Regulatory Effects of Senescent Mesenchymal Stem Cells: Endotheliocyte Reaction.

Currently, there is a growing focus on aging and age-related diseases. The processes of aging are based on cell senescence, which results in changes in intercellular communications and pathological alterations in tissues. In the present study, we investigate the influence of senescent mesenchymal stem cells (MSCs) on endothelial cells (ECs). In order to induce senescence in MSCs, we employed a method of stress-induced senescence utilizing mitomycin C (MmC). Subsequent experiments involved the interaction of ECs with MSCs in a coculture or the treatment of ECs with the secretome of senescent MSCs. After 48 h, we assessed the EC state. Our findings revealed that direct interaction led to a decrease in EC proliferation and migratory activity of the coculture. Furthermore, there was an increase in the activity of the lysosomal compartment, as well as an upregulation of the genes P21, IL6, IL8, ITGA1, and ITGB1. Treatment of ECs with the "senescent" secretome resulted in less pronounced effects, although a decrease in proliferation and an increase in ICAM-1 expression were observed. The maintenance of high levels of typical "senescent" cytokines and growth factors after 48 h suggests that the addition of the "senescent" secretome may have a prolonged effect on the cells. It is noteworthy that in samples treated with the "senescent" secretome, the level of PDGF-AA was higher, which may explain some of the pro-regenerative effects of senescent cells. Therefore, the detected changes may underlie both the negative and positive effects of senescence. The findings provide insight into the effects of cell senescence in vitro, where many of the organism's regulatory mechanisms are absent.

Senescence-Associated Alterations in Matrisome of Mesenchymal Stem Cells.

The process of aging is intimately linked to alterations at the tissue and cellular levels. Currently, the role of senescent cells in the tissue microenvironment is still being investigated. Despite common characteristics, different cell populations undergo distinctive morphofunctional changes during senescence. Mesenchymal stem cells (MSCs) play a pivotal role in maintaining tissue homeostasis. A multitude of studies have examined alterations in the cytokine profile that determine their regulatory function. The extracellular matrix (ECM) of MSCs is a less studied aspect of their biology. It has been shown to modulate the activity of neighboring cells. Therefore, investigating age-related changes in the MSC matrisome is crucial for understanding the mechanisms of tissue niche ageing. This study conducted a broad proteomic analysis of the matrisome of separated fractions of senescent MSCs, including the ECM, conditioned medium (CM), and cell lysate. This is the first time such an analysis has been conducted. It has been established that there is a shift in production towards regulatory molecules and a significant downregulation of the main structural and adhesion proteins of the ECM, particularly collagens, fibulins, and fibrilins. Additionally, a decrease in the levels of cathepsins, galectins, S100 proteins, and other proteins with cytoprotective, anti-inflammatory, and antifibrotic properties has been observed. However, the level of inflammatory proteins and regulators of profibrotic pathways increases. Additionally, there is an upregulation of proteins that can directly cause prosenescent effects on microenvironmental cells (SERPINE1, THBS1, and GDF15). These changes confirm that senescent MSCs can have a negative impact on other cells in the tissue niche, not only through cytokine signals but also through the remodeled ECM.

Hypoxic Extracellular Matrix Preserves Its Competence after Expansion of Human MSCs under Physiological Hypoxia In Vitro.

Tissue-relevant O2 levels are considered as an important tool for the preconditioning of multipotent mesenchymal stromal cells (MSCs) for regenerative medicine needs. The present study investigated the quality and functions of the extracellular matrix (ECM) of MSCs under low O2 levels. Human adipose tissue-derived MSCs were continuously expanded under normoxia (20% O2, N) or "physiological" hypoxia (5% O2, Hyp). Decellularized ECM (dcECM) was prepared. The structure of the dcECM was analyzed using confocal laser and scanning electron microscopy. Collagen, dcECM-N, and dcECM-Hyp were recellularized with MSC-N and further cultured at normoxia. The efficacy of adhesion, spreading, growth, osteogenic potential, and paracrine activity of recellularized MSC-N were evaluated. At low O2, the dcECM showed an increased alignment of fibrillar structures and provided accelerated spreading of MSC-N, indicating increased dcECM-Hyp stiffness. We described O2-dependent "ECM-education" of MSC-N when cultured on dcECM-Hyp. This was manifested as attenuated spontaneous osteo-commitment, increased susceptibility to osteo-induction, and a shift in the paracrine profile. It has been suggested that the ECM after physiological hypoxia is able to ensure the maintenance of a low-commitment state of MSCs. DcECM, which preserves the competence of the natural microenvironment of cells and is capable of "educating" others, appears to be a prospective tool for guiding cell modifications for cell therapy and tissue engineering.

Phytohormones Affect Differentiation Status of Human Skin Fibroblasts via UPR Activation.

Normalization of secretory activity and differentiation status of mesenchymal cells, including fibroblasts, is an important biomedical problem. One of the possible solutions is modulation of unfolded protein response (UPR) activated during fibroblast differentiation. Here, we investigated the effect of phytohormones on the secretory activity and differentiation of cultured human skin fibroblasts. Based on the analysis of expression of genes encoding UPR markers, abscisic acid (ABA) upregulated expression of the GRP78 and ATF4 genes, while gibberellic acid (GA) upregulated expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA promoted secretion and synthesis of procollagen I and synthesis of fibronectin, as well as the total production of collagen and non-collagen proteins of the extracellular matrix (ECM). ABA also stimulated the synthesis of smooth muscle actin α (α-SMA), which is the marker of myofibroblasts, and increased the number of myofibroblasts in the cell population. On the contrary, GA increased the level of fibronectin secretion, but reduced procollagen I synthesis and the total production of the ECM collagen proteins. GA downregulated the synthesis of α-SMA and decreased the number of myofibroblasts in the cell population. Our results suggest that phytohormones modulate the biosynthetic activity of fibroblasts and affect their differentiation status.

Preparation and In Vitro Evaluation of Chitosan-g-Oligolactide Based Films and Macroporous Hydrogels for Tissue Engineering.

In the current study, novel matrices based on chitosan-g-oligo (L,L-/L,D-lactide) copolymers were fabricated. In particular, 2D films were prepared by solvent casting, while 3D macroporous hydrogels were obtained by lyophilization of copolymer solutions. Copolymers of chitosan (Chit) with semi-crystalline oligo (L,L-lactide) (Chit-LL) or amorphous oligo (L,D-lactide) (Chit-LD) were obtained by solid-state mechanochemical synthesis. The structure of the hydrogels was found to be a system of interconnected macropores with an average size of 150 µm. In vitro degradation of these copolymer-based matrices was shown to increase in the case of the Chit-LL-based hydrogel by 34% and decrease for the Chit-LD-based hydrogel by 23% compared to the parameter of the Chit sample. Localization and distribution of mouse fibroblast L929 cells and adipose tissue-derived mesenchymal stromal cells (MSCs) within the hydrogels was studied by confocal laser scanning microscopy (CLSM). Moreover, cellular response, namely cell adhesion, spreading, growth, proliferation, as well as cell differentiation in vitro were also evaluated in the hydrogels for 10-14 days. Both the Chit-LL and Chit-LD matrices were shown to support cell growth and proliferation, while they had improved swelling compared to the Chit matrix. Osteogenic MSCs differentiation on the copolymer-based films was studied by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Maximal expression levels of osteogenesis markers (alkaline phosphatase (ALPL), bone transcription factor (Runx2), and osteopontin (SPP1) were revealed for the Chit-LD films. Thus, osteodifferentiation was demonstrated to depend on the film composition. Both Chit-LL and Chit-LD copolymer-based matrices are promising for tissue engineering.

Real and Simulated Microgravity: Focus on Mammalian Extracellular Matrix.

The lack of gravitational loading is a pivotal risk factor during space flights. Biomedical studies indicate that because of the prolonged effect of microgravity, humans experience bone mass loss, muscle atrophy, cardiovascular insufficiency, and sensory motor coordination disorders. These findings demonstrate the essential role of gravity in human health quality. The physiological and pathophysiological mechanisms of an acute response to microgravity at various levels (molecular, cellular, tissue, and physiological) and subsequent adaptation are intensively studied. Under the permanent gravity of the Earth, multicellular organisms have developed a multi-component tissue mechanosensitive system which includes cellular (nucleo- and cytoskeleton) and extracellular (extracellular matrix, ECM) "mechanosensory" elements. These compartments are coordinated due to specialized integrin-based protein complexes, forming a distinctive mechanosensitive unit. Under the lack of continuous gravitational loading, this unit becomes a substrate for adaptation processes, acting as a gravisensitive unit. Since the space flight conditions limit large-scale research in space, simulation models on Earth are of particular importance for elucidating the mechanisms that provide a response to microgravity. This review describes current state of art concerning mammalian ECM as a gravisensitive unit component under real and simulated microgravity and discusses the directions of further research in this field.