Armenuhi Moghrovyan, Lilya Parseghyan, Gohar Sevoyan, Anna Darbinyan, Naira Sahakyan, Monica Gaboyan, Zaruhi Karabekian, and Armen Voskanyan. Korean J Pain 2022;35:140-51. https://doi.org/10.3344/kjp.2022.35.2.140

Background: Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells provide a unique opportunity to modify the original source of differentiated cells in order to minimize their rejection by non-autologous hosts. Hypothesis: Immune rejection of non-autologous human embryonic stem cells (hESC) derivatives can be reduced by down-regulating HLA-class I molecules, without affecting the ability of these cells to differentiate into specific lineages. Methods and Results: Beta2-microglobulin expression was decreased by lentiviral transduction using human anti HLA class I light chain beta2-microglobulin (B2M) shRNA. mRNA levels of B2M were decreased by 90% in a RUES2 modified hESC line, as determined by qRT-PCR analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as cell targets in a Mixed Lymphocyte Reaction (MLR) assay, transduced hESC and their differentiated derivatives did not stimulate allogeneic T cell proliferation. Using a cardiac differentiation protocol, transduced hESC formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESC cells were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages. Conclusions: HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from non-autologous hESC sources with improved immunocompatibility.

This study presents manufacturing of Poly(ethylene glycol) diacrylate (PEGDA)- Hydroxyapatite (HA) composite by Digital Light Processing (DLP) technology. PEGDA along with HA extracted from bovine was mixed with a photoinitiator (PI) and water to achieve optimized rheology for DLP. The printing parameters for DLP were optimized. Chemical composition, microstructure and mechanical properties of the composite were characterized. The compressive strength of PEGDA-HA composite achieved a maximum of 20 MPa in Z direction. The flexural strength was 56 MPa and 34 MPa for horizontal and vertical orientations, correspondingly, indicating anisotropic behavior of the samples. The cytotoxicity assessments were performed. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and confocal imaging indicated that the scaffold powder exhibits minimal cytotoxicity at low concentrations (0.001 g/ml), with high cell viability and minimal cell death or membrane damage.

Plants of the genus Alchemilla are well known for their pharmacological effects. Alchemilla smirnovii Juz., also known as lady's mantle possessed high growth-inhibiting properties against cancer cells according to earlier reports. We aimed to explore the anticancer modulatory effect of A. smirnovii extract toward 5-fluorouracil and elucidate the possible mechanism of its cytotoxic action in the HeLa cancer cell model. Synergistic interactions of plant extract with the chemotherapeutic agent were assessed by MTT assay. Based on obtained data the ethanol extract of A. smirnovii exhibits synergistic effects, enhancing the growth-inhibiting properties of 5-fluorouracil when used together. Additionally, the extract leads to a reduction in arginase activity and an elevation in the level of MDA․ This implies that A. smirnovii induces oxidative stress, and can potentially cause apoptosis in cancer cells.

At present, fully decellularized animal organ scaffolds are widely used in research for cell biology, tissue engineering, and regenerative medicine. These scaffolds serve as enhanced 3D models, providing natural microenvironments that support and guide cell interactions. In this study, the authors aimed to generate a reproducible 3D in vitro liver model for drug metabolism and toxicity testing that is based on decellularized rat liver scaffolds and to validate the model's suitability for toxicity tests. To restore liver-specific functions, various rat and human cell lines were used, including hepatocytes and macrophages. To date, the authors have achieved high reproducibility in the preparation of the scaffolds and demonstrated low cytotoxicity of liver scaffold material. Immunogenicity assays and experiments on recellularization of rat liver scaffolds are ongoing. The model's metabolic functionality is to be confirmed by assessing liver-specific markers. The development of such a model should enable efficient assessment of drug candidates while reducing the need for animal testing.