov–Smirnov Test for two samples).From a molecular point of view, typical radiation-induced oxidation profiles were observed in all the tested polyethylene samples, but the overall degradation was more significant in the fixed bearing inserts and this is likely to play a role on the wear performances.

Tribocorrosion behavior of DLC-coated CoCrMo alloy in simulated biological environment

Due to renewed interest in hard-on-hard prosthetic joint replacements, especially metal-on-metal (MOM) and ceramic-on-metal joints (COM), concerns about debris and released metal ions from wear and mechanical-enhanced electrochemical (tribocorrosion) processes have raised. In order to reduce the metal debris quantity and the release of metal ions, a diamond like carbon (DLC) film is deposited on CoCrMo alloy by using a filtered cathodic vacuum arc technique (FCVA). The tribocorrosion behavior of DLC coated CoCrMo alloy is studied in bovine calf serum and 0.9% NaCl solution by using a linear reciprocating pin-on-plate tribometer with an integrated electrochemical cell. The open circuit potential (OCP) and the polarization test are monitored during the reciprocating pin-on-plate test. The results show that the absorbed protein layer on DLC surface play a positive role in reducing wear and metal ion release. The presence of DLC layer improves the tribocorrosion resistance of CoCrMo alloy. And The DLC film has a potential application on hard-on-hard prosthetic joint replacements and it can effectively reduce the metal debris quantity and the release of metal ions.

PAGE4 Positivity Is Associated with Attenuated AR Signaling and Predicts Patient Survival in Hormone-Naive Prostate Cancer

Aberrant activation of the androgen receptor (AR) plays a key role during prostate cancer (PCa) development and progression to castration-resistant prostate cancer (CR-PCa) after androgen deprivation therapy, the mainstay systemic treatment for PCa. New strategies to abrogate AR activity and biomarkers that predict aggressive tumor behavior are essential for improved therapeutic intervention. PCa tissue microarrays herein reveal that prostate-associated gene 4 (PAGE4), an X-linked cancer/testis antigen, is highly up-regulated in the epithelium of preneoplastic lesions compared with benign epithelium, but subsequently decreases with tumor progression. We show that AR signaling is attenuated in PAGE4-expressing cells both in vitro and in vivo, most likely via impaired androgen-induced AR nuclear translocation and subsequently reduced AR protein stabilization and phosphorylation at serines 81 and 213. Consistently, epithelial PAGE4 protein levels inversely correlated with AR activation status in hormone-naive and CR-PCa clinical specimens. Moreover, PAGE4 impaired the development of CR-PCa xenografts, and strong PAGE4 immunoreactivity independently predicted favorable patient survival in hormone-naive PCa. Collectively, these data suggest that dysregulation of epithelial PAGE4 modulates AR signaling, thereby promoting progression to advanced lethal PCa and highlight the potential value of PAGE4 as a prognostic and therapeutic target.

An investigation into PEEK-on-PEEK as a bearing surface candidate for cervical total disc replacement

Total disc replacement (TDR) is a relatively new reconstructive non-fusion technology for the spine that aims to overcome some of the limitations of fusion technology. The first generation artificial discs were mainly based on well-known material combinations from total hip replacement. To evaluate the feasibility of PEEK-on-PEEK as a bearing surface material for use in cervical TDR. In vitro biotribological study including the assessment of different parameters, including the influence of radial clearance, axial load, and angular motion on the wear performance of a PEEK-on-PEEK bearing. PEEK-on-PEEK bearing couples with ball-and-socket articulation were prepared using standard production methods. Two groups of bearing couples, one with large radial clearances and the other with small radial clearances, were manufactured for testing. Wear tests were performed using a servo-hydraulic wear simulator that allowed controlled motions to be applied in three axes corresponding to flexion-extension, lateral bending, and axial rotation. In addition, a dynamic axial compressive load was applied to simulate the weight of the head. All tests were performed at 37°C (±2°C) in bovine calf serum with a 30 g/l protein concentration. In the first test series, the influence of the radial clearance on the wear behavior was evaluated using the load and motion parameters for cervical TDR as defined in the ISO standard 18192-1. Subsequent tests were performed to check if surface degradation was influenced by thermal effects. Finally, in a third series of tests, the effect of load and motion profiles on wear performance was investigated by applying increased loads and corresponding motion parameters for lumbar TDR as defined in the ISO standard 18192-1. The measured wear rates for a PEEK-on-PEEK bearing under cervical test conditions were considerably larger than those of similar testing using UHMWPE-on-CoCr and seemed to depend on initial radial clearances. The PEEK bearing surfaces exhibited severe surface degradation for large and small clearances, but the group with smaller clearances seemed to have less severe damage. Thermal effects were not found to play a role in the surface degradation, as the temperature near the bearing surface was measured and found to vary only a few degrees during testing. A change of the wear test parameters to simulate lumbar loading resulted in a considerable wear rate reduction as well as in the preservation of the bearing surfaces. It was found that a cervical TDR using a PEEK-on-PEEK bearing may be subjected to severe long-term degradation of the bearing surfaces. In this study, the use of PEEK as an articulation material for cervical TDR was found to be sensitive to loading conditions as well as to the initial clearance of the bearing surfaces. Despite the excellent properties of PEEK as an implant material, its use for articulating surfaces for cervical TDR must be critically reviewed.

Self-assembled polymeric nanoparticles based on oleic acid-grafted chitosan oligosaccharide: biocompatibility, protein adsorption and cellular uptake

Oleic acid-grafted chitosan oligosaccharide (OA-g-CSO) was synthesized to prepare self-assembled nanoparticles by sonication at physiological pH value (7.4). The nanoparticles appeared to be spherical in shape with a diameter of 158.1±64.3nm. The biocompatibility of OA-g-CSO nanoparticles was evaluated in vitro via MTT assay and hemolysis test. The nanoparticles showed no cytotoxicity to mouse embryo fibroblasts and the hemolysis rates came well within permissible limits (<2%) in the tested conditions. When incubated with bovine calf serum, the protein adsorption on the surface of OA-g-CSO nanoparticles was concentration-dependent, and the amount of bovine serum albumin was in the highest proportion of the total amount of adsorbed proteins. Cellular uptake rate was evaluated by incubating fluorescence labeled OA-g-CSO nanoparticles with human lung carcinoma cells (A549). OA-g-CSO nanoparticles could be taken up by A549 cells, and the uptake rates increased with incubation time and particle concentration.

Guidelines on the investigation and management of antiphospholipid syndrome

This guidance updates and replaces the previous guideline on the investigation and management of antiphospholipid syndrome (APS) published in 2000 (Greaves et al, 2000), though where there have not been changes we refer back to them when appropriate. The guidance is updated with reference to relevant publications since 2000. Publications known to the writing group were supplemented with additional papers identified by searching PubMed for publications in the last 11 years using the key words: lupus anticoagulant, anticardiolipin, antiphospholipid, b2–glycoprotein I, antiprothrombin and limits (clinical trial, randomized control trial, meta-analysis, humans, core clinical journals, English language). The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 50 UK haematologists, the Royal College of Obstetricians and Gynaecologists (RCOG), and the British Committee for Standards in Haematology (BCSH) Committee and comments incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found at http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMEN DATION/43_GRADE.html. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of patients with antiphospholipid syndrome though individual patient circumstances may dictate an alternative approach.

Synthesis of AIZS@SiO2core–shell nanoparticles for cellular imaging applications

I–III–VI2 semiconductor nanoparticles such as AgInS2 have been developed for cell imaging applications in recent years. However, there are few published works reporting the phase transfer (from hydrophobic to hydrophilic) of the AgInS2 nanoparticles for biomedical application purposes. In this work, we report the successful silica coating for the as-synthesized Zn-doped AgInS2 (AIZS) nanoparticlesvia a templated coating route. The silica shell thickness of the obtained AIZS@SiO2 core–shell nanoparticles could be well controlled within 10–20 nm and their overall size was less than 50 nm. As compared to the AIZS nanoparticles stabilized by cetyltrimethylammonium bromide (CTAB), the obtained AIZS@SiO2 nanoparticles showed much improved photoluminescence (PL) stability in water. The obtained core–shell nanoparticles were further conjugated with the PEG functional group, and the resultant PEGylated nanoparticles showed excellent colloidal stability in various media such as water, phosphate buffered saline (PBS) and bovine calf serum (BCS). The cellular imaging application of the obtained AIZS@SiO2 nanoparticles was also demonstrated successfully.

Follicular fluid supplementation during in vitro maturation promotes sperm penetration in bovine oocytes by enhancing cumulus expansion and increasing mitochondrial activity in oocytes

The aim of this study was to examine the effects of bovine follicular fluid (bFF) on mitochondrial activity in in vitro-matured (IVM) oocytes and to assess its importance for fertilisation and embryo development. Bovine follicular oocytes were subjected to IVM in medium supplemented either with polyvinylpyrrolidone, bovine serum albumin, calf serum or bFF. Nuclear maturation, cumulus expansion, mitochondrial distribution and ATP content in oocytes were compared between groups along with subsequent in vitro fertilisation (IVF) and embryo development. Compared with other supplements, bFF generated significantly enhanced re-distribution of active mitochondria in oocytes and this effect was associated with elevated intracellular ATP content. Furthermore, bFF significantly improved cumulus expansion, which was associated with improved fertilisation rates when cumulus-enclosed oocytes were subjected to IVF; however, its promoting effect was neutralised when denuded oocytes were inseminated. Elevating ATP content in oocytes by bFF did not affect maturation or embryo development but promoted fertilisation when mitochondrial electron transport was blocked in oocytes before IVF by Rotenone. In conclusion, supplementation of IVM medium with bFF promotes sperm penetration both by the improvement of cumulus expansion and by enhancing ATP levels in oocytes, which maintains their ability to be fertilised after mitochondrial stress.

In vitro wear performance of standard, crosslinked, and vitamin‐E‐blended UHMWPE

Crosslinked vitamin-E-stabilized polyethylene acetabular cups were compared with both commercially available conventional and custom-crosslinked polyethylene acetabular cups in terms of wear behavior, in a hip joint simulator for five millions cycles, using bovine calf serum as lubricant. We correlated the wear experiments results with the chemical characterization of the investigated materials: Fourier transformed infrared (FTIR) spectroscopic analyses, differential scanning calorimetry, and crosslink density measurements were used to assess the chemical characteristics of the pristine materials. In addition, further FTIR analyses and cyclohexane extraction were carried out after the simulator experiments. Lipids absorption was observed in all tested specimens and it has been shown to strongly affect the results of the wear test. Corrected gravimetric wear measurements showed that vitamin-E blended, crosslinked polyethylene wore more than the traditional crosslinked polyethylene but exhibited a much lower wear than conventional ultrahigh-molecular weight polyethylene. The chemical analyses showed that the addition of vitamin E reduced the crosslinking efficiency. Given the correlation between crosslink density and wear resistance, this gave an explanation for the observed wear performances.

Development of an inactivated iridovirus vaccine against turbot viral reddish body syndrome

Turbot (Scophthalmus maximus L.) reddish body iridovirus (TRBIV) was propagated in turbot fin cells (TF cells) and inactivated as the TRBIV vaccine with its protection efficiency evaluated in this study. TF cells were cultured in 10% bovine calf serum (BCS)-containing MEM medium (pH7.0) at 22°C, in which TRBIV propagated to a titer as high as 105.6 TCID50 mL−1. The TRBIV was inactivated with 0.1% formalin and formulated with 0.5% aluminum hydroxide. The inactivated vaccine caused neither cytopathogenic effect (CPE) on TF cells nor pathogenic effect on turbots. After being administered with the vaccine twice via muscle injection, the turbot developed high-tittered TRBIV neutralizing antibodies in a dose-dependent manner. The vaccine protected the turbot from dying with an immunoprotection rate of 83.3% as was determined via subcutaneous vaccination in the laboratory and 90.5% via bath vaccination in turbot farms, respectively. The inactivated vaccine was very immunogenic, efficiently preventing turbot from death. It holds the potential of being applied in aquaculture.

Indium as a central metal enhances the photosensitizing efficacy of benzoporphyrin derivatives

Compared to benzoporphyrin derivative-dimethyl ester (BPD-DME) and its 8-(1'-hexyloxy)ethyl analog the corresponding In(III) complexes showed enhanced in vitro photosensitizing efficacy in Colon26 tumor cells, which could be due to their higher singlet oxygen producing ability. In both organic (methanol) and aqueous Bovine Calf Serum (17% BCS) solutions the metalated analogs were significantly more stable than the parent photosensitizers. Presence of Indium as a central metal gave 13-25 nm hypsochromic shift to the long wavelength absorption band with reduced absorption and fluorescence intensity. The insertion of metal did not produce any difference in intracellular localization of the photosensitizers and were mainly localized in mitochondria.

Tribological characterisation of Zr-based bulk metallic glass in simulated physiological media

Due to their excellent wear resistant properties and high strength, as well as a low Young's modulus, Zr-based bulk metallic glasses (BMGs) are potentially suitable biomaterials for low-friction arthroplasty. The wear characteristics of the Zr60.14Cu22.31Fe4.85Al9.7Ag3 bulk amorphous alloy against ultra-high-molecular-weight polyethylene (UHMWPE) compared to a CoCrMo/UHMWPE combination were investigated in two different wear screening test devices, reciprocating and unidirectional. Hank's solution and sterile calf bovine serum were selected as the lubricant fluid media. It was found that different fluid media had insignificant effect on polyethylene wear against BMG counterfaces. The wear behaviour obtained on both test devices demonstrated that Zr-based BMG achieved UHMWPE counterface wear rates superior to conventional cast CoCrMo alloy, where the wear rate of UHMWPE is decreased by over 20 times. The tribological performance of these joints is superior to that of conventional metal-on-polymer designs. Contact angle measurements suggested that the advantage of BMG over a CoCrMo alloy counterface is attributed to its highly hydrophilic surfaces.

Novel magnetic bovine serum albumin imprinted polymers with a matrix of carbon nanotubes, and their application to protein separation

Novel magnetic multi-walled carbon nanotubes@Fe(3)O(4) molecularly imprinted polymers (MWNTs@Fe(3)O(4)-MIPs) intended for bovine serum albumin (BSA) recognition were successfully developed. The MWNTs@Fe(3)O(4)-MIPs were characterized with scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR). Scanning electron microscopy images showed that the Fe(3)O(4) nanoparticles (diameter: 50-60 nm) were coated with a layer of MIPs with an average thickness of 25-30 nm. The magnetic material was easily dispersed and retrieved through the application of an external magnetic field. Adsorption experiments showed that the estimated maximum amount of BSA that could be adsorbed onto the MWNTs@Fe(3)O(4)-MIPs was 52.8 mg/g, and the time taken to reach equilibrium was about 40 min. Meanwhile, the MWNTs@Fe(3)O(4)-MIPs exhibited excellent selectivity towards (i.e., recognition of) BSA. The feasibility of the use of the MWNTs@Fe(3)O(4)-MIPs as a solid-phase extraction (SPE) sorbent was evaluated, and the results showed that the MWNTs@Fe(3)O(4)-MIPs were able to separate the template protein BSA from a binary protein solution. The proposed sorbent based on MWNTs@Fe(3)O(4)-MIPs for BSA separation exhibited satisfactory recoveries ranging from 92.0% to 97.3% in real samples. It was also successfully used for the purification of BSA from bovine calf serum.

A Role for Prolyl 3-Hydroxylase 2 in Post-translational Modification of Fibril-forming Collagens

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.

A laboratory exercise illustrating the sensitivity and specificity of Western blot analysis

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the separated polypeptides from the gel is created on the membrane, which is then probed with antibodies or other ligands to identify specific polypeptide(s). Here, we report an undergraduate laboratory exercise involving Western blotting. During the 3-week laboratory exercise, students investigated the likelihood of the presence of a serum albumin in fruit fly (Drosophila melanogaster) homogenate, bovine calf serum (BCS), and fetal bovine serum (FBS). Students isolate proteins from fruit fly larvae and perform sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic blotting, and immunoassay comparing those proteins with BCS and FBS proteins. Their results indicate that serum albumin is present in BCS and FBS but is absent in fruit flies. In the process, the specificity and sensitivity of Western blot analysis is demonstrated. The laboratory exercise can be easily incorporated into any college-level biochemistry or molecular techniques laboratory. The procedure used can be easily adapted to study other proteins.

Tribocorrosion behavior of CoCrMo alloy for hip prosthesis as a function of loads: A comparison between two testing systems

Metal-on-metal (MOM) hip prosthesis bearings have enjoyed renewed popularity, but concerns remain with wear debris and metal ion release causing a negative response in the surrounding tissues. Further understanding into the wear and corrosion mechanisms occurring in MOM hips is therefore essential.The purpose of this study was to evaluate the tribocorrosion behaviour, or interplay between corrosion and wear, of a low-carbon CoCrMo alloy as a function of loading. The tribocorrosion tests were performed using two tribometer configurations. In the first configuration, "System A", a linearly reciprocating alumina ball slid against the flat metal immersed in a phosphate buffer solution (PBS). In the second configuration, "System B", the flat end of a cylindrical metal pin was pressed against an alumina ball that oscillated rotationally, using bovine calf serum (BCS) as the lubricant and electrolyte. System B was custom-built to emulate in vivo conditions. The tribocorrosion tests were performed under potentiostatic conditions at -0.345V, with a sliding duration of 1800 seconds and a frequency of 1Hz. In System A the applied loads were 0.05, 0.5, and 1N (138, 296 and 373MPa, respectively) and in System B were 16, 32, and 64N (474, 597, and 752MPa, respectively). Electrochemical impedance spectroscopy (EIS) and polarization resistance were estimated. The total mass loss (K(wc)) in the CoCrMo was determined. The mass loss due to wear (K(w)) and that due to corrosion (K(c)) were determined. The dominant wear regime for the CoCrMo alloy subjected to sliding changes from wear-corrosion to mechanical wear as the contact stress increases. An attempt was made to compare both system, in their tribochemical responses and formulate some insights in the total degradation processes. Our results also suggest that the proteins in the serum lubricant assist in the generation of a protective layer against corrosion during sliding. The study highlights the need of adequate methodology/guidelines to compare the results from different test systems and translating in solving the practical problems.

Construction of a tribocorrosion test apparatus for the hip joint: Validation, test methodology and analysis

a b s t r a c t In biomedical research, because of the critical nature of corrosion within the environment of motion and interactions of weight bearing surfaces, scientists and engineers have shown a growing interest in the area of tribocorrosion. Fundamentally, tribocorrosion combines the disciplines of tribology and corrosion and their concurrent exchanges. Principles of tribocorrosion are applied in a range of industries including the offshore, space and biomedical (for example, dental and orthopedic) industries. One of the challenges in tribocorrosion research is the lack of standard testing equipment and method- ologies, particularly in its bio-medical applications. Many investigators and research laboratories are engaged in modifying their existing classical tribology apparatus, whether they are commercially avail- able or customized tribometers, to integrate electrochemical monitoring. The current work is focused on developing a tribocorrosion test apparatus to model the contact situation of artificial hip joints with particular attention on possible erroneous issues that could arise in such a system. The test system facilitates studies on the tribocorrosion behavior of implant metals or coatings as a function of diverse tribological and electrochemical parameters, and allows monitoring temperature, pH and dissolved oxygen level during testing. Initial results on the HC CoCrMo alloy in bovine calf serum (BCS), indicate that the proposed system is reliable and useful for the investigation of tribocorrosion behavior of implant materials. Electrochemical impedance spectroscopy tests (EIS) are employed before and after the sliding tests. The usefulness of such tests in understanding the changes in the surface chemistry and corrosion kinetics are explained, with the help of optimum EIS models. An attempt has also been made to highlight the relevance on wear quantification methods and possible ways to analyse the synergistic interactions of wear and corrosion for the biomedical tribosystems in orthopedics and

Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony-forming cells

A hierarchy of endothelial colony-forming cells (ECFC) with different levels of proliferative potential has been identified in human circulating blood and blood vessels. ECFC has recently become an attractive target for new vascular regenerative therapies; however, in vitro expansion of ECFC typically depends on the presence of fetal bovine serum (FBS) or fetal calf serum (FCS) in the culture medium, which is not appropriate for its therapeutic application. To identify optimal conditions for in vitro expansion of ECFC, the effects of human endothelial serum-free medium (SFM) supplemented with six pro-angiogenic cytokines and human umbilical cord blood plasma (HCP) were investigated. The in vitro morphology, proliferation, surface antigen expression and in vivo vessel-forming ability were utilized for examining the effects of medium on ECFC. This novel formulation of endothelial cell culture medium allows us, for the first time, to isolate and expand human ECFC efficiently in vitro with a low concentration of HCP (1.5%) and without bovine serum additives. In this serum-reduced medium (SRM), human ECFC colony yields remained quantitatively similar to those cultured in a high concentration (10%) of bovine serum-supplemented medium. SRM-cultured ECFC displayed a robust clonal proliferative ability in vitro and human vessel-forming capacity in vivo. The present study provides a novel method for the expansion of human ECFC in vitro and will help to advance approaches for using the cells in human therapeutic trials.

Isolation, characterization, and gene expression analysis of Wharton&amp;rsquo;s jelly-derived mesenchymal stem cells under xeno-free culture conditions

Mesenchymal stem cells (MSCs) have become an attractive tool for tissue engineering and targets in clinical transplantation due to their regeneration potential and immuno-suppressive capacity. Although MSCs derived from bone marrow are the most widely used, their harvest requires an invasive procedure. The umbilical cord, which is discarded at birth, can provide an inexhaustible source of stem cells for therapy. The Wharton’s jelly-derived MSCs (WJ-MSCs), from the umbilical cord, have been shown to have faster proliferation rates and greater expansion capability compared with adult MSCs. The standard isolation and in vitro culture protocol for WJ-MSCs utilizes fetal bovine serum (FBS) or calf serum as a nutrient supplement. However, FBS raises potential safety concerns such as transmission of viral/prion disease and may initiate xenogeneic immune reactions against bovine antigens. Therefore, for therapeutic applications, there is an urgent requirement to establish an alternative nutrient supplement which would favor cell proliferation, retain MSC characteristics, and prove safe in human subjects. In the present study, we isolated and expanded WJ-MSCs in 5% pooled, allogeneic human serum (HS) supplemented with 2 ng/mL of basic fibroblast growth factor. For cell dissociation, porcine trypsin was replaced with TrypLE, a recombinant enzyme, and a protease-free protocol was adapted for isolation of MSCs from WJ. We determined their growth kinetics, in vitro differentiation potential, surface marker expression, and colony-forming unit potential and compared them against standard WJ-MSC cultures expanded in 10% FBS. All these parameters matched quite well between the two MSC populations. To test whether there is any alteration in gene expression on switching from FBS to HS, we analyzed a panel of stem cell and early lineage markers using Taqman® low density array. No significant deviation in gene expression was observed between the two populations. Thus we established an efficient, complete xeno-free protocol for propagation of human WJ-MSCs.

RNA Polymerase 1-driven Transcription as a Mediator of BDNF-induced Neurite Outgrowth

Neurite outgrowth is essential for development of the nervous system. Neurotrophins including BDNF are among extracellular signals that regulate neurite outgrowth. The ERK1/2 pathway contributes to intracellular signaling networks transducing the pro-neuritic effects of BDNF. In the nucleolus, RNA polymerase-1 (Pol1)-mediated transcription regulates ribosomal biogenesis, enabling cellular protein synthesis and growth. Hence, we tested the possibility that Pol1 is an effector for pro-neuritic signals such as BDNF. We report that Pol1-mediated nucleolar transcription was increased by BDNF in an ERK1/2-dependent manner in rat forebrain neurons. Conversely, in cultured hippocampal neurons, knockdown of a Pol1 coactivator, transcription initiation factor 1A (TIF1A), attenuated BDNF- or ERK1/2-induced neurite outgrowth. Also, upon overexpression, a constitutively active mutant of TIF1A strongly promoted neurite outgrowth, including increases in total neurite length and branching. Finally, overexpression of wild-type TIF1A enhanced the pro-neuritic effects of ERK1/2 activation. These observations indicate that the Pol1-mediated nucleolar transcription regulates neurite outgrowth and serves as a major pro-neuritic effector of the BDNF-activated ERK1/2 pathway. Thus, development of the nervous system appears critically dependent on the nucleolus.