Comparison of Various Extraction Approaches for Optimized Preparation of Intracellular Metabolites from Human Mesenchymal Stem Cells and Fibroblasts for NMR-Based Study.

Metabolomics has proven to be a sensitive tool for monitoring biochemical processes in cell culture. It enables multi-analysis, clarifying the correlation between numerous metabolic pathways. Together with other analysis, it thus provides a global view of a cell's physiological state. A comprehensive analysis of molecular changes is also required in the case of mesenchymal stem cells (MSCs), which currently represent an essential portion of cells used in regenerative medicine. Reproducibility and correct measurement are closely connected to careful metabolite extraction, and sample preparation is always a critical point. Our study aimed to compare the efficiencies of four harvesting and six extraction methods. Several organic reagents (methanol, ethanol, acetonitrile, methanol-chloroform, MTBE) and harvesting approaches (trypsinization vs. scraping) were tested. We used untargeted nuclear magnetic resonance spectroscopy (NMR) to determine the most efficient method for the extraction of metabolites from human adherent cells, specifically human dermal fibroblasts adult (HDFa) and dental pulp stem cells (DPSCs). A comprehensive dataset of 29 identified and quantified metabolites were determined to possess statistically significant differences in the abundances of several metabolites when the cells were detached mechanically to organic solvent compared to when applying enzymes mainly in the classes of amino acids and peptides for both types of cells. Direct scraping to organic solvent is a method that yields higher abundances of determined metabolites. Extraction with the use of different polar reagents, 50% and 80% methanol, or acetonitrile, mostly showed the same quality. For both HDFa and DPSC cells, the MTBE method, methanol-chloroform, and 80% ethanol extractions showed higher extraction efficiency for the most identified and quantified metabolites Thus, preparation procedures provided a cell sample processing protocol that focuses on maximizing extraction yield. Our approach may be useful for large-scale comparative metabolomic studies of human mesenchymal stem cell samples.

Comparison of pulsed and continuous electromagnetic field generated by WPT system on human dermal and neural cells.

In recent decades, we have seen significant technical progress in the modern world, leading to the widespread use of telecommunications systems, electrical appliances, and wireless technologies. These devices generate electromagnetic radiation (EMR) and electromagnetic fields (EMF) most often in the extremely low frequency or radio-frequency range. Therefore, they were included in the group of environmental risk factors that affect the human body and health on a daily basis. In this study, we tested the effect of exposure EMF generated by a new prototype wireless charging system on four human cell lines (normal cell lines-HDFa, NHA; tumor cell lines-SH-SY5Y, T98G). We tested different operating parameters of the wireless power transfer (WPT) device (87-207 kHz, 1.01-1.05 kW, 1.3-1.7 mT) at different exposure times (pulsed 6 × 10 min; continuous 1 × 60 min). We observed the effect of EMF on cell morphology and cytoskeletal changes, cell viability and mitotic activity, cytotoxicity, genotoxicity, and oxidative stress. The results of our study did not show any negative effect of the generated EMF on either normal cells or tumor cell lines. However, in order to be able to estimate the risk, further population and epidemiological studies are needed, which would reveal the clinical consequences of EMF impact.

Modelling Duchenne muscular dystrophy in vitro with newly generated, blood cell-derived induced pluripotent stem cell line ORIONi003-A.

Here, we present newly derived in vitro model for modeling Duchenne muscular dystrophy. Our new cell line was derived by reprogramming of peripheral blood mononuclear cells (isolated from blood from pediatric patient) with Sendai virus encoding Yamanaka factors. Derived iPS cells are capable to differentiate in vitro into three germ layers as verified by immunocytochemistry. When differentiated in special medium, our iPSc formed spontaneously beating cardiomyocytes. As cardiomyopathy is the main clinical complication in patients with Duchenne muscular dystrophy, the cell line bearing the dystrophin gene mutation might be of interest to the research community.

In vitro modeling of amyotrophic lateral sclerosis with induced pluripotent stem cell technology-derived cell line ORIONi002-A.

We present here a new iPS cell line for modeling sporadic form of ALS. Cell line was generated by reprogramming skin fibroblasts isolated with explant culture technology from skin biopsy, donated by ALS patient. For reprogramming, polycistronic self-replicating RNA vector was used and derived iPS cells were characterized by immunocytochemistry and FACS (pluripotent factors expression), karyotyping, STR fingerprinting analysis and in vitro differentiation assay. New cell line showed normal (46, XY) karyotype and differentiated in vitro into cells from three germ layers. STR analysis proved the origin and originality of the cell line.

Establishment of PANDA - a new human pancreatic ductal adenocarcinoma cell line with 3D cell culture technology.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive type of malignancy with one of the worst prognoses amongst any type of cancer. Surgery is applicable only to the limited number of patients with locally resectable tumors and currently represents the only curative treatment option. Treatment with chemotherapy and radiotherapy can only extend patient survival. Despite advances in conventional therapies, the five-year survival of PDAC remained largely unchanged. New in vitro and in vivo models are therefore urgently needed to investigate this type of cancer. Here, we present the establishment and characterization of a novel pancreatic cancer cell line, isolated from a patient with PDAC. Cell line abbreviated as PANDA (PANncreatic Ductal Adenocarcinoma) was established with an optimized 3D culture protocol published previously by our group. The new cancer cell line "PANDA" represents a novel in vitro approach for PDAC cancer research and new therapy testing.

Induced Pluripotency: A Powerful Tool for In Vitro Modeling.

One of the greatest breakthroughs of regenerative medicine in this century was the discovery of induced pluripotent stem cell (iPSC) technology in 2006 by Shinya Yamanaka. iPSCs originate from terminally differentiated somatic cells that have newly acquired the developmental capacity of self-renewal and differentiation into any cells of three germ layers. Before iPSCs can be used routinely in clinical practice, their efficacy and safety need to be rigorously tested; however, iPSCs have already become effective and fully-fledged tools for application under in vitro conditions. They are currently routinely used for disease modeling, preparation of difficult-to-access cell lines, monitoring of cellular mechanisms in micro- or macroscopic scales, drug testing and screening, genetic engineering, and many other applications. This review is a brief summary of the reprogramming process and subsequent differentiation and culture of reprogrammed cells into neural precursor cells (NPCs) in two-dimensional (2D) and three-dimensional (3D) conditions. NPCs can be used as biomedical models for neurodegenerative diseases (NDs), which are currently considered to be one of the major health problems in the human population.

Generation of ORIONi001-A induced pluripotent stem cell line for in vitro modeling of sporadic form of amyotrophic lateral sclerosis.

We generated new in vitro model for sporadic form of amyotrophic lateral sclerosis by reprogramming isolated skin fibroblasts into iPSCs. Fibroblasts were reprogrammed with commercially available synthetic polycistronic, self-replicating RNA vector. As verified by FISH, an early passages of a new iPSC line showed mosaic karyotype (cells with normal and abnormal karyotype 46,XY,t(2;14)(q13;p12) were present), while late passages contained only cells with abnormal karyotype. New iPSCs differentiated into all three germ layers and formed a teratoma in nude mice. Our iPSC line represents a new model for therapy testing and drug development in the field of ALS research.

A Comparative In Vitro Analysis of the Osteogenic Potential of Human Dental Pulp Stem Cells Using Various Differentiation Conditions.

Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification-OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.