miR-125b-5p impacts extracellular vesicle biogenesis, trafficking, and EV subpopulation release in the porcine trophoblast by regulating ESCRT-dependent pathway

Abstract Intercellular communication is a critical process that ensures cooperation between distinct cell types at the embryo–maternal interface. Extracellular vesicles (EVs) are considered to be potent mediators of this communication by transferring biological information in their cargo (e.g., miRNAs) to the recipient cells. miRNAs are small non-coding RNAs that affect the function and fate of neighboring and distant cells by regulating gene expression. Focusing on the maternal side of the dialog, we recently revealed the impact of embryonic signals, including miRNAs, on EV-mediated cell-to-cell communication. In this study, we show the regulatory mechanism of the miR-125b-5p ESCRT-mediated EV biogenesis pathway and the further secretion of EVs by trophoblasts at the time when the crucial steps of implantation are taking place. To test the ability of miR-125b-5p to influence the expression of genes involved in the generation and release of EV subpopulations in porcine conceptuses, we used an ex vivo approach. Next, in silico and in vitro analyses were performed to confirm miRNA–mRNA interactions. Finally, EV trafficking and release were assessed using several imaging and particle analysis tools. Our results indicated that conceptus development and implantation are accompanied by changes in the abundance of EV biogenesis and trafficking machinery. ESCRT-dependent EV biogenesis and the further secretion of EVs were modulated by miR-125b-5p, specifically impacting the ESCRT-II complex (via VPS36) and EV trafficking in primary porcine trophoblast cells. The identified miRNA–ESCRT interplay led to the generation and secretion of specific subpopulations of EVs. miRNA present at the embryo–maternal interface governs EV-mediated communication between the mother and the developing conceptus, leading to the generation, trafficking, and release of characteristic subpopulations of EVs

Stress conditions affect the immunomodulatory potential of Candida albicans extracellular vesicles and their impact on cytokine release by THP-1 human macrophages

Human immune cells possess the ability to react complexly and effectively after contact with microbial virulence factors, including those transported in cell-derived structures of nanometer sizes termed extracellular vesicles (EVs). EVs are produced by organisms of all kingdoms, including fungi pathogenic to humans. In this work, the immunomodulatory properties of EVs produced under oxidative stress conditions or at host concentrations of $CO_{2}$ by the fungal pathogen Candida albicans were investigated. The interaction of EVs with human pro-monocytes of the U-937 cell line was established, and the most notable effect was attributed to oxidative stress-related EVs. The immunomodulatory potential of tested EVs against human THP-1 macrophages was verified using cytotoxicity assay, ROS-production assay, and the measurement of cytokine production. All fungal EVs tested did not show a significant cytotoxic effect on THP-1 cells, although a slight pro-oxidative impact was indicated for EVs released by C. albicans cells grown under oxidative stress. Furthermore, for all tested types of EVs, the pro-inflammatory properties related to increased IL-8 and TNF-$\alpha$ production and decreased IL-10 secretion were demonstrated, with the most significant effect observed for EVs released under oxidative stress conditions

Anti-inflammatory, Anti-fibrotic and pro-cardiomyogenic effects of genetically engineered extracellular vesicles enriched in miR-1 and miR-199a on human cardiac fibroblasts

Rationale Emerging evidence indicates that stem cell (SC)- derived extracellular vesicles (EVs) carrying bioactive miRNAs are able to repair damaged or infarcted myocardium and ameliorate adverse remodeling. Fibroblasts represent a major cell population responsible for scar formation in the damaged heart. However, the effects of EVs on cardiac fibroblast (CFs) biology and function has not been investigated. Objective To analyze the biological impact of stem cell-derived EVs (SC-EVs) enriched in miR-1 and miR-199a on CFs and to elucidate the underlying molecular mechanisms. Methods and Results Genetically engineered human induced pluripotent stem cells (hiPS) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) expressing miR-1 or miR-199a were used to produce miR-EVs. Cells and EVs were thoughtfully analyzed for miRNA expression using RT-qPCR method. Both hiPS-miRs-EVs and UC-MSC-miRs-EVs effectively transferred miRNAs to recipient CFs, however, hiPS-miRs-EVs triggered cardiomyogenic gene expression in CFs more efficiently than UC-MSC-miRs-EVs. Importantly, hiPS-miR-1-EVs exhibited cytoprotective effects on CFs by reducing apoptosis, decreasing levels of pro-inflammatory cytokines (CCL2, IL-$1\beta$, IL-8) and downregulating the expression of a pro-fibrotic gene – $\alpha$-smooth muscle actin ($\alpha$-SMA). Notably, we identified a novel role of miR-199a-3p delivered by hiPS-EVs to CFs, in triggering the expression of cardiomyogenic genes (NKX2.5, TNTC, MEF2C) and ion channels involved in cardiomyocyte contractility (HCN2, SCN5A, KCNJ2, KCND3). By targeting SERPINE2, miR-199a-3p may reduce pro-fibrotic properties of CFs, whereas miR-199a-5p targeted BCAM and TSPAN6, which may be implicated in downregulation of inflammation. Conclusions hiPS-EVs carrying miR-1 and miR-199a attenuate apoptosis and pro-fibrotic and pro-inflammatory activities of CFs, and increase cardiomyogenic gene expression. These finding serve as rationale for targeting fibroblasts with novel EV-based miRNA therapies to improve heart repair after myocardial injury

Heat shock protein A2 is a novel extracellular vesicle-associated protein

70-kDa Heat Shock Proteins (HSPA/HSP70) are chaperones playing a central role in the proteostasis control mechanisms. Their basal expression can be highly elevated as an adaptive response to environmental and pathophysiological stress conditions. HSPA2, one of poorly characterised chaperones of the HSPA/HSP70 family, has recently emerged as epithelial cells differentiation-related factor. It is also commonly expressed in cancer cells, where its functional significance remains unclear. Previously, we have found that proteotoxic stress provokes a decrease in HSPA2 levels in cancer cells. In the present study we found that proteasome inhibition-related loss of HSPA2 from cancer cells neither is related to a block in the gene transcription nor does it relate to increased autophagy-mediated disposals of the protein. Proteotoxic stress stimulated extracellular release of HSPA2 in extracellular vesicles (EVs). Interestingly, EVs containing HSPA2 are also released by non-stressed cancer and normal cells. In human urinary EVs levels of HSPA2 were correlated with the levels of TSG101, one of the main EVs markers. We conclude that HSPA2 may constitute basic components of EVs. Nevertheless, its specific role in EVs and cell-to-cell communication requires further investigation

Electric field as a potential directional cue in homing of bone marrow-derived mesenchymal stem cells to cutaneous wounds

AbstractBone marrow-derived cells are thought to participate and enhance the healing process contributing to skin cells or releasing regulatory cytokines. Directional cell migration in a weak direct current electric field (DC-EF), known as electrotaxis, may be a way of cell recruitment to the wound site. Here we examined the influence of electric field on bone marrow adherent cells (BMACs) and its potential role as a factor attracting mesenchymal stem cells to cutaneous wounds. We observed that in an external EF, BMAC movement was accelerated and highly directed with distinction of two cell populations migrating toward opposite poles: mesenchymal stem cells migrated toward the cathode, whereas macrophages toward the anode. Analysis of intracellular pathways revealed that macrophage electrotaxis mostly depended on Rho family small GTPases and calcium ions, but interruption of PI3K and Arp2/3 had the most pronounced effect on electrotaxis of MSCs. However, in all cases we observed only a partial decrease in directionality of cell movement after inhibition of certain proteins. Additionally, although we noticed the accumulation of EGFR at the cathodal side of MSCs, it was not involved in electrotaxis. Moreover, the cell reaction to EF was very dynamic with first symptoms occurring within <1min. In conclusion, the physiological DC-EF may act as a factor positioning bone marrow cells within a wound bed and the opposite direction of MSC and macrophage movement did not result either from utilizing different signalling or redistribution of investigated cell surface receptors

Impact of graphene-based surfaces on the basic biological properties of human umbilical cord mesenchymal stem cells : implications for ex vivo cell expansion aimed at tissue repair

The potential therapeutic applications of mesenchymal stem/stromal cells (MSCs) and biomaterials have attracted a great amount of interest in the field of biomedical engineering. MSCs are multipotent adult stem cells characterized as cells with specific features, e.g., high differentiation potential, low immunogenicity, immunomodulatory properties, and efficient in vitro expansion ability. Human umbilical cord Wharton&rsquo;s jelly-derived MSCs (hUC-MSCs) are a new, important cell type that may be used for therapeutic purposes, i.e., for autologous and allogeneic transplantations. To improve the therapeutic efficiency of hUC-MSCs, novel biomaterials have been considered for use as scaffolds dedicated to the propagation and differentiation of these cells. Nowadays, some of the most promising materials for tissue engineering include graphene and its derivatives such as graphene oxide (GO) and reduced graphene oxide (rGO). Due to their physicochemical properties, they can be easily modified with biomolecules, which enable their interaction with different types of cells, including MSCs. In this study, we demonstrate the impact of graphene-based substrates (GO, rGO) on the biological properties of hUC-MSCs. The size of the GO flakes and the reduction level of GO have been considered as important factors determining the most favorable surface for hUC-MSCs growth. The obtained results revealed that GO and rGO are suitable scaffolds for hUC-MSCs. hUC-MSCs cultured on: (i) a thin layer of GO and (ii) an rGO surface with a low reduction level demonstrated a viability and proliferation rate comparable to those estimated under standard culture conditions. Interestingly, cell culture on a highly reduced GO substrate resulted in a decreased hUC-MSCs proliferation rate and induced cell apoptosis. Moreover, our analysis demonstrated that hUC-MSCs cultured on all the tested GO and rGO scaffolds showed no alterations of their typical mesenchymal phenotype, regardless of the reduction level and size of the GO flakes. Thus, GO scaffolds and rGO scaffolds with a low reduction level exhibit potential applicability as novel, safe, and biocompatible materials for utilization in regenerative medicine

An influence of extracellular vesicles released by human induced pluripotent stem cells on the functional properties of cord blood-derived hematopoietic stem cells

Krew pępowinowa (KP), jako bogate źródło komórek macierzystych (KM) - w szczególności KM krwiotwórczych (ang. hematopoietic stem cells; HSCs), stanowi obiecującą alternatywę dla przeszczepów szpiku kostnego u pacjentów z koniecznością rekonstytucji układu krwiotwórczego. Jej wykorzystanie w hematologii transplantacyjnej niesie ze sobą dodatkowe korzyści wynikające z nieinwazyjnego pobrania, mniej restrykcyjnych wymagań dopasowania antygenów zgodności tkankowej, a także zmniejszonego ryzyka wystąpienia choroby przeszczep przeciwko gospodarzowi. Z drugiej jednak strony, zastosowanie KP u dorosłych pacjentów wiąże się z ryzykiem opóźnionego wszczepienia u biorcy, będącego efektem niedostatecznej liczby podanych komórek HSCs obecnych w materiale, limitowanej objętością pobranej podczas porodu KP. Stąd też, skuteczne wykorzystanie preparatów KP w transplantologii, w tym u osób dorosłych, wciąż wymaga poszukiwania nowych strategii ekspansji komórek HSCs ex vivo, z jednoczesnym zwiększeniem ich potencjału hematopoetycznego i efektywności zasiedlania nisz szpikowych po przeszczepie. Z badań przeprowadzonych dotychczas w naszym zespole wynika, że zastosowanie pęcherzyków zewnątrzkomórkowych (ang. extracellular vesicles; EVs) - obłonionych struktur o średnicy od 30 nm do 1 μm, wydzielanych przez indukowane komórki macierzyste pluripotencjalne (ang. human induced pluripotent stem cells; hiPS) może zwiększać proliferację i modulować szereg funkcji innych komórek. EVs zawierają aktywne biologicznie molekuły, w tym receptory, białka cytoplazmatyczne, czynniki transkrypcyjne oraz kwasy nukleinowe (w szczególności mRNA oraz miRNA), które mogą być przenoszone pomiędzy komórkami, stanowiąc istotne parakrynne czynniki wymiany informacji. Stąd też, sukcesywnie wzrasta zainteresowanie badaczy próbami zastosowania EVs do modyfikacji funkcji biologicznych komórek docelowych. Dotychczasowe, nieliczne badania nad zastosowaniem EVs w modulacji potencjału regeneracyjnego komórek HSCs z KP koncentrują się głównie na pęcherzykach wydzielanych przez komórki stromalne niszy, a wciąż niewiele wiadomo na temat wpływu EVs pochodzących z innych typów KM na właściwości biologiczne komórek krwiotwórczych. W kontekście tych prac, interesującym było zbadanie czy EVs z komórek hiPS (hiPS-EVs) będą także wykazywać funkcjonalny efekt na komórki krwiotwórcze z KP, w kontekście możliwości ich potencjalnego zastosowania w hematologii transplantacyjnej. W związku z powyższym, celem niniejszej rozprawy doktorskiej było zbadanie wpływu hiPS-EVs na właściwości biologiczne i potencjał hematopoetyczny komórek HSCs z KP, zarówno w warunkach in vitro, jak i in vivo w modelu rekonstytucji szpikowej u myszy szczepu NOD/SCID. Przeprowadzone badania wykazały m.in., że hiPS-EVs mogą skutecznie oddziaływać z wysokooczyszczonymi frakcjami komórek KP, wzbogaconymi w HSCs i poddanymi ekspansji ex vivo. Pomimo braku znaczącego wpływu na tempo proliferacji, stymulacja za pomocą hiPS-EVs zwiększała aktywność metaboliczną komórek HSCs oraz ich różnicowanie w kierunku poszczególnych linii komórek hematopoetycznych in vitro. Wykazano również, że potencjał klonogenny komórek HSCs z KP ulega zwiększeniu po kontakcie z hiPS-EVs in vitro, a wyniki te korelowały ze wzrostem ekspresji genów związanych z hematopoezą w tych komórkach. Ponadto, przeprowadzone doświadczenia wskazują, że hiPS-EVs wykazują właściwości cytoprotekcyjne wobec komórek HSCs poddanych działaniu czynników cytotoksycznych. Dalsze analizy wykazały także wzrost właściwości adhezyjnych komórek HSCs oraz ich chemotaktycznej migracji in vitro, poddanych działaniu czynników obecnych w hiPS-EVs. W komórkach HSCs poddanych działaniu hiPS-EVs, zaobserwowano też istotne zmiany aktywności szeregu kinaz białkowych zaangażowanych w ważne szlaki sygnałowe, uczestniczące w regulacji wymienionych procesów. Uzyskane wyniki badań in vitro świadczą o zwiększeniu aktywności biologicznej oraz potencjału hematopoetycznego komórek HSCs z KP w wyniku działania bioaktywnej zawartości hiPS-EVs. Wyniki badań in vivo potwierdzają obserwacje uzyskane w badaniach in vitro. W szczególności, wykazano pozytywny wpływ hiPS-EVs na zdolność komórek HSCs z KP do zasiedlania nisz szpikowych i rekonstytucji układu krwiotwórczego u myszy NOD/SCID, poddanych uprzednio radiacyjnej mieloablacji. Podsumowując, w niniejszej rozprawie doktorskiej po raz pierwszy wykazano, że hiPS-EVs mogą modulować aktywność biologiczną komórek HSCs izolowanych z KP, a tym samym wpływać na ich cechy funkcjonalne istotne z punktu widzenia zastosowania tych komórek w hematologii transplantacyjnej. Otrzymane rezultaty badań stanowią podstawę do dalszych prac badawczych nad próbą zastosowania hiPS-EVs do zwiększenia potencjału regeneracyjnego tych komórek, a tym samym potencjalnego poszerzenia możliwości zastosowania KP w praktyce klinicznej.Umbilical cord blood (CB) as a rich source of stem cells (SCs), especially hematopoietic stem cells (HSCs), is a promising material alternative for bone marrow transplantation in patients requiring the hematopoietic system reconstitution. Its utilization in the hematology provides additional benefits resulting from non-invasive collection, less stringent requirements for the donor matching as well as a reduced risk of graft-versus host disease. On the other hand, the use of CB cells in adult patients is associated with the risk of delayed engraftment resulting from the insufficient number of HSCs present in the transplanted material, which may be limited by the volume of CB unit collected during the delivery. Therefore, effective utilization of CB in the transplantology still requires the development of novel strategies of HSCs expansion ex vivo, with simultaneous increase in their hematopoietic potential and efficiency to engraft into the bone marrow niches after the transplantation. So far, the research conducted in our laboratory have shown that the use of extracellular vesicles (EVs) - vesicular structures with a diameter ranging from 30 nm to 1 μm, secreted by human induced pluripotent stem cells (hiPS) - can increase proliferation and modulate several functions of target cells. EVs are known to contain biologically active molecules including receptors, cytoplasmic proteins, transcription factors and nucleic acids (in particular mRNA and miRNA) that can be transferred between the cells mediating the exchange of biological information via transferring several paracrine factors. Hence, the scientific interest attempting the use of EVs to modify the biological functions of target cells is gradually increasing. Currently, few studies conducted on the use of EVs in modulation of the regenerative potential of CB-derived HSCs are mainly focused on vesicles secreted by stromal cells. Importantly, still little is known about the impact of EVs from other SCs types on the biological properties of hematopoietic cells. Thus, it was interesting to investigate whether EVs from hiPS cells (hiPS-EVs) will also possess a functional effect on CB-derived HSCs, in the context of their potential use in the hematology. Therefore, the aim of this study was to investigate the effect of hiPS-EVs on biological properties and hematopoietic potential of HSCs isolated from CB, both in vitro as well as in vivo, in the murine model of bone marrow reconstitution after mieloablative irradiation. The results obtained in this study showed that hiPS-EVs can effectively interact with highly purified CB-derived CD34+ fraction enriched in HSCs, subjected to ex vivo expansion. Despite the lack of a significant effect on the proliferation rate, the stimulation with hiPS-EVs significantly increased the metabolic activity of HSCs and their capacity to differentiate toward hematopoietic lineages in vitro. It was also shown that the clonogenic potential of CB-derived HSCs was enhanced after the contact with hiPS-EVs in vitro, and these results correlated with the increase in the expression of hematopoietic genes in those cells. In addition, performed experiments revealed that hiPS-EVs have a great cytoprotective effect on HSCs exposed to cytotoxic agents. Further analyzes have also shown an increase in the adhesion efficacy and chemotactic migration of CB-HSCs in vitro following the treatement with factors present in hiPS-EVs. Additionally, we also observed significant changes in the activity of a number of protein kinases involved in important signaling pathways in HSCs cells after the treatment with hiPS-EVs. Thus, obtained results of in vitro studies indicated that bioactive content of hiPS-EVs increased biological activity and hematopoietic potential of CB-derived HSCs. In next step, I also performed in vivo study supporting the results obtained from experiments conducted in vitro. In particular, it has been demonstrated that hiPS-EVs may have the positive effect on the ability of HSCs isolated from CB to home and engraft to bone marrow niches, as well as on the effectiveness of reconstitution of the hematopoietic system after the myeloablation in NOD/SCID mice. In conclusion, for the first time we demonstrated that hiPS-EVs can modulate the biological activity of CB-derived HSCs impacting on their functional properties important for their use in hematology. Our results constitute important basis for further research on potential employment of hiPS-EVs as effective agents increasing the regenerative potential of hematopoietic cells and in consequence enhancing the possibility for clinical applications of CB samples