Cellular In Vitro Responses Induced by Human Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Obtained from Suspension Culture.

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-ß and IL-1ß was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.

A peptide dataset for target analysis of human complement system proteins.

The targeted LC-MS/MS method has been widely applied for peptide quantification, offering sensibility, specificity, and reproducibility to the analysis. However, it requires the prior selection of targets, including the construction of a spectral library. Here, we present a dataset comprising peptide mass spectra for targeted LC-MS/MS method setup, applied to a set of human complement system proteins. Additionally, we selected a group of peptides and demonstrated their stability and reproducibility in quantification. This dataset is invaluable for studies aiming at the quantification of the complement system proteins by targeted LC-MS/MS, as it provides data for spectral library construction and a list of selected peptides.

Generation of an induced pluripotent stem cell line from a patient with epileptic encephalopathy caused by the CYFIP2 R87C variant.

Induced pluripotent stem cells (iPSCs) opened the possibility to use patient cells as a model for several diseases. iPSCs can be reprogrammed from somatic cells collected in a non-invasive way, and then differentiated into any other cell type, while maintaining the donor´s genetic background. CYFIP2 variants were associated with the onset of an early form of epileptic encephalopathy. Studies with patients showed that the R87C variant seems to be one of the variants that causes more severe disease, however, to date there are no studies with a human cell model that allows investigation of the neuronal phenotype of the R87C variant. Here, we generated an iPSC line from a patient with epileptic encephalopathy caused by the CYFIP2 R87C variant. We obtained iPSC clones by reprogramming urinary progenitor cells from a female patient. The generated iPSC line presented a pluripotent stem cell morphology, normal karyotype, expressed pluripotency markers and could be differentiated into the three germ layers. In further studies, this cell line could be used as model for epileptic encephalopathy disease and drug screening studies.

Selecting Genetic Variants and Interactions Associated with Amyotrophic Lateral Sclerosis: A Group LASSO Approach.

Amyotrophic lateral sclerosis (ALS) is a multi-system neurodegenerative disease that affects both upper and lower motor neurons, resulting from a combination of genetic, environmental, and lifestyle factors. Usually, the association between single-nucleotide polymorphisms (SNPs) and this disease is tested individually, which leads to the testing of multiple hypotheses. In addition, this classical approach does not support the detection of interaction-dependent SNPs. We applied a two-step procedure to select SNPs and pairwise interactions associated with ALS. SNP data from 276 ALS patients and 268 controls were analyzed by a two-step group LASSO in 2000 iterations. In the first step, we fitted a group LASSO model to a bootstrap sample and a random subset of predictors (25%) from the original data set aiming to screen for important SNPs and, in the second step, we fitted a hierarchical group LASSO model to evaluate pairwise interactions. An in silico analysis was performed on a set of variables, which were prioritized according to their bootstrap selection frequency. We identified seven SNPs (rs16984239, rs10459680, rs1436918, rs1037666, rs4552942, rs10773543, and rs2241493) and two pairwise interactions (rs16984239:rs2118657 and rs16984239:rs3172469) potentially involved in nervous system conservation and function. These results may contribute to the understanding of ALS pathogenesis, its diagnosis, and therapeutic strategy improvement.

Molecular Dynamics of CYFIP2 Protein and Its R87C Variant Related to Early Infantile Epileptic Encephalopathy.

The CYFIP2 protein (cytoplasmic FMR1-interacting protein 2) is part of the WAVE regulatory complex (WRC). CYFIP2 was recently correlated to neurological disorders by the association of the R87C variant with early infantile epileptic encephalopathy (EIEE) patients. In this set of syndromes, the epileptic spasms and seizures since early childhood lead to impaired neurological development in children. Inside the WRC, the variant residue is at the CYFIP2 and WAVE1 protein interface. Thus, the hypothesis is that the R87C modification weakens this interaction, allowing the WRC complex's constant activation. This work aimed to investigate the impacts of the mutation on the structure of the WRC complex through molecular dynamics simulation. For that, we constructed WRC models containing WAVE1-NCKAP1 proteins complexed with WT or R87C CYFIP2. Our simulations showed a flexibilization of the loop comprising residues 80-110 due to the loss of contacts between internal residues in the R87C CYFIP2 as well as the key role of residues R/C87, E624, and E689 in structural modification. These data could explain the mechanism by which the mutation impairs the stability and proper regulation of the WRC.

Safety and long-term improvement of mesenchymal stromal cell infusion in critically COVID-19 patients: a randomized clinical trial.

BACKGROUND: COVID-19 is a multisystem disease that presents acute and persistent symptoms, the postacute sequelae (PASC). Long-term symptoms may be due to consequences from organ or tissue injury caused by SARS-CoV-2, associated clotting or inflammatory processes during acute COVID-19. Various strategies are being chosen by clinicians to prevent severe cases of COVID-19; however, a single treatment would not be efficient in treating such a complex disease. Mesenchymal stromal cells (MSCs) are known for their immunomodulatory properties and regeneration ability; therefore, they are a promising tool for treating disorders involving immune dysregulation and extensive tissue damage, as is the case with COVID-19. This study aimed to assess the safety and explore the long-term efficacy of three intravenous doses of UC-MSCs (umbilical cord MSCs) as an adjunctive therapy in the recovery and postacute sequelae reduction caused by COVID-19. To our knowledge, this is one of the few reports that presents the longest follow-up after MSC treatment in COVID-19 patients. METHODS: This was a phase I/II, prospective, single-center, randomized, double-blind, placebo-controlled clinical trial. Seventeen patients diagnosed with COVID-19 who require intensive care surveillance and invasive mechanical ventilation-critically ill patients-were included. The patient infusion was three doses of 5 × 105 cells/kg UC-MSCs, with a dosing interval of 48 h (n = 11) or placebo (n = 6). The evaluations consisted of a clinical assessment, viral load, laboratory testing, including blood count, serologic, biochemical, cell subpopulation, cytokines and CT scan. RESULTS: The results revealed that in the UC-MSC group, there was a reduction in the levels of ferritin, IL-6 and MCP1-CCL2 on the fourteen day. In the second month, a decrease in the levels of reactive C-protein, D-dimer and neutrophils and an increase in the numbers of TCD3, TCD4 and NK lymphocytes were observed. A decrease in extension of lung damage was observed at the fourth month. The improvement in all these parameters was maintained until the end of patient follow-up. CONCLUSIONS: UC-MSCs infusion is safe and can play an important role as an adjunctive therapy, both in the early stages, preventing severe complications and in the chronic phase with postacute sequelae reduction in critically ill COVID-19 patients. Trial registration Brazilian Registry of Clinical Trials (ReBEC), UTN code-U1111-1254-9819. Registered 31 October 2020-Retrospectively registered, https://ensaiosclinicos.gov.br/rg/RBR-3fz9yr.

Modulation and function of Pumilio proteins in cancer.

Post-transcriptional regulation is involved in tumorigenesis, and in this control, RNA-binding proteins are the main protagonists. Pumilio proteins are highly conserved RNA-binding proteins that regulate many aspects of RNA processing. The dysregulation of Pumilio expression is associated with different types of cancer. This review summarizes the roles of Pumilio 1 and Pumilio 2 in cancer and discusses the factors that account for their distinct biological functions. Pumilio levels seem to be related to tumor progression and poor prognoses in some kinds of tumors, such as lung, pancreatic, prostate, and cervical cancers. Pumilio 1 is associated with cancer proliferation, migration, and invasion, and so is Pumilio 2, although there are contradictory reports regarding the latter. Furthermore, the circular RNA, circPUM1, has been described as a miRNAs sponge, regulating miRNA involved in the cell cycle. The expression and function of Pumilio proteins depend on the fine adjustment of a set of modulators, including miRNAs, lncRNAs, and circRNAs; this demonstrates that Pumilio plays an important role in tumorigenesis through a variety of regulatory axes.

Interleukin 21 Receptor Affects Adipogenesis of Human Adipose-Derived Stem/Stromal Cells.

Dysfunctions in adipose tissue cells are responsible for several obesity-related metabolic diseases. Understanding the process of adipocyte formation is thus fundamental for understanding these diseases. The adipocyte differentiation of adipose-derived stem/stromal cells (ADSCs) showed a reduction in the mRNA level of the interleukin 21 receptor (IL21R) during this process. Although the receptor has been associated with metabolic diseases, few studies have examined its function in stem cells. In this study, we used confocal immunofluorescence assays to determine that IL21R colocalizes with mitochondrial protein ATP5B, ALDH4A1, and the nucleus of human ADSCs. We demonstrated that silencing and overexpression of IL21R did not affect the cell proliferation and mitochondrial activity of ADSCs. However, IL21R silencing did reduce ADSC adipogenic capacity. Further studies are needed to understand the mechanism involved between IL21R and the adipogenic differentiation process.

Accessing the Human Pluripotent Stem Cell Translatome by Polysome Profiling.

Polysome profiling is a technique that uses sucrose density gradient ultracentrifugation to separate complexes of mRNAs associated with one or more ribosomes. Here we describe polysome profiling analysis in human pluripotent stem cells (hPSCs) using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR or large-scale sequencing analysis, used to establish the translational status of specific mRNAs and identify the role of noncoding RNA in translation.

Alternative Methods as Tools for Obesity Research: In Vitro and In Silico Approaches.

The study of adipogenesis is essential for understanding and treating obesity, a multifactorial problem related to body fat accumulation that leads to several life-threatening diseases, becoming one of the most critical public health problems worldwide. In this review, we propose to provide the highlights of the adipogenesis study based on in vitro differentiation of human mesenchymal stem cells (hMSCs). We list in silico methods, such as molecular docking for identification of molecular targets, and in vitro approaches, from 2D, more straightforward and applied for screening large libraries of substances, to more representative physiological models, such as 3D and bioprinting models. We also describe the development of physiological models based on microfluidic systems applied to investigate adipogenesis in vitro. We intend to identify the main alternative models for adipogenesis evaluation, contributing to the direction of preclinical research in obesity. Future directions indicate the association of in silico and in vitro techniques to bring a clear picture of alternative methods based on adipogenesis as a tool for obesity research.