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.

The secretome from embryonic stem cell cardiomyogenesis: Same signals, different cellular feedbacks.

Ischemic heart diseases are a global health problem that requires the search for alternative therapies to the current treatments. Thus, an understanding of how cardiomyogenic signals can affect cellular behavior would allow us to create strategies to improve the cell recovery in damaged tissues. In this study, we aimed to evaluate the effects of the conditioned medium (CM), collected at different time points during in vitro cardiomyogenesis of human embryonic stem cells (hESCs), to direct cell behavior. We assayed different cell types to demonstrate noncytotoxic effects from the collected CM and that the CM obtained at initial time points of cardiomyogenic differentiation could promote the cell proliferation. Otherwise, the secretome derived from cardiac committed cells during cardiomyogenesis was unable to improve angiogenesis or migration in endothelial cells, and ineffective to stimulate the differentiation of cardioblasts or increase the differentiation efficiency of hESC. Therefore, we demonstrated that the effectiveness of the CM response varies depending on the cell type and the differentiation step of hESC-derived cardiomyocytes.

Morphological and pathogenic investigation of the emerging fungal threat Emergomyces africanus.

Emergomyces africanus is a highly fatal fungal pathogen affecting individuals with advanced HIV disease. Molecular patterns and ultrastructural aspects of E. africanus are unknown, and pathogenic models have not been investigated in detail. Since the cell wall of fungi is a determinant for interaction with the host and antifungal development, we characterized the ultrastructural aspects of E. africanus and the general properties of cell wall components under different conditions of growth in vitro and in vivo. We also tested the pathogenic potential of E. africanus in a Galleria mellonella model of infection. Transmission electron microscopy revealed the common intracellular, ultrastructural features of fungi in association with a thick cell wall. Scanning electron microscopy revealed a smooth cell surface, with no apparent decorative structures. Yeast cultures of E. africanus showed the distribution of chitin, chitooligomers, and mannoproteins commonly observed in fungi. However, in mixed microenvironments containing yeast and filamenting forms of E. africanus, the detection of chitooligomers was increased in comparison with isolated yeast cells, while the detection of these components in filamenting forms was markedly reduced. These observations were suggestive of the ability of E. africanus to change its cell wall composition in response to different microenvironments. Although E. africanus was unable to kill G. mellonella, this infection model allowed us to isolate infected hemocytes for further analysis of mannoproteins, chitin, and chitooligomers. Once again, the detection of E. africanus chitooligomers was markedly increased. These results reveal previously unknown ultrastructural features of E. africanus and suggest a high plasticity in the cell wall of this lethal pathogen. IMPORTANCE: The epidemiology of fungal infections is very dynamic, and novel health emergencies are hard to predict. New fungal pathogens have been continuously emerging for the last few decades, and Emergomyces africanus is one of these threats to human health. This complex scenario points to the need for generating knowledge about emerging pathogens so that new therapeutic strategies can be designed. In this study, we characterized the general cellular and pathogenic properties of the emerging fungal pathogen E. africanus. Our results reveal that E. africanus manifests some of the typical properties of fungal cells but also exhibits some unique characteristics that might be helpful for the future development of therapeutic strategies.

Adipogenesis, Osteogenesis, and Chondrogenesis of Human Mesenchymal Stem/Stromal Cells: A Comparative Transcriptome Approach.

Adipogenesis, osteogenesis and chondrogenesis of human mesenchymal stem/stromal cells (MSC) are complex and highly regulated processes. Over the years, several studies have focused on understanding the mechanisms involved in the MSC commitment to the osteogenic, adipogenic and/or chondrogenic phenotypes. High-throughput methodologies have been used to investigate the gene expression profile during differentiation. Association of data analysis of mRNAs, microRNAs, circular RNAs and long non-coding RNAs, obtained at different time points over these processes, are important to depict the complexity of differentiation. This review will discuss the results that were highlighted in transcriptome analyses of MSC undergoing adipogenic, osteogenic and chondrogenic differentiation. The focus is to shed light on key molecules, main signaling pathways and biological processes related to different time points of adipogenesis, osteogenesis and chondrogenesis.

Influence of donor age on the differentiation and division capacity of human adipose-derived stem cells.

BACKGROUND: Human adipose-derived stromal/stem cells (hASCs) are one of the most useful types of mesenchymal stromal/stem cells, which are adult multipotent cells with great therapeutic potential for the treatment of several diseases. However, for successful clinical application, it is critical that high-quality cells can be obtained. Diverse factors seem to be able to influence cell quality and performance, especially factors related to donors' intrinsic characteristics, such as age. Nevertheless, there is no consensus regarding this characteristic, and there is conflicting information in the literature. AIM: To investigate the growth kinetics and differentiation potential of adipose-derived stem cells isolated from the lipoaspirates of elderly and young donors. METHODS: hASCs were harvested from liposuctioned adipose tissue obtained from female donors (aged 20-70 years). Cells were distributed into two groups according to age range: old hASCs (oASCs, ≥ 55 years, n = 9) and young hASCs (yASCs, ≤ 35 years, n = 9). For each group, immunophenotypic characterization was performed by flow cytometry. Population doubling time was assessed over seven days. For adipogenic potential evaluation, lipid deposits were assessed after 7 d, 14 d and 21 d of adipogenic induction. Osteogenic potential was verified by analyzing cell mineralization after 14 d, 21 d and 28 d of osteogenic induction. mRNA expression of PPARγ2, CEBPA and Runx2 were detected by quantitative reverse transcription polymerase chain reaction. RESULTS: hASCs were successfully obtained, cultured, and grouped according to their age: yASCs (26.33 ± 4.66 years old) and oASCs (64.78 ± 4.58 years old). After maintenance of the cells in culture, there were no differences in morphology between cells from the young and old donors. Additionally, both groups showed classical immunophenotypic characteristics of mesenchymal stem/stromal cells. The average doubling time indicated that yASCs (4.09 ± 0.94 d) did not significantly differ from oASCs (4.19 ± 1.29 d). Concerning differentiation potential, after adipogenic and osteogenic induction, yASCs and oASCs were able to differentiate to greater levels than the noninduced control cells. However, no differences were found in the differentiation efficiency of yASCs and oASCs in adipogenesis or osteogenesis. Additionally, the mRNA expression of PPARγ2, CEBPA and Runx2 were similar in yASCs and oASCs. CONCLUSION: Our findings suggest that age does not seem to significantly affect the cell division or adipogenic or osteogenic differentiation ability of adipose-derived stem cells isolated from lipoaspirates.

Cardiomyogenesis Modeling Using Pluripotent Stem Cells: The Role of Microenvironmental Signaling.

Pluripotent stem cells (PSC) can be used as a model to study cardiomyogenic differentiation. In vitro modeling can reproduce cardiac development through modulation of some key signaling pathways. Therefore, many studies make use of this strategy to better understand cardiomyogenesis complexity and to determine possible ways to modulate cell fate. However, challenges remain regarding efficiency of differentiation protocols, cardiomyocyte (CM) maturation and therapeutic applications. Considering that the extracellular milieu is crucial for cellular behavior control, cardiac niche studies, such as those identifying secreted molecules from adult or neonatal tissues, allow the identification of extracellular factors that may contribute to CM differentiation and maturation. This review will focus on cardiomyogenesis modeling using PSC and the elements involved in cardiac microenvironmental signaling (the secretome - extracellular vesicles, extracellular matrix and soluble factors) that may contribute to CM specification and maturation.