An efficient method for isolation of murine bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been isolated based on the ability of adherence to plastic surfaces. The potential of these cells to differentiate along multiple lineages is the key to identifying stem cell populations in the absence of molecular markers. Here we describe a homogenous population of MSCs from mouse bone marrow isolated using a relatively straightforward and novel approach. This method is based on the combination of frequent medium change (FMC) and treatment of the primary cultures with trypsin. Cells isolated using this method demonstrated the MSCs characteristics including their ability to differentiate into mesenchymal lineages. MSCs retained the differentiation potentials in expanded cultures up to 10 passages. Isolated MSCs were reactive to the CD44, Sca-1, and CD90 cell surface markers. MSCs were negative for the hematopoietic surface markers such as CD34, CD11b, CD45, CD31, CD106, CD117 and CD135. The data presented in this report indicated that this method can result in efficient isolation of homogenous populations of MSCs from mouse bone marrow.     

Multivariate optimisation of microwave-assisted extraction of capsaicin from Capsicum frutescens L. and quantitative analysis by 1H-NMR

A simple and rapid microwave-assisted extraction (MAE) procedure combined with 1H-NMR spectrometry was developed and optimised for the extraction and quantitative determination of capsaicin in Capsicum frutescens. The influence of experimental variables, including irradiation power, extraction temperature and dynamic extraction time before reaching the selected extraction temperature, on the performance of the extraction procedure was systematically studied using a Box-Behnken experimental design followed by a conventional central composite design approach. Statistical treatment of the results together with results from some additional experiments suggested optimum extraction conditions as 120 degrees C and 150 W, for 15 min with acetone as extractant. The optimised MAE method provides extracts that can be analysed quantitatively using 1H-NMR without any preliminary clean-up or derivatisation steps. In the 1H-NMR spectrum of the crude extracts the doublet signal in the delta range 4.349-4.360 ppm was well separated from other resonances in deuterated chloroform. The quantity of the compound was calculated from the relative ratio of the integral value of the target peak to that of a known amount of dimethylformamide as internal standard. In comparison with traditional Soxhlet extraction, the proposed method is less labour-intensive and provides a drastic reduction of extraction time and solvent consumption. In addition, MAE showed higher extraction yield and selectivity, with comparable reproducibility and recovery, relative to both conventional Soxhlet and sonication methods.     

Expression of Thy 1.2 surface antigen increases significantly during the murine mesenchymal stem cells cultivation period

This study sought to investigate the absence or expression of some surface antigens on murine mesenchymal stem cells (mMSCs) during the cultivation period of primary culture to passage 3 (equivalent to about 15 or 16 population doubling number). For this purpose, bone marrow cells from 6-8-week-old mice (either NMRI or Balb/c) were cultivated in 75-cm(2) culture flask for three successive passages, in each of which the culture was examined for the expression of CD135, CD44, CD31, Thy1.2, CD11b, CD45, CD34, Vcam1, Sca-1, and c-Kit antigens, using flow cytometry. Passage-3 cells from each strain can easily be differentiated into bone and fat, which was indicative of their mesenchymal nature. Our results demonstrated that for each given antigen, the percentages of the cells expressing that antigen had been changed by subcultures. The statistical analysis showed that nearly all differences between the passages were statistically significant. In this term, the expressional changes of Thy 1.2 seemed to be very significant in such a way that the expression increased to about half of the whole population in passage 3. In conclusion, it seems that this antigen could be considered as an enriching antigen for mMSCs population from bone marrow adherent cell culture.     

Comparison of different protocols for neural differentiation of human induced pluripotent stem cells

Although embryonic stem cells (ESCs) have enormous potentials due to their pluripotency, their therapeutic use is limited by ethical, biological and safety issues. Compared to ESCs, induced pluripotent stem cells (iPSCs) can be obtained from mouse or human fibroblasts by reprogramming. Numerous studies have established many protocols for differentiation of human iPSCs (hiPSCs) into neural lineages. However, the low differentiation efficiency of such protocols motivates researchers to design new protocols for high yield differentiation. Herein, we compared neural differentiation potential of three induction media for conversion of hiPSCs into neural lineages. In this study, hiPSCs-derived embryoid bodies were plated on laminin coated dishes and were treated with three induction media including (1) bFGF, EGF (2) RA and (3) forskolin, IBMX. Immunofluorescence staining and quantitative real-time PCR (qPCR) analysis were used to detect the expression of neural genes and proteins. qPCR analysis showed that the expression of neural genes in differentiated hiPSCs in forskolin, IBMX supplemented media was significantly higher than undifferentiated cells and those in induction media containing bFGF, EGF or RA. In conclusion, our results indicated a successful establishment protocol with high efficiency for differentiation of hiPSCs into neural lineages.     

Nuclear and chloroplast DNA variability and phylogeny of Iranian apples (Malus domestica)

This study was conducted to reveal genetic diversity among 23 local apple genotypes using nuclear (RAPD) and chloroplast DNA (PCR-cpRFLP) markers. Eleven RAPD primers and four cpDNA primer combinations were used in this study. RAPD primers produced a total of 77 polymorphic fragments with an average of seven bands per primer. The percentage of polymorphic bands (68.14 %) showed the efficiency of used RAPD primers in distinguishing all the genotypes considered. Genetic similarity between studied genotypes varied from 0.38 to 0.72 and cluster analysis showed the abundant diversity, indicating high intraspecific genetic variation between Iranian apple genotypes. From the four universal chloroplast primer pairs, three primer pairs amplified the fragments and their combinations showed polymorphic patterns and revealed intraspecific chloroplast variation. The information will facilitate germplasm identification, conservation and new cultivar development.     

Prostanoids and pain: unraveling mechanisms and revealing therapeutic targets

Advances in our understanding of the synthesis, regulation and function of prostanoids have led to a new appreciation of their actions in health and disease. Prostanoid synthesis is essential for the generation of inflammatory pain and this depends not only on prostanoid production at the site of inflammation, but also on the actions of prostanoids synthesized within the central nervous system (CNS). Moreover, central prostanoid synthesis is controlled both by neural and humoral signals, the latter being a novel form of input to the CNS. Diverse compounds that act along the pathway of prostanoid synthesis and action, both in the periphery and in the CNS, might provide increased benefit for treating inflammatory pain hypersensitivity and its associated sickness syndrome, with a reduced risk of adverse effects.     

Circadian gene variants and susceptibility to type 2 diabetes: a pilot study

Background

Disruption of endogenous circadian rhythms has been shown to increase the risk of developing type 2 diabetes, suggesting that circadian genes might play a role in determining disease susceptibility. We present the results of a pilot study investigating the association between type 2 diabetes and selected single nucleotide polymorphisms (SNPs) in/near nine circadian genes. The variants were chosen based on their previously reported association with prostate cancer, a disease that has been suggested to have a genetic link with type 2 diabetes through a number of shared inherited risk determinants.

Methodology/Principal Findings

The pilot study was performed using two genetically homogeneous Punjabi cohorts, one resident in the United Kingdom and one indigenous to Pakistan. Subjects with (N = 1732) and without (N = 1780) type 2 diabetes were genotyped for thirteen circadian variants using a competitive allele-specific polymerase chain reaction method. Associations between the SNPs and type 2 diabetes were investigated using logistic regression. The results were also combined with in silico data from other South Asian datasets (SAT2D consortium) and white European cohorts (DIAGRAM+) using meta-analysis. The rs7602358G allele near PER2 was negatively associated with type 2 diabetes in our Punjabi cohorts (combined odds ratio [OR] = 0.75 [0.66–0.86], p = 3.18×10⁻⁵), while the BMAL1 rs11022775T allele was associated with an increased risk of the disease (combined OR = 1.22 [1.07–1.39], p = 0.003). Neither of these associations was replicated in the SAT2D or DIAGRAM+ datasets, however. Meta-analysis of all the cohorts identified disease associations with two variants, rs2292912 in CRY2 and rs12315175 near CRY1, although statistical significance was nominal (combined OR = 1.05 [1.01–1.08], p = 0.008 and OR = 0.95 [0.91–0.99], p = 0.015 respectively).

Conclusions/significance

None of the selected circadian gene variants was associated with type 2 diabetes with study-wide significance after meta-analysis. The nominal association observed with the CRY2 SNP, however, complements previous findings and confirms a role for this locus in disease susceptibility.     

Elucidating the protein cold-adaptation: Investigation of the parameters enhancing protein psychrophilicity

To investigate the role of the critical parameters in adaptation of proteins to low temperatures, a comparative systematic analysis was performed. Several parameters were proposed to have contribution to cold adaptation of proteins. Among proposed parameters, total values of residual structure states, secondary structure states and oligomeric states were alike in both psychrophilic and mesophilic proteins. In addition, our results provided new quantitative information about the trends in the substitution preference of Ile, Phe, Tyr, Lys, Arg, His, Glu and Leu with most of amino acids and substitution avoidance of Gly, Thr and Ala with most of amino acids. These findings would help future efforts propose a strategy for designing psychrophilic proteins.     

Characterization of fibroblast-like cells from the rat olfactory bulb

The isolation and characterization of stem cells from an alternative tissue is a subject of intensive investigation. In the present study, we have focused on the characterization of fibroblastic cells in olfactory bulb tissue of the rat. To this end, 4-6 week old rats were killed and their olfactory bulb tissue was dissected out. Olfactory bulb derived fibroblast-like cells were recovered by adhesion to cell culture plastic. The plastic adherent cultivated cells were induced to differentiate along osteoblastic, adipogenic and chondrogenic lineages. Furthermore, the expression of some surface antigens was investigated. We obtained purified cells with spindle shaped morphology in primary culture, which differentiated into mesenchymal lineages. These cells expressed CD29 and CD90 (Thy1.1) surface antigens, but not CD31, CD34 and CD45. Our results indicate that fibroblast-like cells from the olfactory bulb are mesenchymal stem cells in nature. Taken together, our data suggest that olfactory bulb tissue may constitute a new source of mesenchymal stem cells and could be used for the treatment of injury.