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Simple Summary Triple-negative breast cancer (TNBC) is the deadliest subtype of breast cancer, with limited treatment options. This review summarizes the most recent applications of extracellular vesicles (EVs) in TNBC as diagnostic/prognostic biomarkers, nanoscale drug delivery systems (NDDS) and immunotherapeutic agents, as well as the associated challenges and future directions of EV applications in cancer immunotherapy. Abstract Triple-negative breast cancer (TNBC) is the most aggressive and refractory subtype of breast cancer, often occurring in younger patients with poor clinical prognosis. Given the current lack of specific targets for effective intervention, the development of better treatment strategies remains an unmet medical need. Over the last decade, the field of extracellular vesicles (EVs) has grown tremendously, offering immense potential for clinical diagnosis/prognosis and therapeutic applications. While TNBC-EVs have been shown to play an important role in tumorigenesis, chemoresistance and metastasis, they could be repurposed as potential biomarkers for TNBC diagnosis and prognosis. Furthermore, EVs from various cell types can be utilized as nanoscale drug delivery systems (NDDS) for TNBC treatment. Remarkably, EVs generated from specific immune cell subsets have been shown to delay solid tumour growth and reduce tumour burden, suggesting a new immunotherapy approach for TNBC. Intrinsically, EVs can cross the blood–brain barrier (BBB), which holds great potential to treat the brain metastases diagnosed in one third of TNBC patients that remains a substantial clinical challenge. In this review, we present the most recent applications of EVs in TNBC as diagnostic/prognostic biomarkers, nanoscale drug delivery systems and immunotherapeutic agents, as well as discuss the associated challenges and future directions of EVs in cancer immunotherapy.

Zika virus (ZIKV) infection is a serious public threat with cases reported in about 70 countries and territories. One of the most serious consequences of ZIKV infection is congenital microcephaly in babies. Congenital microcephaly has been suggested to result from infection of neural progenitor cells (NPCs) in the developing fetal brain. However, the molecular and cellular mechanisms underlying microcephaly development remains to be fully elucidated. In this study, we employed quantitative proteomics to determine protein expression profile that occur during viral replication in NPCs. Bioinformatics analysis of the protein expression changes resulted in the identification of a wide range of cell signaling pathways. Specifically, pathways involved in neurogenesis and embryonic development were markedly altered, along with those associated with cell cycle, apoptosis, lipid metabolism and oxidative stress. Notably, the differential regulation of Ephrin Receptor and PPAR signaling pathways, as revealed by quantitative proteomics and validated by qPCR array, underscores the need to explore these pathways in disease development. Collectively, these results indicate that ZIKV-induced pathogenesis involves complex virus-host reactions; the findings reported here could help shed light on the mechanisms underlying ZIKV-induced microcephaly and ZIKV replication in NPCs.

Respiratory syncytial virus (RSV) infection is a severe threat to young children and the elderly. Despite decades of research, no vaccine has been approved. Notably, instead of affording protection, a formalin-inactivated RSV vaccine induced severe respiratory disease including deaths in vaccinated children in a 1960s clinical trial; however, recent studies indicate that other forms of experimental vaccines can also induce pulmonary pathology in pre-clinical studies. These findings suggest that multiple factors/pathways could be involved in the development of enhanced respiratory diseases. Clearly, a better understanding of the mechanisms underlying such adverse reactions is critically important for the development of safe and efficacious vaccines against RSV infection, given the exponential growth of RSV vaccine clinical trials in recent years. By employing an integrated systems biology approach in a pre-clinical cotton rat model, we unraveled a complex network of pulmonary canonical pathways leading to disease development in vaccinated animals upon subsequent RSV infections. Cytokines including IL-1, IL-6 GRO/IL-8, and IL-17 in conjunction with mobilized pulmonary inflammatory cells could play important roles in disease development, which involved a wide range of host responses including exacerbated pulmonary inflammation, oxidative stress, hyperreactivity, and homeostatic imbalance between coagulation and fibrinolysis. Moreover, the observed elevated levels of MyD88 implicate the involvement of this critical signal transduction module as the central node of the inflammatory pathways leading to exacerbated pulmonary pathology. Finally, the immunopathological consequences of inactivated vaccine immunization and subsequent RSV exposure were further substantiated by histological analyses of these key proteins along with inflammatory cytokines, while hypercoagulation was supported by increased pulmonary fibrinogen/fibrin accompanied by reduced levels of plasma D-dimers. Enhanced respiratory disease associated with inactivated RSV vaccine involves a complex network of host responses, resulting in significant pulmonary lesions and clinical manifestations such as tachypnea and airway obstruction. The mechanistic insight into the convergence of different signal pathways and identification of biomarkers could help facilitate the development of safe and effective RSV vaccine and formulation of new targeted interventions.

Multipotent mesenchymal stromal cell (MSC) transplantation is proposed as a novel therapy for treating diabetes by promoting the regeneration of damaged islets. The clinical promise of such treatments may be hampered by a high degree of donor-related variability in MSC function and a lack of standards for comparing potency. Here, we set out to identify markers of cultured human MSCs directly associated with islet regenerative function. Stromal cultures from nine separate bone marrow donors were demonstrated to have differing capacities to reduce hyperglycemia in the NOD/SCID streptozotocin-induced diabetic model. Regenerative (R) and non-regenerative (NR) MSC cultures were directly compared using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. A total of 1,410 proteins were quantified resulting in the identification of 612 upregulated proteins and 275 downregulated proteins by ± 1.2-fold in R-MSC cultures. Elastin microfibril interface 1 (EMILIN-1), integrin-linked protein kinase (ILK), and hepatoma-derived growth factor (HDGF) were differentially expressed in R-MSCs, and Ingenuity Pathway Analyses revealed each candidate as known regulators of integrin signaling. Western blot validation of EMILIN-1, ILK, and HDGF not only showed significantly higher abundance levels in R-MSCs, as compared with NR-MSCs, but also correlated with passage-induced loss of islet-regenerative potential. Generalized estimating equation modeling was applied to examine the association between each marker and blood glucose reduction. Both EMILIN-1 and ILK were significantly associated with blood glucose lowering function in vivo. Our study is the first to identify EMILIN-1 and ILK as prospective markers of islet regenerative function in human MSCs.

financial support for this work was obtained from: Health Canada (H4084-112281) to PM and MR-M. The European Research Council (CoG-2014-646903) to PM. The Spanish Ministry of Economy and Competitiveness (SAF-SAF2013-43065), to PM. The Generalitat de Catalunya (SGR330 and PERIS 2017-2019 Program) to P.M. The Asociacion Espanola Contra el Cancer (AECC-CI-2015) and Fero Foundation to CB. The "Fundacion Hay Esperanza'' to E.A. The Health Institute Carlos III (ISCIII/FEDER/PI14-01191) to CB. PM also acknowledges the institutional support from The Obra Social La Caixa-Fundacio Josep Carreras. PM is investigator of the Spanish Cell Therapy cooperative network (TERCEL).

Respiratory Syncytial Virus (RSV) infects almost all children under the age of one and is the leading cause of hospitalization among infants. It also causes high rates of infection in the elderly and immunocompromised patient populations. Despite several decades of research with dozens of candidate vaccines being vigorously evaluated in pre-clinical and clinical studies, there is no licensed vaccine available to date. A clinical trial conducted in the early 1960s testing a formaldehyde-inactivated RSV (FIRSV) vaccine resulted in enhanced respiratory disease, following subsequent RSV infection, leading to hospitalization of 80% of the participants and 2 deaths. Since then, other forms of RSV vaccine have also been found to induce enhanced disease in preclinical animal studies. Here, we aimed to develop a vaccine that can effectively protect mice from RSV infection and help identify facets of FIRSV-induced enhanced disease. In this study, BALB/c mice were immunized with an adenoviral vector containing the RSV fusion protein (F) fused with CD40 ligand where the CD40 ligand serves two vital functions as a molecular adjuvant and an antigen-targeting molecule. In contrast to the FIRSV vaccine, the vectored vaccine effectively protected animals against RSV without inducing enhanced respiratory disease. This protection involved a robust induction of neutralizing antibodies and memory CD8 T cells, which were not observed in the FIRSV group. Finally, the vectored vaccine was able to elicit long-lasting protection against RSV, which was mediated by increased levels of effector memory CD8 T cell 3 months post-vaccination.

Background. Human mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity. Methods. In order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. One transfection method using TransIT-2020 was selected and tested with an emphasis on cell quality (viability, identity, and yield), as well as efficacy with a human placental growth factor (PlGF) plasmid. Results. TransIT-2020 yielded the highest fluorescence signal per cell out of the methods that did not decrease cell recovery. Transfecting GFP to 5 hBM-MSC donors using TransIT-2020 yielded 24–36% GFP-expressing cells with a viability of 85–96%. hBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection. When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. Both growth and secretion of PlGF in overexpressing hBM-MSC were sustained over 7 days, confirming the sustainability and applicability of the TransIT-2020 transfection system. Discussion. We report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics.

Gene regulatory networks in AML may be influenced by microRNAs (miRs) contained in exosomes derived from bone marrow mesenchymal stromal cells (MSCs). We sequenced miRs from exosomes isolated from marrow-derived MSCs from patients with AML (n = 3) and from healthy controls (n = 3; not age-matched). Known targets of mIRs that were significantly different in AML-derived MSC exosomes compared to controls were identified. Of the five candidate miRs identified by differential packaging in exosomes, only miR-26a-5p and miR-101-3p were significantly increased in AML-derived samples while miR-23b-5p, miR-339-3p and miR-425-5p were significantly decreased. Validation of the predicted change in gene expression of the potential targets was investigated by interrogating gene expression levels from public datasets of marrow-derived CD34-selected cells from patients with AML (n = 69) and healthy donors (n = 40). Two molecules with decreased gene expression in AML (EZH2 and GSK3β) were predicted by the miR profiling and have been previously implicated in AML while three molecules were increased in AML-derived cells and have not been previously associated with leukemogenesis (KRBA2, RRBP1 and HIST2H 2BE). In summary, profiling miRs in exosomes from AML-derived MSCs allowed us to identify candidate miRs with potential relevance in AML that could yield new insights regarding leukemogenesis or new treatment strategies. Electronic supplementary material The online version of this article (10.1007/s12015-017-9762-0) contains supplementary material, which is available to authorized users.

Background— Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular endothelial cells (ECs). Hereditary PAH (HPAH) is often caused by mutations in the bone morphogenetic protein receptor type 2 gene ( BMPR2 ). However, the mechanisms by which these mutations cause PAH remain unclear. Therefore, we screened for dysregulated proteins in blood-outgrowth ECs of HPAH patients with BMPR2 mutations compared with healthy control subjects. Methods and Results— A total of 416 proteins were detected with 2-dimensional PAGE in combination with liquid chromatography/tandem mass spectrometry analysis, of which 22 exhibited significantly altered abundance in blood-outgrowth ECs from patients with HPAH. One of these proteins, translationally controlled tumor protein (TCTP), was selected for further study because of its well-established role in promoting tumor cell growth and survival. Immunostaining showed marked upregulation of TCTP in lungs from patients with HPAH and idiopathic PAH, associated with remodeled vessels of complex lesions. Increased TCTP expression was also evident in the SU5416 rat model of severe and irreversible PAH, associated with intimal lesions, colocalizing with proliferating ECs and the adventitia of remodeled vessels but not in the vascular media. Furthermore, silencing of TCTP expression increased apoptosis and abrogated the hyperproliferative phenotype of blood-outgrowth ECs from patients with HPAH, raising the possibility that TCTP may be a link in the emergence of apoptosis-resistant, hyperproliferative vascular cells after EC apoptosis. Conclusion— Proteomic screening identified TCTP as a novel mediator of endothelial prosurvival and growth signaling in PAH, possibly contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.

Acute myeloid leukemia (AML) is a blood malignancy resulting in abnormal hematopoiesis that is reported to be associated with alterations in the bone marrow microenvironment (BME). Current treatments for this heterogeneous disease, which target the leukemic cells but not the BME, are largely unsuccessful for the majority of AML subtypes. By better understanding the mechanisms by which the BME contributes to leukemogenesis, it may be possible to introduce more effective treatments for AML. Mesenchymal stem cells (MSCs) are an essential component of the BME that have been shown to support normal hematopoiesis. Therefore, MSCs may have several roles in the alteration of the BME, leukemogenesis, and AML relapse and can provide an excellent model for studying the BME in vitro. While some studies have characterized AML-derived MSCs (AML-MSCs), their exact role in the disease remains unclear. Our RNAseq analysis of AML-MSCs (n=30), and healthy donor MSCs (HD-MSCs) (n=8) identified that, among 7655 genes, 21 genes were significantly differentially expressed in AML-MSCs. Through in silico analysis of this gene set, genes of interest were identified as having the potential to directly alter the BME and affect AML pathogenesis through BMP/TGF-β pathways. Current work is focusing on investigation of the effects of selected genes with biological relevance on MSCs intrinsic and extrinsic functional properties. This study will improve our understanding of the role of MSCs in AML BME and help in the discovery of new therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Background: Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified. Methods: Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 ( BMPR2 ) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells. Results: Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1 , and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo. Conclusions: Pulmonary vascular and circulating progenitor endothelial cells isolated from patients with PAH demonstrate downregulation of miR-124, leading to the metabolic and proliferative abnormalities in PAH ECs via PTPB1 and PKM1/PKM2. Therefore, the manipulation of this miRNA or its targets could represent a novel therapeutic approach for the treatment of PAH.

Thalidomide is an immunomodulatory drug (IMiD) with proven therapeutic action in several autoimmune/inflammatory diseases; however, its inherent high toxicity has led to the development of more powerful and safer thalidomide analogs, including lenalidomide and pomalidomide. These are new generation IMiDs that exhibit direct antitumor activity as well as anti-inflammatory/immunomodulatory properties, and are FDA-approved for the treatment of several hematological malignances. Here we investigated the potential therapeutic effects of lenalidomide and pomalidomide in several experimental murine models of autoimmune/inflammatory diseases: 2,4,6-trinitrobenzene sulfonic acid- and dextran sulfate sodium-induced inflammatory bowel disease and type II collagen-induced arthritis. Lenalidomide displayed a strong therapeutic effect in all these models of autoimmune/inflammatory diseases, while the effect of pomalidomide was less pronounced. In vitro experiments confirmed the immunosuppressive effect of both IMiDs on the proliferative response of stimulated human lymphocytes and on the balance of secreted cytokines toward an anti-inflammatory profile. We conclude that lenalidomide may offer a therapeutic opportunity against autoimmune/inflammatory diseases., This work was supported by Health Canada (contract: H4080-144541) to PM and MR-M based on funding from Health Canada’s Genomics Research and Development Initiative Phase VI (2014-2019) obtained by MR-M and JRL. PM also acknowledges the financial support from The Obra Social La Caixa-Fundaciò Josep Carreras and The Generalitat de Catalunya (SGR330). PM is investigator of the Spanish Cell Therapy cooperative network (TERCEL). We are indebted to Dominique Patry from Health Canada for his assistance with the Luminex experiments.

International audience; Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene, leading to progressive and unrelenting muscle loss. Although the genetic basis ofDMD is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that secondary mechanisms, as the alteration of key signaling pathways, may play an important role. In order to identify reliable biomarkers and potential therapeutic targets, and taking advantage of the clinically relevant Golden Retriever Muscular Dystrophy (GRMD) dog model, a proteomic study was performed. Isotope-coded affinity tag (ICAT) profiling was used to compile quantitative changes in protein expression profiles of the vastus lateralis muscles of 4-month old GRMD vs healthy dogs. Interestingly, the set of under-expressed proteins detected appeared primarily composed of metabolic proteins, many of which have been shown to be regulated by the transcriptional peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1R). Subsequently, we were able to showed that PGC1-R expression is dramatically reduced inGRMDcompared to healthy muscle. Collectively, these results provide novel insights into the molecular pathology of the clinically relevant animal model of DMD, and indicate that defective energy metabolism is a central hallmark of the disease in the canine model.

In this study, we obtained evidence indicating that annexin 1 is a new target of the p38/MAPKAP kinase-2 pathway and that it regulates endothelial cell migration in response to vascular endothelial growth factor (VEGF). These conclusions are supported by a series of substantiating experiments. First, by two-dimensional gel electrophoresis and mass spectrometry, we identified annexin 1 as a protein whose phosphorylation is induced by VEGF and is impaired by inhibiting p38. Second, using in vitro kinase assays and in vivo phosphorylation assays, we found that VEGF-mediated activation of LIM kinase 1 downstream of the p38 pathway triggers the phosphorylation of annexin 1. Third, VEGF-induced cell migration and tube formation in Matrigel are inhibited following small interfering RNA-mediated knockdown of annexin 1. Fourth, both processes are rescued in cells expressing an annexin 1 construct insensitive to the small interfering RNA knockdown. Finally, the VEGF/annexin 1-mediated cell migration is impaired by inhibiting p38. We therefore conclude that phosphorylation of annexin 1 regulates the angiogenic effect that is associated with the activation of the p38/LIM kinase 1 axis by VEGF.

Progenitor cell therapy is hindered in patients with diabetes mellitus (DM) due to cellular senescence. Glycogen synthase kinase-3β (GSK3β) activity is increased in DM, potentially exacerbating impaired cell-based therapies. Thus, we aimed to determine if and how GSK3β inhibitors (GSKi) can improve therapeutic efficacy of endothelial progenitor cells (EPC) from patients with DM. Patients with DM had fewer EPCs and increased rates of apoptosis. DM EPCs also exhibited higher levels of GSK3β activity resulting in increased levels of phosphorylated β-catenin. Proteomic profiling of DM EPCs treated with GSKi identified 37 nonredundant, differentially regulated proteins. Cathepsin B (cathB) was subsequently confirmed to be differentially regulated and showed 40% less baseline activity in DM EPCs, an effect reversed by GSKi treatment. Finally, in vivo efficacy of cell-based therapy was assessed in a xenotransplant femoral wire injury mouse model. Administration of DM EPCs reduced the intima-to-media ratio, an effect that was further augmented when DM EPCs were pretreated with GSKi yet absent when cathB was antagonized. In DM, increased basal GSK3β activity contributes to accelerated EPC cellular senescence, an effect reversed by small molecule antagonism of GSK3β, which enhanced cell-based therapy after vascular injury.

Mesenchymal stem cells (MSC) show great promise in a wide array of therapeutic applications due mainly to their capacity to suppress immune and inflammatory reactions and instigate normal tissue repair processes. The secretion of bioactive factors is thought to play a predominant role in the mechanisms of action for these clinically relevant functions. As such, a large body of MSC research has focussed on characterization of the MSC secretome; including both soluble factors and factors released in extracellular vesicles (e.g., exosomes and microvesicles). This review provides an overview of our current knowledge of the MSC secretome in the context of determining the clinical relevance of these cells. In addition, the review summarizes various approaches that have been utilized to identify proteins secreted by MSC and discusses the advantages and limitations of different proteomic methods. Finally, we discuss issues that must be addressed before the clinical relevance of research into the MSC secretome can be realized.

Introduction Pulmonary arterial hypertension (PAH) is a rare desease characterised by profound vascular abnormalities in the peripheral arteries of the lung, leading to a progressive increase in pulmonary vascular resistance, right heart failure and death. The disease exists in several forms including a heritable form (HPAH) caused primarily by mutations in bone morphogenetic protein receptor type 2 (BMPR2) and an idiopathic form (IPAH). Endothelial cell (EC) dysfunction is considered a critical initiating factor in the pathobiology of PAH, manifested by increased susceptibility to apoptosis, heightened permeability and enhanced endothelial proliferation. Substantial changes in bioenergetics of ECs, including higher rates of glycolysis, have been reported in PAH patients. However, the mechanisms underlying alterations in energy production have not been identified. Methods We measured glycolysis in blood outgrowth endothelial cells (BOECs) from HPAH patients carrying mutations in BMPR2 and IPAH patients to confirm the metabolic abnormalities previously. We also employed an unbiased genome-wide microarray and proteomic screening approach to detect miRNAs and proteins dysregulated in the same groups to determine the mechanisms underlying abnormal endothelial glycolysis. Results HPAH and IPAH BOECs recapitulated the metabolic phenotype previously observed in PAECs. These alterations were found to be associated with the downregulation of miR-124 and the upregulation of its known target, splicing factor polypyrimidine-tract-binding protein (PTBP1). We also demonstrated that increased PTBP1 promotes the switching in expression of two forms of pyruvate kinase, PKM1 and PKM2, resulting in an increase of aerobic glycolysis, consequently increasing cell proliferation (mechanism schematized in Figure 1). Overexpression of miR-124, or siRNA silencing of PTPB1, restoring normal expression levels of PKM2, also restored normal proliferation and glycolysis in HPAH BOECs. Finally, we observed reduced miR-124 and increased PTPB1 and PKM2 expression in a well-established rat model of PAH, characterised by endothelial proliferation, supporting the presence of this mechanism in vivo. Conclusions Loss of function of BMPR2 results in the downregulation of miR-124 and consequently in the glycolytic abnormalities reported in PAH ECs. Therefore, the manipulation of this miRNA, or its targets, could represent a novel and effective strategy to achieve clinical benefits in the treatment of PAH.

Induced-pluripotent stem cells (iPSCs) are a potential alternative cell source in regenerative medicine, which includes the use of differentiated iPSCs for cell therapies to treat coronary artery and/or peripheral arterial diseases. Late-outgrowth endothelial progenitor cells (late-EPCs) are a unique primary cell present in peripheral blood that exhibit high proliferative capacity, are being used in a wide variety of clinical trials, and have the ability to differentiate into mature endothelial cells. The objective of this study was to reprogram peripheral blood-derived late-EPCs to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs that were retrovirally transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal disease, characterized by excessive proliferation of pulmonary vascular cells. Hereditary PAH is mainly caused by “lossof-function” mutations in the bone morphogenetic protein type II receptor (Bmpr2). However, the mechanisms by which these mutations cause PAH remain unclear. AIM: To identify dysregulated proteins in blood-derived endothelial cells of PAH patients with Bmpr2 mutations compared with healthy controls that may contribute to the pathogenesis of this disease. METHODS: Blood-outgrowth endothelial cells (ECs) were expanded ex vivo from peripheral blood mononuclear cell samples of four patients with heritable PAH and four healthy subjects. Protein isolates were subjected to 2D gel electrophoresis and stained for total proteins with Sypro Ruby. Gels were scanned and subjected to quantitative computer-assisted analysis (PDQuest software) and differentially regulated proteins determined by statistical tests (p-value 0.05) were identified by mass spectrometry (LC-MS/MS). RESULTS: Of the 416 proteins detected, 11 were significantly downregulated in PAH cells, including proteins in pathways previously implicated in PAH (i.e. PKA and guanine nucleotide-binding protein). 11 proteins were significantly upregulated, including a number of cell growth regulatory proteins, notably translationally controlled tumor protein (TCTP). TCTP has previously been shown to be enriched in EC apoptotic nanovesicles and to mediate pro-survival signaling in adjacent cells. Increased expression of TCTP in PAH compared to healthy control cells was demonstrated in patient-derived blood outgrowth ECs in vitro. In addition, the potential role of TCTP in PAH was studied in vivo in the SU5416 (SU) rat model of severe, angioproliferative PAH. SU is a VEGFR2inhibitor which induces EC apoptosis followed by reactive pulmonary vascular cell proliferation causing obliterative pulmonary vascular lesions closely mimicking the human condition. Immunofluorescence staining revealed high expression of TCTP in arteriolar ECs of PAH lungs tightly localized to proliferating cells within occlusive intimal lesions; whereas, only minimal TCTP expression was seen in vascular ECs of normal lungs. CONCLUSIONS: Bmpr2 mutant ECs exhibited dysregulation of key proteins controlling vasodilation and EC growth. TCTP could play an important role in PAH, possibly mediating prosurvival signaling in smooth muscle cells in response to EC apoptosis and thereby contributing to the process of pulmonary vascular remodeling. CIHR, FRQS, Cambridge NIHR Biomedical Research Centre, UK