Upstream Process Protocol for MSCs Isolated from Different Human-Based Tissue Origins.

This chapter introduces the increasing significance of mesenchymal stromal/stem cell (MSC) production in regenerative medicine and cellular therapeutics, outlines the growing interest in MSCs for various medical applications, and highlights their potential in advanced therapy medicinal products (ATMPs) and the advancements in cell culture technologies that have facilitated large-scale MSC production under Good Manufacturing Practices (GMP), ensuring safety and efficacy. This chapter describes an optimized upstream protocol for laboratory-scale MSC production from different tissue sources. This protocol, conducted in flasks, controls critical parameters and lays the foundation for downstream processing to generate ATMPs. This comprehensive approach underscores the potential of MSCs in clinical applications and the importance of tailored production processes.

Coffee-Derived Exosome-Like Nanoparticles: Are They the Secret Heroes?

BACKGROUND: Regular coffee consumption has beneficial and preventative effects on liver and chronic neurodegenerative diseases. However, the studies performed with the ingredients found in coffee beverages have not clarified the responsible mechanisms. Exosomes are small, membrane-coated cargo packages secreted by prokaryote and eukaryote cells. Exosomes regulate intercellular communication and affect cellular metabolic activities even among different species. In this study, we aimed to isolate and characterize the edible plant-derived exosome-like nanoparticles from roasted hot coffee beverages, hypothesizing that the edible plant-derived exosome-like nanoparticles were responsible for the beneficial effects of coffee. METHODS: Size exclusion chromatography and commercial kits were used for the isolation process. Efficient coffee edible plant-derived exosome-like nanoparticle fractions were determined by an ultraviolet-visible spectrophotometer. Harvested coffee edible plant-derived exosome-like nanoparticles were characterized by transmission electron microscopy. The quantification procedure was performed using a commercial kit. Coffee edible plant-derived exosome-like nanoparticles' proliferative effects on human hepatic stellate cells and human hepatocellular carcinoma cells were studied using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Whole-exosome RNA sequencing was performed. RESULTS: Transmission electron microscopy scanning analysis indicated round-shaped nanoparticles with sizes ranging from 40 to 100 nm. Both size exclusion chromatography and kit-isolated edible plant-derived exosome-like nanoparticle samples showed maximum absorbance at 227.5 nm in ultraviolet-visible spectrophotometer analysis. Regarding the quantitation results, kit isolation was more efficient than the size exclusion chromatography method when the harvested particle numbers were compared. An important MTT assay finding confirmed the observed beneficial effects of coffee beverages: coffee edible plant-derived exosome-like nanoparticles significantly suppressed hepatocellular carcinoma cell proliferation. As a result of sequencing, we identified 15 mature miRNAs. A MapReduce-based MicroRNA Target Prediction Method (The DIANA tools' MR-microT algorithm) highlighted 2 genes specifically associated with the miRNAs that we obtained: KMT2C and ZNF773. CONCLUSION: For the first time in the literature, coffee edible plant-derived exosome-like nanoparticles were identified. These nanoparticles may have therapeutic effects on chronic liver diseases. Experimental studies, therefore, should be performed on disease models to demonstrate their efficacy.

Evaluation of IL-8 nasal lavage levels and the effects of nasal involvement on disease severity in patients with stable chronic obstructive pulmonary disease.

Interleukin 8 (IL-8) is used to evaluate the severity of inflammation in the airways. The aim of this study was to evaluate patients with chronic obstructive pulmonary disease (COPD) for the presence of upper respiratory tract involvement by questioning patients regarding nasal symptoms and by measuring levels of IL-8 in nasal lavage material. A total of 47 COPD patients and 23 healthy controls were enrolled in this study. Pulmonary function tests were performed for all participants who were asked to complete a Sinonasal Outcome Test-20 (SNOT-20) questionnaire on the same day, as a measure of quality of life. Median IL-8 level in nasal lavage specimens of COPD patients with stable disease was higher than that of healthy controls. An increase in cigarette pack-years was significantly associated with an increase in nasal IL-8 levels. Similarly, IL-8 levels correlated positively with stage of COPD. A significant link between number of visits to the emergency department and stage of disease was observed. Patients with COPD had a significantly higher mean SNOT-20 severity score compared to healthy controls. Proper management of sinonasal disease may help to decrease the number of COPD attacks and consequently improve quality of life.

Continual expression throughout the cell cycle and downregulation upon adipogenic differentiation makes nucleostemin a vital human MSC proliferation marker.

Nucleostemin (NS) is a nucleolar protein expressed in stem and cancer cells. In combination with nuclear/nucleolar proteins, NS has been demonstrated to be involved in cell-cycle regulation and telomere maintenance. NS expression reflects the cell's proliferation state indicating that the cell is active in the cell cycle, whereas NS signals disappear upon differentiation. This study analyzes the spatio-temporal (nucleolar/nuclear localization during interphase and M-phase) NS remodeling in two distinct human mesenchymal stem cell (MSC) populations to discriminate the NS differences, if any, throughout their stem cell and differentiation states. Beside its prominent multilobular nucleolar localization in interphase cells, coexistence of NS with chromosome arms during mitosis was also observed. Disruption of mitotic microtubules induced dissociation of NS from the chromosome arms and scattered it into the cytoplasm. Compared to deciduous dental pulp MSCs, NS mRNA expression gradually decreased upon aging in umbilical cord stroma-derived MSCs as culture time increased. Following adipogenic differentiation of the latter, NS signals gradually disappeared in both dividing and non-dividing cells, even before the morphological and functional signs of adipogenic transformation appeared. Quantitative NS mRNA measurements showed that MSCs from two sources exhibit a strong nucleostemin expression similar to embryonic stem cells. In conclusion, apart from its novel chromosomal localization shown in this study, nucleolar NS can be considered as a marker that indicates the proliferation/differentiation states in human MSCs. Moreover, differences in the relative NS protein and mRNA levels may reflect the degree of proliferation and can be used to characterize in vitro expansion capabilities.