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The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: a systematic review

Mesenchymal stem cells (MSCs) represent an invaluable asset for the feld of cell therapy. Human Bone marrowderived MSCs (hBM-MSCs) are one of the most commonly used cell types in clinical trials. They are currently being studied and tested for the treatment of a wide range of diseases and conditions. The future availability of MSCs therapies to the public will require a robust and reliable delivery process. Cryopreservation represents the gold standard in cell storage and transportation, but its efect on BM-MSCs is still not well established. A systematic review was conducted to evaluate the impact of cryopreservation on BM-MSCs and to attempt to uncover the reasons behind some of the controversial results reported in the literature. Forty-one in vitro studies were analysed, and their results organised according to the cell attributes they assess. It was concluded that cryopreservation does not afect BMMSCs morphology, surface marker expression, diferentiation or proliferation potential. However, mixed results exist regarding the efect on colony forming ability and the efects on viability, attachment and migration, genomic stability and paracrine function are undefned mainly due to the huge variabilities governing the cryopreservation process as a whole and to the lack of standardised assay

The effect of low-resistance high-repetition resistance training on longer-term functional adaptations and total athletic score.

Purpose: To determine the effect of 52 weeks of low-load high-repetition resistance training (BODYPUMPTM) on broader athletic performance in healthy adults. To identify if any relationship between the performance within the training program and athletic performance exists when measured independently. Methods: Twenty-six, apparently healthy adults: four males (age = 51.6 ± 4.0 years; height 178.8 ± 13cm and body mass = 82.4kg ± 6.5kg) and twenty-twofemales (age = 38.3 ± 7.0 years; height 163.5 ± 6cm and body mass = 58.6kg ± 4.5kg)participated in and completed the yearlong longitudinal cross-sectional study. All participants had trained with BODYPUMP™ for ≥ 1 year; this was their solemethod of training which they attended on average a minimum of twice a week.Isometric mid-thigh pull (IMTP) peak force (PF), 10m sprint time, counter movement jump (CMJ) height and the total score of athleticism (TSA)were all assessed. Results: Testing revealed that after 1-year total load (kg) relative to body weight (BW) has a strong correlation with isometric mid-thigh pull (IMTP) peak force (PF) (r=0.767), 10m Sprint (r=0.712), counter movement jump (CMJ) height (r=0.719) and the total score of athleticism (TSA) (r=0.721) as assessed by Pearson’s correlation p <0.05. Conclusions: This research demonstrates that BODYPUMP™ does have athletic carryover for some components of fitness (strength, power and speed). This study has shown that thetotal score of athleticism (TSA) is strongly correlated to lifting ability (r=0.721) within the training program

Determining conditions for successful culture of multi-cellular 3D tumour spheroids to investigate the effect of mesenchymal stem cells on breast cancer cell invasiveness

Mesenchymal stem cells have been widely implicated in tumour development and metastases. Moving from the use of two-dimensional (2D) models to three-dimensional (3D) to investigate this relationship is critical to facilitate more applicable and relevant research on the tumour microenvironment. We investigated the effects of altering glucose concentration and the source of foetal bovine serum (FBS) on the growth of two breast cancer cell lines (T47D and MDA-MB-231) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) to determine successful conditions to enable their co-culture in 3D tumour spheroid models. Subsequently, these 3D multicellular tumour spheroids were used to investigate the effect of hBM-MSCs on breast cancer cell invasiveness. Findings presented herein show that serum source had a statistically significant effect on two thirds of the growth parameters measured across all three cell lines, whereas glucose only had a statistically significant effect on 6%. It was determined that the optimum growth media composition for the co-culture of 3D hBM-MSCs and breast cancer cell line spheroids was 1 g/L glucose DMEM supplemented with 10% FBS from source A. Subsequent results demonstrated that co-culture of hBMMSCs and MDA-MB-231 cells dramatically reduced invasiveness of both cell lines (F(1,4) = 71.465, p = 0.001) when embedded into a matrix comprising of growth-factor reduced base membrane extract (BME) and collagen

The role of dissolved oxygen levels on human Mesenchymal Stem Cell culture success, regulatory compliance and therapeutic potential

Most cells in the human body, including human mesenchymal stem cells (hMSCs), have evolved to survive and function in a low, physiological, oxygen (O2) environment. Investigators have become increasingly aware of the effects of O2 levels on hMSC biology and culture and are mimicking the natural niche of these cells in vitro to improve cell culture yields. This presents many challenges in relation to hMSC identity and function and in the maintenance of a controlled O2 environment for cell culture. The aim of this review is to discuss a “hMSC checklist” as a guide to establishing which identity and potency assays to implement when studying hMSCs. The checklist includes markers, differentiation potential, proliferation & growth, attachment & migration, genomic stability and paracrine activity. Evidence drawn from the current literature demonstrates that low O2 environments could improve most “hMSC checklist” attributes. However, there are substantial inconsistencies around both the terminology and the equipment used in low O2 studies. Therefore, “hypoxia” as a term and as a culture condition are discussed. The biology of short (acute) vs long-term (chronic) hypoxia is considered and a nascent hypothesis to explain the behaviour of hMSCs in long-term hypoxia is presented. It is hoped that by establishing an ongoing discourse, and driving towards a regulatory recognisable “hMSC checklist”, we may be better able to provide the patient population with safe and efficacious regenerative treatments

Genetic analysis of novel Alu insertion polymorphisms in selected Indian populations.

Indian subpopulations (Chenchu, Koya, and Lobana Sikh) were analyzed at the genetic level for 12 Alu polymorphisms. These markers were then utilized to establish levels of genetic identity between the Indian populations and more widely between the Indian populations and a European population.Previously collected blood samples were extracted using the phenol-chloroform method. The samples were utilized as templates for PCR using Alu specific primers and then analyzed by agarose gel electrophoresis for the presence and absence of the approximately 300 bp insertions. Allele frequencies were calculated by the gene counting method and were tested for Hardy-Weinberg equilibrium, heterozygosities, inbreeding coefficient, and GST to assess the level of genetic differentiation.All of the Alu loci were polymorphic in the three Indian populations studied and their average observed heterozygosity ranged from 0.294 (Lobana Sikh) to 0.357 (Koya). Allele and genotype frequency variation at the 2b, 9a, and ACE loci led to statistically significant pairwise differences among the three study populations. Overall population heterogeneity was observed for 7 out of 12 Alu polymorphisms.The overall results show that these Indian samples, though displaying significant genetic variation and differences among themselves, form an Indian cluster, which as expected, is distinct from the European sample (Russian). As Alus are easily analyzed and quantified by standard and cost-effective methodologies, this finding further reinforces their utility as effective population genetic markers. Am. J. Hum. Biol., 2016. © 2016 Wiley Periodicals, Inc

The effect of chronic high insulin exposure upon metabolic and myogenic markers in C2C12 skeletal muscle cells and myotubes.

Skeletal muscle is an insulin sensitive tissue and accounts for approximately 80% of post-prandial glucose disposal. This study describes the effects of insulin, delivered for 72 hours, to skeletal muscle myoblasts during differentiation or to skeletal muscle myotubes. After chronic treatment, cultures were acutely stimulated with insulin and analysed for total and phosphorylated Akt (Ser473), mRNA expression of metabolic and myogenic markers and insulin-stimulated glucose uptake. Skeletal muscle cells differentiated in the presence of insulin chronically, reduced acute insulin stimulated phosphorylation of Akt Ser473. In addition, there was a reduction in mRNA expression of Hexokinase II (HKII), GLUT4 and PGC-1α. Insulin-stimulated glucose uptake was attenuated when cells were differentiated in the presence of insulin. In contrast, myotubes exposed to chronic insulin showed no alterations in phosphorylation of Akt Ser473. Both HKII and GLUT4 mRNA expression were reduced by chronic exposure to insulin; while PGC-1α was not different between culture conditions and was increased by acute insulin stimulation. These data suggest that there are differential responses in insulin signalling, transcription and glucose uptake of skeletal muscle cells when cultured in either the presence of insulin during differentiation or in myotube cultures

The effect of a 12-week resistance training intervention on leukocyte telomere length

Telomere dynamics are an active biological process and positive lifestyle factors such as exercise are proposed to potentiate their length. The aim of this study was to investigate the effect of a low-resistance, high-repetition resistance training intervention on leukocyte telomere length (LTL) and associated health parameters. 23 sedentary middle-aged adults volunteered for this study (16 female/7 male; age = 51.5 ± 4.9 years) and performed two one-hour sessions of Les Mills BODYPUMP™ per week for 12 weeks. Outcome measures were taken at baseline, after the training intervention and at 12-month follow-up. LTL remained unchanged following the training intervention (pre 0.819 ± 0.121 vs post 0.812 ± 0.114, p = 0.420), despite a borderline significant increase in hTERT expression (p = 0.050). Circulating levels of tumour necrosis factor alpha were reduced after the intervention (p = 0.001). At 12-month follow-up, subjects who returned to a sedentary lifestyle (n = 10) displayed shorter telomeres compared to their pre (p = 0.036) values. In conclusion, no changes were observed in LTL following the 12-week training intervention, despite improvements in molecular parameters associated with telomere dynamics. It appears continued long-term exercise (>12 months) is necessary to preserve LTL in previously sedentary individuals

Association of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms with coronary artery disease (CAD) in a North Indian population

There is significant variation in reported associations of the MTHFR C677T (rs1801133) and A1298C (rs1801131) polymorphisms and coronary artery disease (CAD) in different global populations. This study aims to identify any individual or combined associations between the 1298 and 677 loci of MTHFR and CAD in a North Indian population. A total of 159 patients and 166 controls were genotyped using validated TaqMan assays. Odds ratio analysis identified associations at crude level and multiple logistic regression controlled for confounding variables. Linkage disequilibrium between the loci was assessed along with haplotype association analysis. At the C677T locus, homozygosity of the T allele identified a significantly protective association (OR = 0.38, CI: 0.24–0.60). For the A1298C locus the AC genotype had a protective effect in codominant model (OR = 0.53, CI: 0.32–0.85) and CC genotype showed a susceptible association in recessive model when controlled for age, sex and lipids (OR = 2.70, CI: 1.27–5.77). This study identified that, independently, both heterozygous genotypes show a protective association with CAD. In addition the CC genotype of A1298C in recessive model was a susceptible genotype. The combined associations of MTHFR are protective (primarily due to the effects of C677T locus) suggesting an interaction between the loci and their associations with CAD within this sample

Genetic association of pro-inflammatory cytokine gene polymorphisms with coronary artery disease (CAD) in a North Indian population

Background: Cytokines regulate the expression of inflammatory molecules which destabilize the atheromatic plaques. This study focuses on studying the association of inflammatory cytokine polymorphisms like TNF-α -308 (G/A), TNF-β +252 (A/G), IL-6 -174 (G/C) and IL-6 -597 (G/A), and IFN-ɣ +874 (T/A) with coronary artery disease (CAD) among north Indian patients. Materials and methods: 143 CAD and 137 normal healthy controls were recruited in this study. DNA extraction was carried out by high salting out method. TNF-α -308 (G/A) (rs1800797), TNF-β +252 (A/G) (rs909253), IL-6 -174 (G/C) (rs1800795), IL6 -597 (G/A) (rs1800797), and IFN-ɣ +874 (T/A) (rs2430561) SNPs were genotyped by TaqMan®SNP genotyping assays. Different statistical analyses were performed using SPSS v 22.0 and SNPStats. p≤0.05 was considered significant. Results: Significant risk association with CAD was found for TNF-α -308 (G/A) “A” allele (OR =5.6, CI 1.8-17.4, p=0.001) and TNF-β +252 (A/G) “G” allele (OR=3.4, CI=1.9-6.0, p<0.001). However, no statistical significance was found for IL-6 -174 (G/C) or IL6 -597 (G/A), with CAD. TNF-α -308 (G/A), and TNF-β +252 (A/G) haplotype “GG” “AG” increased CAD risk significantly (GG haplotype, adjusted OR = 2.6, CI 1.4-5.0, p=0.003 and AG haplotype OR =8.5, CI 2.2-33.35, p=0.002) after adjustments for age, sex, TC, TG, HDL, APOB, smoking and diet. Discussion: The present study found significant risk association for TNF-α -308 (G/A), and TNF-β +252 (A/G) genotypes, alleles and haplotypes, with CAD in a North Indian Population