Functional characterization of primary mesenchymal stem cells under proinflammatory and altered oxygenation conditions

Mezenhimske matične ćelije (MMĆ) deo su populacije multipotentnih matičnih ćelija prisutnih u mnogim adultnim tkivima gde učestvuju u održavanju homeostaze. Zahvaljujući sposobnosti samoobnove, potencijalu diferenciranja u specijalizovane ćelije mezodermskog porekla i imunomodultatornim svojstvima, ove nediferencirane ćelije karakteriše širok opseg potencijalne terapijske primene. Regenerativnom kapacitetu MMĆ doprinosi i sposobnost migracije do oštećenih tkiva gde učestvuju u njihovom obnavlјanju što ove ćelije čini pogodnim za ćelijsku terapiju. Mezenhimske MĆ su do sada izolovane iz velikog broja adultnih tkiva i mnoge studije potvrdile su da njihov regenerativni potencijal u velikoj meri zavisi od izvora iz kojeg potiču. Prvobitno su izolovane iz kostne srži koja je do sada najčešće korišćen izvor MMĆ, međutim zbog invazivnih metoda izolacije i malog prinosa MMĆ, stvorena je potreba za alternativnim izvorima. Činjenica da su MMĆ izolovane i iz periferne krvi jedno je od značajnih otkrića iz ove oblasti i smatra se da ove ćelije najverovatnije vode poreklo iz kostne srži, međutim nije dovoljno poznato na koji način migriraju i bivaju privučene u oštećena tkiva. Poslednjih godina se kao povolјan izvor MMĆ sa potencijalnom primenom u regenerativnoj medicini sve češće pominju i perinatalna tkiva (pupčanik i placenta). Prednost pupčanika kao izvora MMĆ ogleda se u lakoj dostupnosti tkiva koje se smatra medicinskim otpadom, neinvazivnoj izolaciji ćelija i činjenici da su izolovane MMĆ po kapacitetu samoobnove i većoj ekspresiji markera pluripotentnost slične embrionalnim MĆ. Iako se već nekoliko decenija MMĆ intenzivno istražuju, uprkos velikom doprinosu nauci i medicini, o njihovom upkupnom terapijskom potencijalu se još uvek nedovoljno zna. Trenutno nije poznat univerzalan mehanizam terapijskog delovanja MMĆ, a mnogobrojni nalazi sve više u poslednje vreme upućuju da ponašanje i uloge MMĆ zavise od konteksta mikrosredine u kojoj obitavaju i/ili deluju, kao mestu integracije spolјnih signala koji određuju sudbinu MMĆ i usmeravaju ih ka određenoj funkciji...present in many adult tissues where they participate in maintaining of homeostasis. Due to their self-renewal capacity, differentiation potential into specialized cells of mesodermal origin and immunomodulatory features, these undifferentiated cells are characterized with wide range of potential therapeutic applications. Their ability to migrate towards damaged tissues where they participate in tissue repair, also contributes to their regenerative potential which makes them suitable for cell therapy. MSCs have so far been isolated from various adult tissues and many studies confirmed that MSCs’ regenerative potential depends largely on the suorce which they originate from. They were originaly isolated from bone marrow which has been the most commonly used source of MSCs so far, however due to invasive methods of isolation and low MSCs yield, there is a need for alternative sources of MSCs. The fact that these cells were isolated from peripheral blood is one of the significant discoveries in this area and it is believed that these MSCs are most likely to originate from bone marrow, but how they migrate and home to damaged tissues has not yet been elucidated completely. Perinatal tissues (umbilical cord and placenta) have been recently increasingly suggested as favourable sources of MSCs with potential applications in regenerative medicine. The advantage of umbilical cord as a source of MSCs is reflected in easy availability of tissue considered as medical waste, non-invasive cell isolation methods, and the fact that MSCs isolated from umbilical cord are similar to embryonic stem cells in the terms of self-renewal capacity and pluripotency markers’ expression. Eventhough MSCs have been extensively investigated for several decades, and despite their great contribution to science and medicine, their overall therapeutic potential is still poorly understood. Currently, there is no universal mechanism of MSCs’ therapeutic action known, and many findings have recently suggested that behaviour and roles of MSCs depend on the context of microenvironment in which they reside and/or act, as sites of integration of external cues that determine the fate of MSCs and govern them towards specific function..

Inflammatory niche: Mesenchymal stromal cell priming by soluble mediators

Mesenchymal stromal/stem cells (MSCs) are adult stem cells of stromal origin that possess self-renewal capacity and the ability to differentiate into multiple mesodermal cell lineages. They play a critical role in tissue homeostasis and wound healing, as well as in regulating the inflammatory microenvironment through interactions with immune cells. Hence, MSCs have garnered great attention as promising candidates for tissue regeneration and cell therapy. Because the inflammatory niche plays a key role in triggering the reparative and immunomodulatory functions of MSCs, priming of MSCs with bioactive molecules has been proposed as a way to foster the therapeutic potential of these cells. In this paper, we review how soluble mediators of the inflammatory niche (cytokines and alarmins) influence the regenerative and immunomodulatory capacity of MSCs, highlighting the major advantages and concerns regarding the therapeutic potential of these inflammatory primed MSCs. The data summarized in this review may provide a significant starting point for future research on priming MSCs and establishing standardized methods for the application of preconditioned MSCs in cell therapy

Tumorigenic Aspects of MSC Senescence—Implication in Cancer Development and Therapy

As an organism ages, many physiological processes change, including the immune system. This process, called immunosenescence, characterized by abnormal activation and imbalance of innate and adaptive immunity, leads to a state of chronic low-grade systemic inflammation, termed inflammaging. Aging and inflammaging are considered to be the root of many diseases of the elderly, as infections, autoimmune and chronic inflammatory diseases, degenerative diseases, and cancer. The role of mesenchymal stromal/stem cells (MSCs) in the inflammaging process and the age-related diseases is not completely established, although numerous features of aging MSCs, including altered immunomodulatory properties, impeded MSC niche supporting functions, and senescent MSC secretory repertoire are consistent with inflammaging development. Although senescence has its physiological function and can represent a mechanism of tumor prevention, in most cases it eventually transforms into a deleterious (para-)inflammatory process that promotes tumor growth. In this review we are going through current literature, trying to explore the role of senescent MSCs in making and/or sustaining a microenvironment permissive to tumor development and to analyze the therapeutic options that could target this process

Dental mesenchymal stromal/stem cells in different microenvironments — implications in regenerative therapy

Current research data reveal microenvironment as a significant modifier of physical functions, pathologic changes, as well as the therapeutic effects of stem cells. When comparing regeneration potential of various stem cell types used for cytotherapy and tissue engineering, mesenchymal stem cells (MSCs) are currently the most attractive cell source for bone and tooth regeneration due to their differentiation and immunomodulatory potential and lack of ethical issues associated with their use. The microenvironment of donors and recipients selected in cytotherapy plays a crucial role in regenerative potential of transplanted MSCs, indicating interactions of cells with their microenvironment indispensable in MSC-mediated bone and dental regeneration. Since a variety of MSC populations have been procured from different parts of the tooth and tooth-supporting tissues, MSCs of dental origin and their achievements in capacity to reconstitute various dental tissues have gained attention of many research groups over the years. This review discusses recent advances in comparative analyses of dental MSC regeneration potential with regards to their tissue origin and specific microenvironmental conditions, giving additional insight into the current clinical application of these cells

Doxycycline Inhibits IL-17-Stimulated MMP-9 Expression by Downregulating ERK1/2 Activation: Implications in Myogenic Differentiation

Interleukin 17 (IL-17) is a cytokine with pleiotropic effects associated with several inflammatory diseases. Although elevated levels of IL-17 have been described in inflammatory myopathies, its role in muscle remodeling and regeneration is still unknown. Excessive extracellular matrix degradation in skeletal muscle is an important pathological consequence of many diseases involving muscle wasting. In this study, the role of IL-17 on the expression of matrix metalloproteinase- (MMP-) 9 inmyoblast cells was investigated. The expression of MMP-9 after IL-17 treatment was analyzed in mouse myoblasts C2C12 cell line. The increase in MMP-9 production by IL-17 was concomitant with its capacity to inhibit myogenic differentiation of C2C12 cells. Doxycycline (Doxy) treatment protected the myogenic capacity of myoblasts from IL-17 inhibition and, moreover, increased myotubes hypertrophy. Doxy blocked the capacity of IL-17 to stimulateMMP-9 production by regulating IL-17-induced ERK1/2 MAPK activation. Our results imply that MMP-9 mediates IL-17's capacity to inhibit myoblast differentiation during inflammatory diseases and indicate that Doxy can modulate myoblast response to inflammatory induction by IL-17

Inflammatory Cytokines Prime Adipose Tissue Mesenchymal Stem Cells to Enhance Malignancy of MCF-7 Breast Cancer Cells via Transforming Growth Factor-beta 1

Mesenchymal stem cells from human adipose tissue (hASCs) are proposed as suitable tools for soft tissue engineering and reconstruction. Although it is known that hASCs have the ability to home to sites of inflammation and tumor niche, the role of inflammatory cytokines in the hASCs-affected tumor development is not understood. We found that interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF-alpha) prime hASCs to produce soluble factors which enhance MCF-7 cell line malignancy in vitro. IFN-gamma and/or TNF-alpha-primed hASCs produced conditioned media (CM) which induced epithelial to mesenchymal transition (EMT) of MCF-7 cells by reducing E-Cadherin and increasing Vimentin expression. Induced EMT was accompanied by increased invasion, migration, and urokinase type-plasminogen activator (uPA) expression in MCF-7 cells. These effects were mediated by increased expression of transforming growth factor-beta 1(TGF-beta 1) in cytokines-primed hASCs, since inhibition of type I TGF-beta 1 receptor on MCF-7 cells and neutralization of TGF-beta 1 disabled the CM from primed hASCs to increase EMT, cell migration, and uPA expression in MCF-7 cells. Obtained data suggested that IFN-gamma and/or TNF-alpha primed hASCs might enhance the malignancy of MCF-7 cell line by inducing EMT, cell motility and uPA expression in these cells via TGF-beta 1-Smad3 signalization, with potentially important implications in breast cancer progression

Detrimental Effect of Various Preparations of the Human Amniotic Membrane Homogenate on the 2D and 3D Bladder Cancer In vitro Models

Despite being among the ten most common cancers with high recurrence rates worldwide, there have been no major breakthroughs in the standard treatment options for bladder cancer in recent years. The use of a human amniotic membrane (hAM) to treat cancer is one of the promising ideas that have emerged in recent years. This study aimed to investigate the anticancer activity of hAM homogenate on 2D and 3D cancer models. We evaluated the effects of hAM homogenates on the human muscle invasive bladder cancer urothelial (T24) cells, papillary cancer urothelial (RT4) cells and normal porcine urothelial (NPU) cells as well as on human mammary gland non-tumorigenic (MCF10a) cells and low-metastatic breast cancer (MCF7) cells. After 24 h, we observed a gradual detachment of cancerous cells from the culture surface, while the hAM homogenate did not affect the normal cells. The most pronounced effect hAM homogenate had on bladder cancer cells; however, the potency of their detachment was dependent on the treatment protocol and the preparation of hAM homogenate. We demonstrated that hAM homogenate significantly decreased the adhesion, growth, and proliferation of human bladder invasive and papillary cancer urothelial cells and did not affect normal urothelial cells even in 7-day treatment. By using light and electron microscopy we showed that hAM homogenate disrupted the architecture of 2D and 3D bladder cancer models. The information provided by our study highlights the detrimental effect of hAM homogenate on bladder cancer cells and strengthens the idea of the potential clinical application of hAM for bladder cancer treatment

Vitamin D3 Stimulates Proliferation Capacity, Expression of Pluripotency Markers, and Osteogenesis of Human Bone Marrow Mesenchymal Stromal/Stem Cells, Partly through SIRT1 Signaling

The biology of vitamin D3 is well defined, as are the effects of its active metabolites on various cells, including mesenchymal stromal/stem cells (MSCs). However, the biological potential of its precursor, cholecalciferol (VD3), has not been sufficiently investigated, although its significance in regenerative medicine—mainly in combination with various biomaterial matrices—has been recognized. Given that VD3 preconditioning might also contribute to the improvement of cellular regenerative potential, the aim of this study was to investigate its effects on bone marrow (BM) MSC functions and the signaling pathways involved. For that purpose, the influence of VD3 on BM-MSCs obtained from young human donors was determined via MTT test, flow cytometric analysis, immunocytochemistry, and qRT-PCR. Our results revealed that VD3, following a 5-day treatment, stimulated proliferation, expression of pluripotency markers (NANOG, SOX2, and Oct4), and osteogenic differentiation potential in BM-MSCs, while it reduced their senescence. Moreover, increased sirtuin 1 (SIRT1) expression was detected upon treatment with VD3, which mediated VD3-promoted osteogenesis and, partially, the stemness features through NANOG and SOX2 upregulation. In contrast, the effects of VD3 on proliferation, Oct4 expression, and senescence were SIRT1-independent. Altogether, these data indicate that VD3 has strong potential to modulate BM-MSCs’ features, partially through SIRT1 signaling, although the precise mechanisms merit further investigation

Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway

Bladder cancer is the 10th most commonly diagnosed cancer with the highest lifetime treatment costs. The human amniotic membrane (hAM) is the innermost foetal membrane that possesses a wide range of biological properties, including anti-inflammatory, antimicrobial and anticancer properties. Despite the growing number of studies, the mechanisms associated with the anticancer effects of human amniotic membrane (hAM) are poorly understood. Here, we reported that hAM preparations (homogenate and extract) inhibited the expression of the epithelial–mesenchymal transition markers N-cadherin and MMP-2 in bladder cancer urothelial cells in a dose-dependent manner, while increasing the secretion of TIMP-2. Moreover, hAM homogenate exerted its antimigratory effect by downregulating the expression of FAK and proteins involved in actin cytoskeleton reorganisation, such as cortactin and small RhoGTPases. In muscle-invasive cancer urothelial cells, hAM homogenate downregulated the PI3K/Akt/mTOR signalling pathway, the key cascade involved in promoting bladder cancer. By using normal, non-invasive papilloma and muscle-invasive cancer urothelial models, new perspectives on the anticancer effects of hAM have emerged. The results identify new sites for therapeutic intervention and are prompt encouragement for ongoing anticancer drug development studies

Uticaj Doksiciklina na regenerativni potencijal mezenhimskih matičnih ćelija periodoncijuma

Parodontopatija je hronično progresivno inflamatorno oboljenje tkiva parodoncijuma uzrokovano bakterijama zubnog plaka. Destrukcija parodoncijuma je posledica prekomerne aktivacije inflamatornog odgovora domaćina na bakterijsku infekciju i posledične produkcije citokina uključujući Interleukin-17 (IL-17). Lečenje parodontopatije podrazumeva i sistemsku primenu oralnih antibiotika poput doksociklina. Ovaj antibiotik iz klase tetraciklina ispoljava kako antibakterijska, tako i antiinflamatorna svojstva, a poznato je da utiče i na reparaciju tkiva i metabolizam alveolarnih kostiju. Cilj ove studije je bio da utvrdimo da li doksociklin utiče na regenerativni potencijal mezenhimskih matičnih ćelija periodoncijuma (PDL-MMĆ) tretiranih sa IL-17. Naši rezultati su pokazali da doksociklin značajno smanjuje stimulativni efekat IL-17 na migraciju i ekspresiju matriksne metaloproteinaze 2 u PDL-MMĆ. Pored toga, doksociklin stimuliše osteogenu diferencijaciju PDL-MMĆ poništavajući inhibitorni efekat IL-17 na osteogenezu ovih ćelija. Analize ćelijske respiracije su otkrile da doksociklin smanjuje stopu potrošnje kiseonika i mitohondrijalnu biogenezu u IL-17-tretiranim PDL-MMĆ. Pokazana pro-regenerativna svojstva doksociklina u inflamatornom okruženju ukazuju na potencijal njegove primene u razvoju novih pristupa za terapiju parodontopatije