Mesenchymal stem cell-derived extracellular vesicles exert pro-angiogenic and pro-lymphangiogenic effects in ischemic tissues by transferring various microRNAs and proteins including ITGa5 and NRP1.

Mesenchymal stem cells/stromal cells (MSCs)-derived extracellular vesicles (EVs) mediate pro-regenerative effects in damaged ischemic tissues by regulating angiogenesis. MSCs-EVs modulate functions of cells including endogenous mature cells, progenitors and stem cells, resulting in restoration of blood flow. However, the mechanisms underlying such MSC-EV activity still remain poorly understood. The present study analyzes biological effects of bone marrow (BM) MSC-EVs on endothelial cells (ECs) in ischemic tissues both in in vitro and in vivo conditions and elucidates the molecular mechanisms underlying the tissue repair. MSC-EVs were isolated from murine BM-derived MSCs and their morphological, antigenic and molecular composition regarding protein and microRNA levels were evaluated to examine their properties. Global proteomic analysis demonstrated the presence in MSC-EVs of proteins regulating pro-regenerative pathways, including integrin α5 (Itgα5) and neuropilin-1 (NRP1) involved in lymphangiogenesis. MSC-EVs were also enriched in microRNAs regulating angiogenesis, TGF-ß signaling and processes guiding cellular adhesion and interactions with extracellular matrix. The functional effects of MSC-EVs on capillary ECs in vitro included the increase of capillary-like tube formation and cytoprotection under normal and inflammatory conditions by inhibiting apoptosis. Notably, MSC-EVs enhanced also capillary-like tube formation of lymphatic ECs, which may be regulated by Itgα5 and NRP1. Moreover, in a mouse model of critical hind limb ischemia, MSC-EVs increased the recovery of blood flow in ischemic muscle tissue, which was accompanied with increased vascular density in vivo. This pro-angiogenic effect was associated with an increase in nitric oxide (NO) production via endothelial NO-synthase activation in ischemic muscles. Interestingly, MSC-EVs enhanced lymphangiogenesis, which has never been reported before. The study provides evidence on pro-angiogenic and novel pro-lymphangiogenic role of MSC-EVs on ECs in ischemic tissue mediated by their protein and miRNA molecular cargos. The results highlight Itgα5 and NRP1 carried by MSC-EVs as potential therapeutic targets to boost lymphangiogenesis.

Induced pluripotent stem cell-derived extracellular vesicles enriched with miR-126 induce proangiogenic properties and promote repair of ischemic tissue.

Emerging evidence suggests that stem cell-derived extracellular vesicles (EVs) may induce pro-regenerative effects in ischemic tissues by delivering bioactive molecules, including microRNAs. Recent studies have also shown pro-regenerative benefits of EVs derived from induced pluripotent stem (iPS) cells. However, the underlying mechanisms of EV benefits and the role of their transferred regulatory molecules remain incompletely understood. Accordingly, we investigated the effects of human iPS-derived EVs (iPS-EVs) enriched in proangiogenic miR-126 (iPS-miR-126-EVs) on functional properties of human endothelial cells (ECs) in vitro. We also examined the outcomes following EV injection in a murine model of limb ischemia in vivo. EVs were isolated from conditioned media from cultures of unmodified and genetically modified human iPS cells overexpressing miR-126. The iPS-miR-126-EVs were enriched in miR-126 when compared with control iPS-EVs and effectively transferred miR-126 along with other miRNAs to recipient ECs improving their functional properties essential for ischemic tissue repair, including proliferation, metabolic activity, cell survival, migration, and angiogenic potential. Injection of iPS-miR-126-EVs in vivo in a murine model of acute limb ischemia promoted angiogenesis, increased perfusion, and enhanced functional recovery. These observations corresponded with elevated expression of genes for several proangiogenic factors in ischemic tissues following iPS-miR-126-EV transplantation. These results indicate that innate pro-regenerative properties of iPS-EVs may be further enhanced by altering their molecular composition via controlled genetic modifications. Such iPS-EVs overexpressing selected microRNAs, including miR-126, may represent a novel acellular tool for therapy of ischemic tissues in vivo.

Evidence of Stem Cells Mobilization in the Blood of Patients with Pancreatitis: A Potential Link with Disease Severity.

A growing number of studies indicate the potential involvement of various populations of bone marrow-derived stem cells (BMSCs) in tissue repair. However, the mobilization of BMSCs to the peripheral blood (PB) in acute and chronic pancreatitis (AP and CP) has not been investigated. A total of 78 patients were assigned into AP, CP, and healthy control groups in this study. Using flow cytometry, we found that VSELs, EPCs, and CD133+SCs were mobilized to the PB of patients with both AP and CP. Interestingly, AP and CP patients exhibited lower absolute number of circulating MSCs in the PB compared to healthy individuals. SC mobilization to the PB was more evident in patients with AP than CP and in patients with moderate/severe AP than mild AP. Using ELISA, we found a significantly increased HGF concentration in the PB of patients with AP and SDF1α in the PB of patients with CP. We noted a significant positive correlation between SDF1α concentration and the mobilized population of CD133+SCs in AP and between C5a and the mobilized population of VSELs moderate/severe AP. Thus, bone marrow-derived SCs may play a role in the regeneration of pancreatic tissue in both AP and CP, and mobilization of VSELs to the PB depends on the severity of AP.

Processing and Ex Vivo Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells for the Development of an Advanced Therapy Medicinal Product for use in Humans.

Adipose tissue (AT) represents a commonly used source of mesenchymal stem/stromal cells (MSCs) whose proregenerative potential has been widely investigated in multiple clinical trials worldwide. However, the standardization of the manufacturing process of MSC-based cell therapy medicinal products in compliance with the requirements of the local authorities is obligatory and will allow us to obtain the necessary permits for product administration according to its intended use. Within the research phase (RD), we optimized the protocols used for the processing and ex vivo expansion of AT-derived MSCs (AT-MSCs) for the development of an Advanced Therapy Medicinal Product (ATMP) for use in humans. Critical process parameters (including, e.g., the concentration of enzyme used for AT digestion, cell culture conditions) were identified and examined to ensure the high quality of the final product containing AT-MSCs. We confirmed the identity of isolated AT-MSCs as MSCs and their trilineage differentiation potential according to the International Society for Cellular Therapy (ISCT) recommendations. Based on the conducted experiments, in-process quality control (QC) parameters and acceptance criteria were defined for the manufacturing of hospital exemption ATMP (HE-ATMP). Finally, we conducted a validation of the manufacturing process in a GMP facility. In the current study, we presented a process approach leading to the optimization of processing and the ex vivo expansion of AT-MSCs for the development of ATMP for use in humans.

Multilineage Differentiation Potential of Human Dental Pulp Stem Cells-Impact of 3D and Hypoxic Environment on Osteogenesis In Vitro.

Human dental pulp harbours unique stem cell population exhibiting mesenchymal stem/stromal cell (MSC) characteristics. This study aimed to analyse the differentiation potential and other essential functional and morphological features of dental pulp stem cells (DPSCs) in comparison with Wharton's jelly-derived MSCs from the umbilical cord (UC-MSCs), and to evaluate the osteogenic differentiation of DPSCs in 3D culture with a hypoxic microenvironment resembling the stem cell niche. Human DPSCs as well as UC-MSCs were isolated from primary human tissues and were subjected to a series of experiments. We established a multiantigenic profile of DPSCs with CD45-/CD14-/CD34-/CD29+/CD44+/CD73+/CD90+/CD105+/Stro-1+/HLA-DR- (using flow cytometry) and confirmed their tri-lineage osteogenic, chondrogenic, and adipogenic differentiation potential (using qRT-PCR and histochemical staining) in comparison with the UC-MSCs. The results also demonstrated the potency of DPSCs to differentiate into osteoblasts in vitro. Moreover, we showed that the DPSCs exhibit limited cardiomyogenic and endothelial differentiation potential. Decreased proliferation and metabolic activity as well as increased osteogenic differentiation of DPSCs in vitro, attributed to 3D cell encapsulation and low oxygen concentration, were also observed. DPSCs exhibiting elevated osteogenic potential may serve as potential candidates for a cell-based product for advanced therapy, particularly for bone repair. Novel tissue engineering approaches combining DPSCs, 3D biomaterial scaffolds, and other stimulating chemical factors may represent innovative strategies for pro-regenerative therapies.

Impact of developmental origin, niche mechanics and oxygen availability on osteogenic differentiation capacity of mesenchymal stem/stromal cells.

Mesenchymal Stem/Stromal Cells (MSCs) have been widely considered as a promising source of cells for tissue regeneration. Among other stem cells, they are characterized by a high osteogenic potential. Intensive studies in this field had shown that even if basic osteogenic differentiation is relatively simple, its clinical application requires more sophisticated approaches to prepare effective and safe cell therapy products. The aim of this review is to underline biological, physical and chemical factors which play a crucial role in osteogenic differentiation of MSCs. Existence of two distinct mechanisms of ossification (intramembranous and endochondral) indicate that choosing a proper source of MSCs may be critical for successful regeneration of a particular bone type. In this context, Dental Pulp Stem Cells representing a group of MSCs and originating from neural crest ( a structure responsible for development of cranial bones) are considered as the most promising for skull bone defect repair. Factors which facilitate osteogenic differentiation of MSCs include changes in forces exerted on cells during development. Thus, culturing of cells in hydrogels or on biocompatible three-dimensional scaffolds improves osteogenic differentiation of MSCs by both, the mechanotransductive and chemical impact on cells. Moreover, atmospheric oxygen concentration routinely used for cell cultures in vitro does not correspond to lower oxygen concentration present in stem cell niches. A decrease in oxygen concentration allows to create more physiological cell culture conditions, mimicking the ones in stem cell niches, which promote the MSCs stemness. Altogether, factors discussed in this review provide exciting opportunities to boost MSCs propagation and osteogenic differentiation which is crucial for successful clinical applications.

Fenofibrate Augments the Sensitivity of Drug-Resistant Prostate Cancer Cells to Docetaxel.

Metronomic agents reduce the effective doses and adverse effects of cytostatics in cancer chemotherapy. Therefore, they can enhance the treatment efficiency of drug-resistant cancers. Cytostatic and anti-angiogenic effects of fenofibrate (FF) suggest that it can be used for the metronomic chemotherapy of drug-resistant prostate tumors. To estimate the effect of FF on the drug-resistance of prostate cancer cells, we compared the reactions of naïve and drug-resistant cells to the combined treatment with docetaxel (DCX)/mitoxantrone (MTX) and FF. FF sensitized drug-resistant DU145 and PC3 cells to DCX and MTX, as illustrated by their reduced viability and invasive potential observed in the presence of DCX/MTX and FF. The synergy of the cytostatic activities of both agents was accompanied by the inactivation of P-gp-dependent efflux, dysfunction of the microtubular system, and induction of polyploidy in DCX-resistant cells. Chemical inhibition of PPARα- and reactive oxygen species (ROS)-dependent pathways by GW6471 and N-acetyl-L-cysteine, respectively, had no effect on cell sensitivity to combined DCX/FF treatment. Instead, we observed the signs of adenosine triphosphate (ATP) deficit and autophagy in DCX/FF-treated drug-resistant cells. Furthermore, the cells that had been permanently propagated under DCX- and DCX/FF-induced stress did not acquire DCX/FF-resistance. Instead, relatively slow proliferation of DCX-resistant cells was efficiently inhibited by FF. Collectively, our observations show that FF reduces the effective doses of DCX by interfering with the drug resistance and energy metabolism of prostate cancer cells. Concomitantly, it impairs the chemotherapy-induced microevolution and expansion of DCX/FF-resistant cells. Therefore, FF can be applied as a metronomic agent to enhance the efficiency of palliative chemotherapy of prostate cancer.

Bradykinin B2 receptor and dopamine D2 receptor cooperatively contribute to the regulation of neutrophil adhesion to endothelial cells.

Leukocyte adhesion to the vascular endothelium contributes to many immunological and inflammatory disorders. These processes have been shown to be mediated by bradykinin receptor type 2 (B2R) and dopamine receptor type 2 (D2R). In a previous study, we reported the formation of a B2R-D2R heterodimer, possibly altering cellular functions. Hence, in the present study, we examined the effect of co-activation of endothelial cells with B2R and D2R agonists on the interaction of these cells with neutrophils. Bradykinin, the main B2R agonist, significantly increased cell adhesion, and this effect was reversed when the endothelial cells were additionally co-treated with a selective D2R agonist, sumanirole. These results were dependent on the incubation time, showing an opposite tendency after prolonged stimulation. Significant changes in the expression of adhesion proteins, such as E-selectin and intercellular adhesion molecule 1 in endothelial cells were observed. Additionally, the cells preincubated with tumor necrosis factor-α showed decreased cell adhesion and IL-8 release after long incubation with both agonists. The modulation of cell adhesion by D2R and B2R seem to be mediated via STAT3 phosphorylation. In summary, this study demonstrated a protective role of D2R in neutrophil-endothelial cell adhesion induced by bradykinin, especially in cytokine-stimulated endothelial cells.

[Role of the bone marrow derived stem cells in pancreatic inflammatory disorders]. / Rola szpikowych komórek macierzystych w chorobach zapalnych trzustki.

Various independent studies indicate involvement of different populations of bone marrow-derived stem cells in the process of tissue regeneration. In inflammatory disorders bone marrow stem cells are mobilized into peripherial blood and further to different organs, where they take part in tissue regeneration. Experimental studies have shown that bone marrow stem cells play a pivotal role in regeneration of endo and egzocrine pancreas and have a role in pathogenesis of pancreatitis, diabetes and pancreatic neoplasms. Our review summarize available scientific data about different populations of bone marrow stem cells and their role in pathogenesis of inflammatory disorders with special focus on the role of these cells in pancreatic regeneration and their influence on development of pancreatitis. Presented data show also therapeutic potential of bone marrow stem cells in pancreatitis.

Diverse impact of xeno-free conditions on biological and regenerative properties of hUC-MSCs and their extracellular vesicles.

Growing evidence indicates that intracellular signaling mediated by extracellular vesicles (EVs) released by stem cells plays a considerable role in triggering the regenerative program upon transplantation. EVs from umbilical cord mesenchymal stem cells (UC-MSC-EVs) have been shown to enhance tissue repair in animal models. However, translating such results into clinical practice requires optimized EV collection procedures devoid of animal-originating agents. Thus, in this study, we analyzed the influence of xeno-free expansion media on biological properties of UC-MSCs and UC-MSC-EVs for future applications in cardiac repair in humans. Our results show that proliferation, differentiation, phenotype stability, and cytokine secretion by UC-MSCs vary depending on the type of xeno-free media. Importantly, we found distinct molecular and functional properties of xeno-free UC-MSC-EVs including enhanced cardiomyogenic and angiogenic potential impacting on target cells, which may be explained by elevated concentration of several pro-cardiogenic and pro-angiogenic microRNA (miRNAs) present in the EVs. Our data also suggest predominantly low immunogenic capacity of certain xeno-free UC-MSC-EVs reflected by their inhibitory effect on proliferation of immune cells in vitro. Summarizing, conscious selection of cell culture conditions is required to harvest UC-MSC-EVs with the optimal desired properties including enhanced cardiac and angiogenic capacity, suitable for tissue regeneration. KEY MESSAGE: Type of xeno-free media influences biological properties of UC-MSCs in vitro. Certain xeno-free media promote proliferation and differentiation ability of UC-MSCs. EVs collected from xeno-free cultures of UC-MSCs are biologically active. Xeno-free UC-MSC-EVs enhance cardiac and angiogenic potential of target cells. Type of xeno-free media determines immunomodulatory effects mediated by UC-MSC-EVs.

Synergistic Cytotoxic and Anti-invasive Effects of Mitoxantrone and Triterpene Saponins from Lysimachia ciliata on Human Prostate Cancer Cells.

Triterpene saponins are secondary metabolites typical for higher plants. They possess a wide range of pharmaceutical and biological activities. These include anti-inflammatory, vasoprotective, expectorant, and antitumor properties. In particular, the ability of saponins to enhance the cytotoxicity of chemotherapeutic drugs has opened new perspectives for their application in combined cancer chemotherapy. In this study, the biological activity of the saponin fraction isolated from Lysimachia ciliata (denoted as CIL-1/2) was evaluated to assess its chemosensitizing activity in prostate cancer cell lines (DU-145, PC-3). No cytotoxic or cytostatic effect of the CIL-1/2 fraction administered at the concentration of 0.5 µg/mL was observed. In contrast, cocktails of CIL-1/2 and mitoxantrone (a drug commonly used in prostate cancer therapy) exerted synergistic cytostatic and proapoptotic effects. Furthermore, the synergy of proapoptotic activities of the analyzed cocktails is accompanied by their synergistic effects on prostate cancer cell movement and invasiveness. The significantly weaker impact of this cocktail on normal prostate cells additionally adds to the significance of our data and confirms that the CIL-1/2 fraction might be considered a potent adjuvant for prostate cancer chemotherapy.

Curcumin augments the cytostatic and anti-invasive effects of mitoxantrone on carcinosarcoma cells in vitro.

Numerous adverse effects limit the applicability of mitoxantrone for the treatment of drug-resistant tumors, including carcinosarcoma. Here, we estimated the additive effects of mitoxantrone and curcumin, a plant-derived biomolecule isolated from Curcuma longa, on the neoplastic and invasive potential of carcinosarcoma cells in vitro. Curcumin augmented the cytostatic, cytotoxic and anti-invasive effects of mitoxantrone on the Walker-256 cells. It also strengthened the inhibitory effects of mitoxantrone on the motility of drug-resistant Walker-256 cells that had retained viability after a long-term mitoxantrone/curcumin treatment. Thus, curcumin reduces the effective doses of mitoxantrone and augments its interference with the invasive potential of drug-resistant carcinosarcoma cells.

Identification of New Rat Bone Marrow-Derived Population of Very Small Stem Cell with Oct-4A and Nanog Expression by Flow Cytometric Platforms.

Very small embryonic-like stem cells (VSELs) represent a unique rare population of adult stem cells (SCs) sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM-) derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I) antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog) on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS), we also optimized sorting protocol for separation of CD45(-)/Lin(-)/CD106(+) rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury.

Monocyte Chemoattractant Protein-Induced Protein 1 (MCPIP1) Enhances Angiogenic and Cardiomyogenic Potential of Murine Bone Marrow-Derived Mesenchymal Stem Cells.

The current evidence suggests that beneficial effects of mesenchymal stem cells (MSCs) toward myocardial repair are largely due to paracrine actions of several factors. Although Monocyte chemoattractant protein-induced protein 1 (MCPIP1) is involved in the regulation of inflammatory response, apoptosis and angiogenesis, whether MCPIP1 plays any role in stem cell-induced cardiac repair has never been examined. By employing retroviral (RV)-transduced overexpression of MCPIP1, we investigated the impact of MCPIP1 on viability, apoptosis, proliferation, metabolic activity, proteome, secretome and differentiation capacity of murine bone marrow (BM) - derived MSCs. MCPIP1 overexpression enhanced angiogenic and cardiac differentiation of MSCs compared with controls as indicated by elevated expression of genes accompanying angiogenesis and cardiomyogenesis in vitro. The proangiogenic activity of MCPIP1-overexpressing MSCs (MCPIP1-MSCs) was also confirmed by increased capillary-like structure formation under several culture conditions. This increase in differentiation capacity was associated with decreased proliferation of MCPIP1-MSCs when compared with controls. MCPIP1-MSCs also expressed increased levels of proteins involved in angiogenesis, autophagy, and induction of differentiation, but not adverse inflammatory agents. We conclude that MCPIP1 enhances endothelial and cardiac differentiation of MSCs. Thus, modulating MCPIP1 expression may be a novel approach useful for enhancing the immune-regulatory, anti-apoptotic, anti-inflammatory and regenerative capacity of BM-derived MSCs for myocardial repair and regeneration of ischemic tissues.

Influence of kinin peptides on monocyte-endothelial cell adhesion.

Adhesion of leukocytes to vascular endothelium in response to proinflammatory mediators is an important component of the overall inflammatory reaction. In the current work, we used a retinoic acid-differentiated human promonocytic cell line, U937 and a human microvascular endothelial cell line, HMEC-1 to analyze the effect of the potent pro-inflammatory bradykinin-related peptides (kinins) on cell adhesion. Bradykinin (BK) and kinin metabolites without the C-terminal arginine residue enhanced adhesion of the monocyte-like cells to fibronectin and to the HMEC-1 cells. Expression of adhesion proteins on the surface of both cell types was altered by the kinin peptides. In the monocyte-like cells, expression of CD11b, a subunit of Mac-1 integrin, was significantly increased whilst in the endothelial cells, a strong increase in the production of intercellular adhesion molecule 1 (ICAM-1) was observed. The positive bradykinin-induced effect on the cell-cell interaction was reversed by a carboxypeptidase inhibitor (MGTA), hence we suspected a significant role of the des-Arg kinin metabolites, which acted through the kinin receptor type 1. Indeed, the expression of this receptor was up-regulated not only by agonists but also by interferon-γ and bradykinin. Kinin peptides also regulated signal transducer and activator of transcription proteins (STATs) activated by cytokines. Taken together, the above observations support our hypothesis that kinins stimulate monocyte adhesion to the vessel wall, especially during pathological states of the circulatory system accompanied by proinflammatory cytokine release.