Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene—fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies

In Vitro Characterization of Poly(Lactic Acid)/ Poly(Hydroxybutyrate)/ Thermoplastic Starch Blends for Tissue Engineering Application

Complex in vitro characterization of a blended material based on Poly(Lactic Acid), Poly(Hydroxybutyrate), and Thermoplastic Starch (PLA/PHB/TPS) was performed in order to evaluate its potential for application in the field of tissue engineering. We focused on the biological behavior of the material as well as its mechanical and morphological properties. We also focused on the potential of the blend to be processed by the 3D printer which would allow the fabrication of the custom-made scaffold. Several blends recipes were prepared and characterized. This material was then studied in the context of scaffold fabrication. Scaffold porosity, wettability, and cell-scaffold interaction were evaluated as well. MTT test and the direct contact cytotoxicity test were applied in order to evaluate the toxic potential of the blended material. Biocompatibility studies were performed on the human chondrocytes. According to our results, we assume that material had no toxic effect on the cell culture and therefore could be considered as biocompatible. Moreover, PLA/PHB/TPS blend is applicable for 3D printing. Printed scaffolds had highly porous morphology and were able to absorb water as well. In addition, cells could adhere and proliferate on the scaffold surface. We conclude that this blend has potential for scaffold engineering

Isolation, Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem Cells

Mesenchymal stem cells (MSCs) represent an attractive source within the field of tissue engineering. However, their harvesting often requires invasive medical procedures. Urine-derived stem cells (UDSCs) display similar properties to MSCs, and their obtention and further processing is non-invasive for the donors as well as low cost. Here, we offer a comprehensive analysis of their biological properties. The goal of this study was to analyze their morphology, stemness, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm2, they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed that two different types of the cell populations have been present. The first type had a rice-grain shape and the second one was characterized by a polyhedral shape. In several cell cultures, dome-shaped cells were observed as well. All examined UDSCs expressed typical MSC-like surface markers, CD73, CD90 and CD105. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL-8, IL-6 and chemokines MCP-1 and GM-CSF. We have also successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Our findings indicate that UDSCs might have immense potential in the regeneration of the damaged tissues

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