Platelet-Rich Plasma Promotes the Proliferation of Human Muscle Derived Progenitor Cells and Maintains Their Stemness

Human muscle-derived progenitor cells (hMDPCs) offer great promise for muscle cell-based regenerative medicine; however, prolonged ex-vivo expansion using animal sera is necessary to acquire sufficient cells for transplantation. Due to the risks associated with the use of animal sera, the development of a strategy for the ex vivo expansion of hMDPCs is required. The purpose of this study was to investigate the efficacy of using platelet-rich plasma (PRP) for the ex-vivo expansion of hMDPCs. Pre-plated MDPCs, myoendothelial cells, and pericytes are three populations of hMDPCs that we isolated by the modified pre-plate technique and Fluorescence Activated Cell Sorting (FACS), respectively. Pooled allogeneic human PRP was obtained from a local blood bank, and the effect that thrombin-activated PRP-releasate supplemented media had on the ex-vivo expansion of the hMDPCs was tested against FBS supplemented media, both in vitro and in vivo. PRP significantly enhanced short and long-term cell proliferation, with or without FBS supplementation. Antibody-neutralization of PDGF significantly blocked the mitogenic/proliferative effects that PRP had on the hMDPCs. A more stable and sustained expression of markers associated with stemness, and a decreased expression of lineage specific markers was observed in the PRP-expanded cells when compared with the FBS-expanded cells. The in vitro osteogenic, chondrogenic, and myogenic differentiation capacities of the hMDPCs were not altered when expanded in media supplemented with PRP. All populations of hMDPCs that were expanded in PRP supplemented media retained their ability to regenerate myofibers in vivo. Our data demonstrated that PRP promoted the proliferation and maintained the multi-differentiation capacities of the hMDPCs during ex-vivo expansion by maintaining the cells in an undifferentiated state. Moreover, PDGF appears to be a key contributing factor to the beneficial effect that PRP has on the proliferation of hMDPCs.     

Comparison of ISSR polymorphism among cattle breeds

Polymorphism analysis of DNA fragments flanked by (AG)₉C and (GA)₉C inverted dinucleotide microsatellite repeats in 766 animals of 19 cattle breeds and one breeding type revealed 66 fragments, of which 64 were polymorphic. The breeds proved to differ in the frequency and presence or absence of amplified DNA fragments at the genomic level, indicating that ISSR fingerprinting is informative for differentiating the PCR product spectra and cattle breeds. Multilocus ISSR polymorphism analysis identified the group of fragments that can be used as Bos taurus and B. indicus species markers to describe the standards of breeds, their genetic profiles, and breed-specific patterns. Based on ISSR polymorphism, a prototypal gene pool of cattle was constructed and the breeds closest to it were identified. Genetic diversity analysis made it possible to assume that an optimal mean heterozygosity is characteristic of cattle breeds and that deviations from this optimum are indicative of various processes occurring in the population (breed).     

Genipin induces cell death via intrinsic apoptosis pathways in human glioblastoma cells

Genipin, a compound derived from Gardenis jasminoides Ellis fruits, was demonstrated to be the specific uncoupling protein 2 (UCP2) inhibitor. UCP2 is a mitochondrial carrier protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) synthesis. Several studies revealed that UCP2 is broadly over-expressed in leukemia, colorectal, lung, ovarian, prostate, testicular, and bladder cancers. However, the effect of genipin still needs to be elucidated in neurological malignancies. In this study, we investigated the anticancer effect of genipin in U87MG and A172 cell lines. The anticancer effect of genipin on these cell lines was measured by microculture tetrazoliumtest (MTT), Trypan blue exclusion, and colony formation assays, in the presence of various concentrations of genipin at different time intervals. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Expression of UCP2 and some of the genes involved in apoptosis was analyzed by real-time quantitative polymerase chain reaction (PCR). Results of the MTT assay showed that genipin moderately reduced metabolic activity of both cell lines in dose- and time-dependent manner. Result of Trypan blue exclusion test indicated that the viable cell count decreased in the treated group in a concentration-dependent manner. Genipin also significantly decreased colony formation ability of these cells in a concentration-dependent manner. Result of morphological changes showed that there were significant differences in cell number and morphology in treated groups as compared with the untreated groups. Flow cytometric analysis of U87MG and A172 cells with annexin V/propidium iodide staining, 48 hours after treatment with genipin, displays 22.4% and 26.1% apoptotic population, respectively, in treated cells, in comparison to 7.42% and 9.31% apoptotic cells of untreated cells. After treatment, UCP2 and B-cell lymphoma 2 (BCL ₂) genes are downregulated, and BCL ₂ associated X protein, BCL ₂ antagonist/killer, BCL ₂ interacting killer, and Cytochrome c genes are upregulated. Genipin treatment increased mitochondrial ROS levels and also induced apoptosis through caspase-3 upregulation. In conclusion, the antiproliferative effects of genipin on the growth of both glioblastoma cell lines have been shown in all of these assays, and genipin profoundly induced apoptosis in both cell lines via the UCP2-related mitochondrial pathway through the induction of intracellular ROS.     

κ-casein gene (<Emphasis Type="Italic">CSN3</Emphasis>) allelic polymorphism in Russian cattle breeds and its information value as a genetic marker

The frequencies of the κ-casein gene (CSN3) alleles and genotypes have been determined in five Russian cattle breeds (Bestuzhev, Kalmyk, Russian Black Pied, Yaroslavl, and Yakut breeds) by means of PCR-RFLP analysis using two independent restriction nucleases (HinfI and TaqI) and by allele-specific PCR. Typing alleles A and B of CSN3 is of practical importance, because allele B is correlated with commercially valuable parameters of milk productivity (protein content and milk yield) and improves the cheese yielding capacity. The frequencies of the B allele of CSN3 in the breeds studied vary from 0.16 to 0.50; and those of the AB and BB genotypes, from 0.27 to 0.60 and from 0.02 to 0.23, respectively. The Yaroslavl breed had the highest frequencies of CSN3 allele B and genotype BB (0.50 and 0.23, respectively). The frequencies of the B allele and BB genotype in other breeds studied varied from 0.25 to 0.32 and from 0.03 to 0.09, respectively. In none of the breeds studied have the observed and expected heterozygosities been found to differ from each other significantly. However, the observed genotype distributions significantly differ from the expected one in some herds (in most such cases, an excess of heterozygotes is observed). Two herds of the Yaroslavl breed dramatically differ from each other in the heterozygosity level: a deficit (D = ?0.14) and an excess (D = 0.20) of heterozygotes have been observed at the Mikhailovskoe and Gorshikha farms, respectively. In general, however, the heterozygosity of the Yaroslavl breed corresponds to the expected level (D = 0.04). Analysis of breeds for homogeneity with the use of Kulback’s test has shown that all cattle breeds studied are heterogeneous, the CSN3 diversity within breeds being higher than that among different breeds, which is confirmed by low F _st values (0.0025–0.0431). Thus, a DNA marker based on CSN3 gene polymorphism is extremely important for breeding practice as a marker of milk quality; however, it is inapplicable to marking differences between breeds or phylogenetic relationships between cattle breeds because of the high diversity with respect to this locus within breeds.     

Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells

Monoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.