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.

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 (iPSC)-Derived Extracellular Vesicles Are Safer and More Effective for Cardiac Repair Than iPSCs.

RATIONALE: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs. OBJECTIVE: To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo. METHODS AND RESULTS: Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV-treated mice exhibited improved left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe. CONCLUSIONS: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.

Perspectives for Future Use of Extracellular Vesicles from Umbilical Cord- and Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells in Regenerative Therapies-Synthetic Review.

Mesenchymal stem/ stromal cells (MSCs) represent progenitor cells of various origin with multiple differentiation potential, representing the most studied population of stem cells in both in vivo pre-clinical and clinical studies. MSCs may be found in many tissue sources including extensively studied adipose tissue (ADSCs) and umbilical cord Wharton's jelly (UC-MSCs). Most of sanative effects of MSCs are due to their paracrine activity, which includes also release of extracellular vesicles (EVs). EVs are small, round cellular derivatives carrying lipids, proteins, and nucleic acids including various classes of RNAs. Due to several advantages of EVs when compare to their parental cells, MSC-derived EVs are currently drawing attention of several laboratories as potential new tools in tissue repair. This review focuses on pro-regenerative properties of EVs derived from ADSCs and UC-MSCs. We provide a synthetic summary of research conducted in vitro and in vivo by employing animal models and within initial clinical trials focusing on neurological, cardiovascular, liver, kidney, and skin diseases. The summarized studies provide encouraging evidence about MSC-EVs pro-regenerative capacity in various models of diseases, mediated by several mechanisms. Although, direct molecular mechanisms of MSC-EV action are still under investigation, the current growing data strongly indicates their potential future usefulness for tissue repair.

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'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.

Adult Bone Marrow Cell Therapy for Ischemic Heart Disease: Evidence and Insights From Randomized Controlled Trials.

RATIONALE: Notwithstanding the uncertainties about the outcomes of bone marrow cell (BMC) therapy for heart repair, further insights are critically needed to improve this promising approach. OBJECTIVE: To delineate the true effect of BMC therapy for cardiac repair and gain insights for future trials through systematic review and meta-analysis of data from eligible randomized controlled trials. METHODS AND RESULTS: Database searches through August 2014 identified 48 eligible randomized controlled trials (enrolling 2602 patients). Weighted mean differences for changes in left ventricular (LV) ejection fraction, infarct size, LV end-systolic volume, and LV end-diastolic volume were analyzed with random-effects meta-analysis. Compared with standard therapy, BMC transplantation improved LV ejection fraction (2.92%; 95% confidence interval, 1.91-3.92; P<0.00001), reduced infarct size (-2.25%; 95% confidence interval, -3.55 to -0.95; P=0.0007) and LV end-systolic volume (-6.37 mL; 95% confidence interval, -8.95 to -3.80; P<0.00001), and tended to reduce LV end-diastolic volume (-2.26 mL; 95% confidence interval, -4.59 to 0.07; P=0.06). Similar effects were noted when data were analyzed after excluding studies with discrepancies in reporting of outcomes. The benefits also persisted when cardiac catheterization was performed in control patients as well. Although imaging modalities partly influenced the outcomes, LV ejection fraction improved in BMC-treated patients when assessed by magnetic resonance imaging. Early (<48 hours) BMC injection after myocardial Infarction was more effective in reducing infarct size, whereas BMC injection between 3 and 10 days proved superior toward improving systolic function. A minimum of 50 million BMCs seemed to be necessary, with limited additional benefits seen with increasing cell numbers. BMC therapy was safe and improved clinical outcomes, including all-cause mortality, recurrent myocardial Infarction, ventricular arrhythmia, and cerebrovascular accident during follow-up, albeit with differences between acute myocardial Infarction and chronic ischemic heart disease subgroups. CONCLUSIONS: Transplantation of adult BMCs improves LV ejection fraction, reduces infarct size, and ameliorates remodeling in patients with ischemic heart disease. These effects are upheld in the analyses of studies using magnetic resonance imaging and also after excluding studies with discrepant reporting of outcomes. BMC transplantation may also reduce the incidence of death, recurrent myocardial Infarction, ventricular arrhythmia, and cerebrovascular accident during follow-up.

Human Induced Pluripotent Stem Cell-Derived Microvesicles Transmit RNAs and Proteins to Recipient Mature Heart Cells Modulating Cell Fate and Behavior.

Microvesicles (MVs) are membrane-enclosed cytoplasmic fragments released by normal and activated cells that have been described as important mediators of cell-to-cell communication. Although the ability of human induced pluripotent stem cells (hiPSCs) to participate in tissue repair is being increasingly recognized, the use of hiPSC-derived MVs (hiPSC-MVs) in this regard remains unknown. Accordingly, we investigated the ability of hiPSC-MVs to transfer bioactive molecules including mRNA, microRNA (miRNA), and proteins to mature target cells such as cardiac mesenchymal stromal cells (cMSCs), and we next analyzed effects of hiPSC-MVs on fate and behavior of such target cells. The results show that hiPSC-MVs derived from integration-free hiPSCs cultured under serum-free and feeder-free conditions are rich in mRNA, miRNA, and proteins originated from parent cells; however, the levels of expression vary between donor cells and MVs. Importantly, we found that transfer of hiPSC components by hiPSC-MVs impacted on transcriptome and proteomic profiles of target cells as well as exerted proliferative and protective effects on cMSCs, and enhanced their cardiac and endothelial differentiation potential. hiPSC-MVs also transferred exogenous transcripts from genetically modified hiPSCs that opens new perspectives for future strategies to enhance MV content. We conclude that hiPSC-MVs are effective vehicles for transferring iPSC attributes to adult somatic cells, and hiPSC-MV-mediated horizontal transfer of RNAs and proteins to injured tissues may be used for therapeutic tissue repair. In this study, for the first time, we propose a new concept of use of hiPSCs as a source of safe acellular bioactive derivatives for tissue regeneration.

Adult bone marrow cell therapy improves survival and induces long-term improvement in cardiac parameters: a systematic review and meta-analysis.

BACKGROUND: Despite rapid clinical translation and widespread enthusiasm, the therapeutic benefits of adult bone marrow cell (BMC) transplantation in patients with ischemic heart disease continue to remain controversial. A synthesis of the available data is critical to appreciate and underscore the true impact of this promising approach. METHODS AND RESULTS: A total of 50 studies (enrolling 2625 patients) identified by database searches through January 2012 were included. Weighted mean differences for changes in left ventricular (LV) ejection fraction, infarct size, LV end-systolic volume, and LV end-diastolic volume were estimated with random-effects meta-analysis. Compared with control subjects, BMC-treated patients exhibited greater LV ejection fraction (3.96%; 95% confidence interval, 2.90-5.02; P<0.00001) and smaller infarct size (-4.03%, 95% confidence interval, -5.47 to -2.59; P<0.00001), LV end-systolic volume (-8.91 mL; 95% confidence interval, -11.57 to -6.25; P<0.00001), and LV end-diastolic volume (-5.23 mL; 95% confidence interval, -7.60 to -2.86; P<0.0001). These benefits were noted regardless of the study design (randomized controlled study versus cohort study) and the type of ischemic heart disease (acute myocardial infarction versus chronic ischemic heart disease) and persisted during long-term follow-up. Importantly, all-cause mortality, cardiac mortality, and the incidence of recurrent myocardial infarction and stent thrombosis were significantly lower in BMC-treated patients compared with control subjects. CONCLUSIONS: Transplantation of adult BMCs improves LV function, infarct size, and remodeling in patients with ischemic heart disease compared with standard therapy, and these benefits persist during long-term follow-up. BMC transplantation also reduces the incidence of death, recurrent myocardial infarction, and stent thrombosis in patients with ischemic heart disease.

Stem cell- derived extracellular vesicles as new tools in regenerative medicine - Immunomodulatory role and future perspectives.

In the last few decades, the practical use of stem cells (SCs) in the clinic has attracted significant attention in the regenerative medicine due to the ability of these cells to proliferate and differentiate into other cell types. However, recent findings have demonstrated that the therapeutic capacity of SCs may also be mediated by their ability to secrete biologically active factors, including extracellular vesicles (EVs). Such submicron circular membrane-enveloped vesicles may be released from the cell surface and harbour bioactive cargo in the form of proteins, lipids, mRNA, miRNA, and other regulatory factors. Notably, growing evidence has indicated that EVs may transfer their bioactive content into recipient cells and greatly modulate their functional fate. Thus, they have been recently envisioned as a new class of paracrine factors in cell-to-cell communication. Importantly, EVs may modulate the activity of immune system, playing an important role in the regulation of inflammation, exhibiting broad spectrum of the immunomodulatory activity that promotes the transition from pro-inflammatory to pro-regenerative environment in the site of tissue injury. Consequently, growing interest is placed on attempts to utilize EVs in clinical applications of inflammatory-related dysfunctions as potential next-generation therapeutic factors, alternative to cell-based approaches. In this review we will discuss the current knowledge on the biological properties of SC-derived EVs, with special focus on their role in the regulation of inflammatory response. We will also address recent findings on the immunomodulatory and pro-regenerative activity of EVs in several disease models, including in vitro and in vivo preclinical, as well as clinical studies. Finally, we will highlight the current perspectives and future challenges of emerging EV-based therapeutic strategies of inflammation-related diseases treatment.

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-1ß, IL-8) and downregulating the expression of a pro-fibrotic gene - α-smooth muscle actin (α-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.

The cardioprotective and anti-inflammatory effect of inhaled nitric oxide during Fontan surgery in patients with single ventricle congenital heart defects: a prospective randomized study.

BACKGROUND: Fontan surgery with cardiopulmonary bypass (CPB) causes tremendous systemic stress and inflammatory responses, affecting postoperative organ function, morbidity, and mortality. Although this reaction triggers partially protective anti-inflammatory responses, it is harmful in patients with single ventricle congenital heart defects. Despite decades of research, an effective anti-inflammatory and stress defense strategy is lacking. This study investigated the influence of inhaled nitric oxide (NO) during CPB on early clinical results, including the duration of postoperative respiratory support as a primary outcome and a panel of laboratory analytes. METHODS: In this study, 115 patients were randomized to the Fontan-NO group (n = 48) and the Fontan group (n = 49). Eighteen patients were excluded from the study. The Fontan-NO group received NO inhaled directly into the oxygenator during CPB. Clinical data were collected, and blood samples were drawn for analysis at repeated intervals. Multiplex assays were used to analyze a proteome profile of molecules involved in stress response, inflammation, metabolic reactions, as well as heart and lung protection. RESULTS: Fontan-NO patients had significantly shorter respiratory support time with a median of 9.3 h (7.0; 13,2) vs 13.9 h (3.7; 18.5) by the absolute difference of 4.6 h [95% confidence interval, - 30.9 to 12.3; (p = 0.03)]. In addition, they have a shorter time in intensive care (p = 0.04) and lower pulmonary artery pressure after CPB discontinuation (p = 0.04), 4 h (p = 0.03) and 8 h (p = 0.03) after surgery. Fontan-NO patients also had a lower concentration of lactates (p = 0.04) and glucose after separation from CPB (p = 0.02) and lower catecholamine index (p = 0.042). Plasma factors analysis has shown a significantly higher concentration of interleukin-10, and a lower concentration of interleukin-6, interleukin-8, interleukin-1ß, pentraxin, matrix metalloproteinase-8, troponin-I, creatine kinase myocardial band (CK-MB), and insulin in Fontan-NO group. CONCLUSIONS: NO inhaled into the oxygenator during CPB can improve short-term clinical outcomes. It shortens intubation time and intensive care time. It reduces inflammatory response, improves myocardial and lung protection, and diminishes metabolic stress in patients with a single ventricle undergoing Fontan surgery. TRIAL REGISTRATION NUMBER: The trial was preregistered, supervised, and supported by The Polish National Science Center ( NCN/01/B/NZ5/04246 ).

Transplantation of expanded bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) improves left ventricular function and remodelling after myocardial infarction.

Adult bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) exhibit a Sca-1(+)/Lin(-)/CD45(-) phenotype and can differentiate into various cell types, including cardiomyocytes and endothelial cells. We have previously reported that transplantation of a small number (1 × 10(6)) of freshly isolated, non-expanded VSEL-SCs into infarcted mouse hearts resulted in improved left ventricular (LV) function and anatomy. Clinical translation, however, will require large numbers of cells. Because the frequency of VSEL-SCs in the marrow is very low, we examined whether VSEL-SCs can be expanded in culture without loss of therapeutic efficacy. Mice underwent a 30 min. coronary occlusion followed by reperfusion and, 48 hrs later, received an intramyocardial injection of vehicle (group I, n = 11), 1 × 10(5) enhanced green fluorescent protein (EGFP)-labelled expanded untreated VSEL-SCs (group II, n = 7), or 1 × 10(5) EGFP-labelled expanded VSEL-SCs pre-incubated in a cardiogenic medium (group III, n = 8). At 35 days after myocardial infarction (MI), mice treated with pre-incubated VSEL-SCs exhibited better global and regional LV systolic function and less LV hypertrophy compared with vehicle-treated controls. In contrast, transplantation of expanded but untreated VSEL-SCs did not produce appreciable reparative benefits. Scattered EGFP(+) cells expressing α-sarcomeric actin, platelet endothelial cell adhesion molecule (PECAM)-1, or von Willebrand factor were present in VSEL-SC-treated mice, but their numbers were very small. No tumour formation was observed. We conclude that VSEL-SCs expanded in culture retain the ability to alleviate LV dysfunction and remodelling after a reperfused MI provided that they are exposed to a combination of cardiomyogenic growth factors and cytokines prior to transplantation. Counter intuitively, the mechanism whereby such pre-incubation confers therapeutic efficacy does not involve differentiation into new cardiac cells. These results support the potential therapeutic utility of VSEL-SCs for cardiac repair.

Extracellular vesicles from human iPSCs enhance reconstitution capacity of cord blood-derived hematopoietic stem and progenitor cells.

Cord blood (CB) represents a source of hematopoietic stem and progenitor cells (CB-HSPCs) for bone marrow (BM) reconstitution, but clinical CB application is limited in adult patients due to the insufficient number of CB-HSCPCs and the lack of effective ex vivo approaches to increase CB-HSPC functionality. Since human-induced pluripotent stem cells (hiPSCs) have been indicated as donor cells for bioactive extracellular vesicles (EVs) modulating properties of other cells, we are the first to employ hiPSC-derived EVs (hiPSC-EVs) to enhance the hematopoietic potential of CB-derived CD45dimLin-CD34+ cell fraction enriched in CB-HSPCs. We demonstrated that hiPSC-EVs improved functional properties of CB-HSPCs critical for their hematopoietic capacity including metabolic, hematopoietic and clonogenic potential as well as survival, chemotactic response to stromal cell-derived factor 1 and adhesion to the model components of hematopoietic niche in vitro. Moreover, hiPSC-EVs enhanced homing and engraftment of CB-HSPCs in vivo. This phenomenon might be related to activation of signaling pathways in CB-HSPCs following hiPSC-EV treatment, as shown on both gene expression and the protein kinases activity levels. In conclusion, hiPSC-EVs might be used as ex vivo modulators of CB-HSPCs capacity to enhance their functional properties and augment future practical applications of CB-derived cells in BM reconstitution.

Graphene-based materials enhance cardiomyogenic and angiogenic differentiation capacity of human mesenchymal stem cells in vitro - Focus on cardiac tissue regeneration.

Cell-based therapies have recently emerged as promising strategies for the treatment of cardiovascular disease. Mesenchymal stem cells (MSCs) are a promising cell type that represent a class of adult stem cells characterized by multipotency, high proliferative capacity, paracrine activity, and low immunogenicity. To improve the functional and therapeutic efficacy of MSCs, novel biomaterials are considered as scaffolds/surfaces that promote MSCs growth and differentiation. One of them are graphene-based materials, including graphene oxide (GO) and reduced graphene oxide (rGO). Due to the unique physical, chemical, and biological properties of graphene, scaffolds comprising GO/rGO have been examined as novel platforms to improve the differentiation potential of human MSCs in vitro. We verified different i) size of GO flakes, ii) reduction level, and iii) layer thickness to select the most suitable artificial niche for MSCs culture. The results revealed that graphene-based substrates constitute non-toxic substrates for MSCs. Surfaces with large flakes of GO as well as low reduced rGO are the most biocompatible for MSCs propagation and do not affect their proliferation and survival. Interestingly, small GO flakes and highly reduced rGO decreased MSCs proliferation and induced their apoptosis. We also found that GO and rGO substrates did not alter the MSCs phenotype, cell cycle progression and might modulate the adhesive capabilities of these cells. Importantly, we demonstrated that both materials promoted the cardiomyogenic and angiogenic differentiation capacity of MSCs in vitro. Thus, our data indicates that graphene-based surfaces represent promising materials that may influence the therapeutic application of MSCs via supporting their pro-regenerative potential.

Optimization of isolation and further characterization of umbilical-cord-blood-derived very small embryonic/ epiblast-like stem cells (VSELs).

Because of their small size and density, umbilical cord blood (UCB)-derived very small embryonic/epiblast-like stem cells (VSELs) are usually lost at various steps of UCB preparation. Accordingly, we noticed that a significant number of these cells, which are smaller than erythrocytes, are lost during gradient centrifugation over Ficoll-Paque as well as during routine volume depletion of UCB units before freezing. To preserve these cells in final UCB preparations, we propose a relatively short and economical three-step isolation protocol that allows recovery of approximately 60% of the initial number of Lin(-)/CD45(-)/CD133(+) UCB-VSELs present in freshly harvested UCB units. In this novel approach (i) UCB is lysed in a hypotonic ammonium chloride solution to deplete erythrocytes; (ii) CD133(+) including VSELs cells are enriched by employing immunomagnetic beads; and subsequently (iii) Lin(-)/CD45(-)/CD133(+) cells are sorted by fluorescence-activated cell sorting. The whole isolation procedure takes approximately 2-3 h per UCB unit and isolated cells are highly enriched for an Oct-4(+) and SSEA-4(+) population of small Lin(-)/CD45(-)/CD133(+) cells.

"Small stem cells" in adult tissues: very small embryonic-like stem cells stand up!

This review summarizes information regarding the rare population of very small embryonic-like stem cells (VSELs) that has been identified in adult tissues, emphasizing both their unique morphological features and potential biological significance. We focus on their pluripotent nature and expression of markers characteristic for embryonic stem cells (ESCs), epiblast (EP)SCs, and primordial germ cells (PGCs). Furthermore, we will discuss their rank in the developmental hierarchy of the SC compartment as well as their relationship to other bone marrow-derived, primitive, nonhematopoietic SCs including: (i) endothelial progenitor cells (EPCs); (ii) mesenchymal (M)SCs; (iii) multipotent adult progenitor cells (MAPCs); (iv) marrow-isolated adult multilineage inducible (MIAMIs) cells; (v) multipotent adult (MA)SCs; and (vi) OmniCytes. We will also present different populations of very "small SCs" that have been recently described in the literature (e.g., spore-like cells and Lin(-)/ALDH(high) long-term repopulating hematopoietic SCs).

Evidence that very small embryonic-like stem cells are mobilized into peripheral blood.

Recently, we identified in murine adult tissues, including bone marrow, a population of very small embryonic-like (VSEL) stem cells. Here, we provide further evidence that under steady-state conditions these cells circulate at very low levels in peripheral blood (PB) ( approximately 100-200 cells/ml) and could be additionally mobilized during pharmacological granulocyte-colony-stimulating factor-induced or stress-related mobilization, as demonstrated in a model of toxic liver or skeletal muscle damage induced by injection of carbon tetrachloride or cardiotoxin, respectively. The number of circulating VSEL stem cells under steady-state conditions in PB of 2-month-old animals was five times higher than that in 1-year-old mice. In conclusion, this study supports a hypothesis that VSEL stem cells are a mobile pool of primitive stem cells that could be released from the stem cell niches into PB. Further studies are needed, however, to see whether the level of these cells circulating in PB could become a prognostic indicator to assess the regenerative potential of an adult organism and/or clinical outcome from an injury. Disclosure of potential conflicts of interest is found at the end of this article.

Transplantation of bone marrow-derived very small embryonic-like stem cells attenuates left ventricular dysfunction and remodeling after myocardial infarction.

Adult bone marrow (BM) contains Sca-1+/Lin-/CD45- very small embryonic-like stem cells (VSELs) that express markers of several lineages, including cardiac markers, and differentiate into cardiomyocytes in vitro. We examined whether BM-derived VSELs promote myocardial repair after a reperfused myocardial infarction (MI). Mice underwent a 30-minute coronary occlusion followed by reperfusion and received intramyocardial injection of vehicle (n= 11), 1 x 10(5) Sca-1+/Lin-/CD45+ enhanced green fluorescent protein (EGFP)-labeled hematopoietic stem cells (n= 13 [cell control group]), or 1 x 10(4) Sca-1+/Lin-/CD45- EGFP-labeled cells (n= 14 [VSEL-treated group]) at 48 hours after MI. At 35 days after MI, VSEL-treated mice exhibited improved global and regional left ventricular (LV) systolic function (echocardiography) and attenuated myocyte hypertrophy in surviving tissue (histology and echocardiography) compared with vehicle-treated controls. In contrast, transplantation of Sca-1+/Lin-/CD45+ cells failed to confer any functional or structural benefits. Scattered EGFP+ myocytes and capillaries were present in the infarct region in VSEL-treated mice, but their numbers were very small. These results indicate that transplantation of a relatively small number of CD45- VSELs is sufficient to improve LV function and alleviate myocyte hypertrophy after MI, supporting the potential therapeutic utility of these cells for cardiac repair. Disclosure of potential conflicts of interest is found at the end of this article.

Erythrocyte-derived microvesicles may transfer phosphatidylserine to the surface of nucleated cells and falsely 'mark' them as apoptotic.

Lysis of erythrocytes using hypotonic solutions is one approach to remove red blood cells (RBCs) from umbilical cord blood (UCB), bone marrow (BM), and peripheral blood (PB) before flow cytometric analysis or sorting of nucleated cells (NCs). Our team employed this separation step to prepare UCB-, BM-, or PB-derived cells to sort very small embryonic-like stem cells (VSELs). We noticed that depletion of RBCs from UCB by hypotonic lysis resulted in a significant increase in the number of NCs including VSELs that bind Annexin-V (Ann-V). Surprisingly, these cells were not apoptotic and displayed normal proliferative potential. To explain this discrepancy, we show that RBC-derived microvesicles (RMV) released during erythrocyte lysis may transfer phosphatidylserine (PS) to the surface of NCs and 'mark' them falsely positive as apoptotic cells. This observation should be considered whenever Ann-V binding viability assays are employed to evaluate the quality of NCs depleted from erythrocytes via hypotonic lysis.