Functional analysis of extracellular vesicles derived from human mesenchymal stromal cells used in cell therapy

As células estromais mesenquimais (MSC) são células heterogêneas com capacidade de controlar a resposta imunológica, atuando como uma ferramenta importante na terapia celular, principalmente no tratamento de doenças inflamatórias e autoimunes. Evidências sugerem que as vesículas extracelulares derivado de células estromais mesenquimais (EV-MSC) apresenta funções semelhantes às MSC, transportando moléculas bioativas para células-alvo, que podem alterar seu fenótipo ou comportamento funcional. O objetivo deste trabalho foi a caracterização morfológica e funcional das vesículas extracelulares derivadas das MSC (EV-MSC) de fontes distintas, cultivadas em meio livre de xenoantígenos, nas condições de hipóxia e normóxia. Para isso, foi padronizado um protocolo eficiente para depleção das vesículas extracelulares do soro AB humano utilizado na produção das EV-MSC, e avaliado o seu efeito sobre as características morfológicas, imunofenotípicas, proliferação, viabilidade e propriedades de diferenciação das MSC. Os resultados obtidos demonstraram que é possível isolar e caracterizar MSC do cordão umbilical (UC-MSC) e tecido adiposo (AT-MSC), em condições de normóxia e hipóxia, livres de xenoantígenos e empobrecido de EV, representando um grande avanço para a produção de EV-MSC. Em seguida, foram isoladas EV do sobrenadante de cultura das UC-MSC e AT-MSC por ultracentrifugação e caracterizadas quanto ao tamanho e concentração, bem como a expressão de proteínas. Também foi avaliado a expressão de genes envolvidos na imunorregulação presentes nas EV e o seu potencial de inibição e interação com os linfócitos T. Como conclusão, este trabalho demonstra que as EV derivadas de MSC apresentam um efeito imunomodulatório em condições livres de xenoantígenos e pré-condicionadas por hipóxia, com potencial aplicação terapêutica.Mesenchymal stromal cells (MSC) are heterogeneous cells capable of controlling the immune response, acting as an important tool in cell therapy, especially in the treatment of inflammatory and autoimmune diseases. Evidence suggests that extracellular vesicles derived from mesenchymal stromal cells (EV-MSC) has similar functions to MSC, transporting bioactive molecules to target cells, which can alter their phenotype or functional behavior. The objective of this work was the morphological and functional characterization of extracellular vesicles derived from MSC (EV-MSC) from different sources, cultured in a medium free of xenoantigens, under hypoxia and normoxia. For this purpose, an efficient protocol for depleting the extracellular vesicles of human AB serum was standardized and will be used in the production of EV-MSC. Then, the effect of depleted EV medium on the morphological, immunophenotypic, proliferation, viability and differentiation properties of MSC was evaluated. The results demonstrated that it is possible to isolate and characterize MSC from umbilical cord (UC-MSC) and adipose tissue (AT-MSC), in normoxic and hypoxic conditions, under conditions free of xenoantigens and depleted of EV, representing a major advance for production of EV-MSC. In addition, EV were isolated from the supernatant of the MSC by ultracentrifugation and characterized in terms of size and concentration, as well as protein expression. The expression of genes involved in immunoregulation present in EVs and their potential for inhibition and interaction with T lymphocytes was also evaluated. In conclusion, this work demonstrates that MSC-derived EVs have an immunomodulatory effect in conditions free of xenoantigens and pre-conditioned by hypoxia, with potential therapeutic application

Dynamics of SARS-CoV-2 Variants of Concern in Vaccination Model City in the State of Sao Paulo, Brazil

From a country with one of the highest SARS-CoV-2 morbidity and mortality rates, Brazil has implemented one of the most successful vaccination programs. Brazil’s first model city vaccination program was performed by the CoronaVac vaccine (Sinovac Biotech) in the town of Serrana, São Paulo State. To evaluate the vaccination effect on the SARS-CoV-2 molecular dynamics and clinical outcomes, we performed SARS-CoV-2 molecular surveillance on 4375 complete genomes obtained between June 2020 and April 2022 in this location. This study included the period between the initial SARS-CoV-2 introduction and during the vaccination process. We observed that the SARS-CoV-2 substitution dynamics in Serrana followed the viral molecular epidemiology in Brazil, including the initial identification of the ancestral lineages (B.1.1.28 and B.1.1.33) and epidemic waves of variants of concern (VOC) including the Gamma, Delta, and, more recently, Omicron. Most probably, as a result of the immunization campaign, the mortality during the Gamma and Delta VOC was significantly reduced compared to the rest of Brazil, which was also related to lower morbidity. Our phylogenetic analysis revealed the evolutionary history of the SARS-CoV-2 in this location and showed that multiple introduction events have occurred over time. The evaluation of the COVID-19 clinical outcome revealed that most cases were mild (88.9%, 98.1%, 99.1% to Gamma, Delta, and Omicron, respectively) regardless of the infecting VOC. In conclusion, we observed that vaccination was responsible for reducing the death toll rate and related COVID-19 morbidity, especially during the gamma and Delta VOC; however, it does not prevent the rapid substitution rate and morbidity of the Omicron VOC