Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

Slow-Freezing Cryopreservation Ensures High Ovarian Tissue Quality Followed by In Vivo and In Vitro Methods and Is Safe for Fertility Preservation.

Background and objectives: Cancer incidence is growing with younger patients diagnosed with this disease every year. Improved cancer diagnostics and treatment lead to better survival of cancer patients. However, after aggressive chemo- or radiotherapy, cancer survivors suffer from various degrees of subfertility or infertility. Several fertility preservation technologies have been developed for young cancer patients: cryopreservation of germ cells, embryos, or reproductive tissues. The best results have been shown by cryopreservation of sperm and embryos. Yet the success of using cryopreserved oocytes or reproductive tissues (ovarian and testicular) is still insufficient. Therefore, this study was designed to assess the vitality, viability, general quality, and safety of frozen-thawed human ovarian tissue for retransplantation using modern molecular tests. Materials and Methods: The new miRNA array test was used to evaluate miRNA expression in thawed ovarian tissue in combination with standard xenotransplantation and pathological examination of microslides. Results: Our results demonstrated that slow freezing is an efficient way (80%) to cryopreserve ovarian tissue with no structural damage afterwards. We have shown that xenotransplantation into immunodeficient mice, histology, and immunohistochemistry could be potentially replaced by more recent molecular methods. Conclusions: The latter method has shown that altered expression of miRNAs might be used as identifiers of normal/damaged tissue after further analysis. Newer, safer, and more specific approaches need to be developed in order to eliminate the risk of disease reoccurrence.

Uptake and distribution of carboxylated quantum dots in human mesenchymal stem cells: cell growing density matters.

BACKGROUND: Human mesenchymal stem cells (MSCs) have drawn much attention in the field of regenerative medicine for their immunomodulatory and anti-inflammatory effects. MSCs possess specific tumor-oriented migration and incorporation highlighting the potential for MSCs to be used as an ideal carrier for anticancer agents. Bone marrow is the main source of MSCs for clinical applications. MSCs tracking in vivo is a critical component of the safety and efficacy evaluation of therapeutic cell products; therefore, cells must be labeled with contrast agents to enable visualization of the MSCs migration in vivo. Due to their unique properties, quantum dots (QDs) are emerging as optimal tools in long-term MSC optical imaging applications. The aim of this study was to investigate the uptake dynamics, cytotoxity, subcellular and extracellular distribution of non-targeted carboxylated quantum dots in human bone marrow MSCs at different cell growing densities. RESULTS: QDs had no negative impact on MSC viability throughout the experiment and accumulated in all observed cells efficiently; however, in some MSCs QDs induced formation of lipid droplets. At low cell growing densities QDs distribute within MSCs cytoplasm already after 1 h of incubation reaching saturation after 6 h. After 24 h QDs localize mainly in the perinuclear region of the cells in endosomes. Interestingly, in more confluent culture QDs localize mostly outside MSCs. QDs abundantly mark MSC long filopodia-like structures attaching neighboring cells. At high cell density cultivation, we for the first time demonstrated that carboxylated QDs localize in human bone marrow MSC extracellular matrix. Moreover, we observed that average photoluminescence lifetime of QDs distributed in extracellular matrix are longer than lifetimes of QDs entrapped in endocytic vesicles; thus, for the first time showing the possibility to identify and distinguish localization of QDs in various extracellular and intracellular structures using fluorescence-lifetime imaging microscopy without additional staining assays. CONCLUSION: Carboxylated QDs can be used as nonspecific and effective dye for staining of human bone marrow MSCs and their specific extracellular structures. These results are promising in fundamental stem cell biology as well as in cellular therapy, anticancer drug delivery and tissue engineering.

Individual and combined effect of TP53, MDM2, MDM4, MTHFR, CCR5, and CASP8 gene polymorphisms in lung cancer.

Lung cancer (LC) is the second common and with the highest mortality oncological disease. Specific biomarkers for its diagnostics, treatment, and prognosis are still under the investigations. Aim of our study was to evaluate the relationship between the polymorphisms of TP53 pathway genes TP53, MDM2, MDM4, the polymorphisms of HPV-associated genes MTHFR, CASP8, CCR5, and HPV infection with survival of LC patients. SNPs were genotyped using PCR-RFLP. qRT-PCR was used to detect, identify, and quantify HPV. No statistically significant differences were detected between individual SNPs and patient survival with stage I-IV LC. Cluster analysis of SNPs in genes MDM4 A/A, CCR5 wt/Δ32, MTHFR C/T, MDM2 T/T showed possible association with the worse survival. Patients who were diagnosed with C/T polymorphic variant of gene MTHFR tend not to survive stage III-IV LC (P = .12). There is a tendency between MDM2 gene T/T variant and worse survival of patients diagnosed with late stage LC (P = .11). HPV infection is very rear among LC patients (3 of 92). Overall, there is a link, although statistically insignificant, between specific SNPs and LC patient survival frequency and time, meanwhile the combination of specific SNPs showed a statistically significant measure. In conclusion, we determined statistically significant (P = .04) link between the poor survival of LC patients after surgery and the combination of polymorphic variants C/T of the MTHFR and T/T of the MDM2 genes, whereas individually these SNPs do not show significant relationship with the survival of patients after surgery.

Microenvironment dependent gene expression signatures in reprogrammed human colon normal and cancer cell lines.

BACKGROUND: Since the first evidence suggesting existence of stem-like cancer cells, the process of cells reprogramming to the stem cell state remains as an attractive tool for cancer stemness research. Current knowledge in the field of cancer stemness, indicates that the microenvironment is a fundamental regulator of cell behavior. With regard to this, we investigated the changes of genome wide gene expression in reprogrammed human colon normal epithelial CRL-1831 and colon carcinoma DLD1 cell lines grown under more physiologically relevant three-dimensional (3D) cell culture microenvironment compared to 2D monolayer. METHODS: Whole genome gene expression changes were evaluated in both cell lines cultured under 3D conditions over a 2D monolayer by gene expression microarray analysis. To evaluate the biological significance of gene expression changes, we performed pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene network analysis was used to study relationships between differentially expressed genes (DEGs) in functional categories by the GeneMANIA Cytoscape toolkit. RESULTS: In total, we identified 3228 and 2654 differentially expressed genes (DEGs) for colon normal and cancer reprogrammed cell lines, respectively. Furthermore, the expression of 1097 genes was commonly regulated in both cell lines. KEGG enrichment analysis revealed that in total 129 and 101 pathways for iPSC-CRL-1831 and for CSC-DLD1, respectively, were enriched. Next, we grouped these pathways into three functional categories: cancer transformation/metastasis, cell interaction, and stemness. ß-catenin (CTNNB1) was confirmed as a hub gene of all three functional categories. CONCLUSIONS: Our present findings suggest common pathways between reprogrammed human colon normal epithelium (iPSC-CRL-1831) and adenocarcinoma (CSC-DLD1) cells grown under 3D microenvironment. In addition, we demonstrated that pathways important for cancer transformation and tumor metastatic activity are altered both in normal and cancer stem-like cells during the transfer from 2D to 3D culture conditions. Thus, we indicate the potential of cell culture models enriched in normal and cancer stem-like cells for the identification of new therapeutic targets in cancer treatment.

Identity, proliferation capacity, genomic stability and novel senescence markers of mesenchymal stem cells isolated from low volume of human bone marrow.

Human bone marrow mesenchymal stem cells (hBM-MSCs) hold promise for treating incurable diseases and repairing of damaged tissues. However, hBM-MSCs face the disadvantages of painful invasive isolation and limited cell numbers. In this study we assessed characteristics of MSCs isolated from residual human bone marrow transplantation material and expanded to clinically relevant numbers at passages 3-4 and 6-7. Results indicated that early passage hBM-MSCs are genomically stable and retain identity and high proliferation capacity. Despite the chromosomal stability, the cells became senescent at late passages, paralleling the slower proliferation, altered morphology and immunophenotype. By qRT-PCR array profiling, we revealed 13 genes and 33 miRNAs significantly differentially expressed in late passage cells, among which 8 genes and 30 miRNAs emerged as potential novel biomarkers of hBM-MSC aging. Functional analysis of genes with altered expression showed strong association with biological processes causing cellular senescence. Altogether, this study revives hBM as convenient source for cellular therapy. Potential novel markers provide new details for better understanding the hBM-MSC senescence mechanisms, contributing to basic science, facilitating the development of cellular therapy quality control, and providing new clues for human disease processes since senescence phenotype of the hematological patient hBM-MSCs only very recently has been revealed.

Quick and effective method of bone marrow mesenchymal stem cell extraction.

BACKGROUND: Mesenchymal stem cells (MSCs) are currently exploited in numerous clinical trials to investigate their potential in immune regulation, hematopoesis or tissue regeneration. The most common source of MSCs for clinical use is human bone marrow. To generate sufficient numbers of cells relevant to clinical use in most cases the high volumes (20-50 ml) of bone marrow aspirates are taken. METHODS: In this pilot study, 8 healthy bone marrow donors were included. Two different MSC extraction methods were evaluated: MSCs extraction from 60 ml of bone marrow using density gradient and MSCs extraction from 6 ml using red blood cell (RBC) lysis. RESULTS: Our results showed that after RBC lysis the efficient amount of human MSCs can be isolated from 10 times less bone marrow volume (6 ml). Moreover, using small volume of bone marrow the adequate therapeutical dose of MSCs could be achieved during similar period of time (3-4 weeks). In conclusion, we have shown that MSCs isolation using RBC lysis is an effective and more advantageous method in comparison to standard MSCs isolation using density-gradient. Using RBC lysis from small volume of bone marrow the same amount of MSCs were obtained as usually using large volume and density-gradient.

Generation of recombinant porcine parvovirus virus-like particles in Saccharomyces cerevisiae and development of virus-specific monoclonal antibodies.

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.