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Endothelial cells (ECs) have essential roles in organ development and regeneration, and therefore they could be used for regenerative therapies. However, generation of abundant functional endothelium from pluripotent stem cells has been difficult because ECs generated by many existing strategies have limited proliferative potential and display vascular instability. The latter difficulty is of particular importance because cells that lose their identity over time could be unsuitable for therapeutic use. Here, we describe a 3-week platform for directly converting human mid-gestation lineage-committed amniotic fluid-derived cells (ACs) into a stable and expandable population of vascular ECs (rAC-VECs) without using pluripotency factors. By transient expression of the ETS transcription factor ETV2 for 2 weeks and constitutive expression the ETS transcription factors FLI1 and ERG1, concomitant with TGF-β inhibition for 3 weeks, epithelial and mesenchymal ACs are converted, with high efficiency, into functional rAC-VECs. These rAC-VECs maintain their vascular repertoire and morphology over numerous passages in vitro, and they form functional vessels when implanted in vivo. rAC-VECs can be detected in recipient mice months after implantation. Thus, rAC-VECs can be used to establish a cellular platform to uncover the molecular determinants of vascular development and heterogeneity and potentially represent ideal ECs for the treatment of regenerative disorders.

Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell-endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal 'crosstalk' between human airway basal cells and endothelial cells that regulates proliferation of basal cells., (© 2015. Published by The Company of Biologists Ltd.)

Turnover and exchange of nucleosomal histones and their variants, a process long believed to be static in post-replicative cells, remains largely unexplored in brain. Here, we describe a novel mechanistic role for HIRA (histone cell cycle regulator) and proteasomal degradation-associated histone dynamics in the regulation of activity-dependent transcription, synaptic connectivity, and behavior. We uncover a dramatic developmental profile of nucleosome occupancy across the lifespan of both rodents and humans, with the histone variant H3.3 accumulating to near-saturating levels throughout the neuronal genome by mid-adolescence. Despite such accumulation, H3.3-containing nucleosomes remain highly dynamic-in a modification-independent manner-to control neuronal- and glial-specific gene expression patterns throughout life. Manipulating H3.3 dynamics in both embryonic and adult neurons confirmed its essential role in neuronal plasticity and cognition. Our findings establish histone turnover as a critical and previously undocumented regulator of cell type-specific transcription and plasticity in mammalian brain., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recent studies in humans and in genetic mouse models have identified Slit- and NTRK-like family (Slitrks) as candidate genes for neuropsychiatric disorders. All Slitrk isotypes are highly expressed in the CNS, where they mediate neurite outgrowth, synaptogenesis, and neuronal survival. However, the molecular mechanisms underlying these functions are not known. Here, we report that Slitrk5 modulates brain-derived neurotrophic factor (BDNF)-dependent biological responses through direct interaction with TrkB receptors. Under basal conditions, Slitrk5 interacts primarily with a transsynaptic binding partner, protein tyrosine phosphatase δ (PTPδ); however, upon BDNF stimulation, Slitrk5 shifts to cis-interactions with TrkB. In the absence of Slitrk5, TrkB has a reduced rate of ligand-dependent recycling and altered responsiveness to BDNF treatment. Structured illumination microscopy revealed that Slitrk5 mediates optimal targeting of TrkB receptors to Rab11-positive recycling endosomes through recruitment of a Rab11 effector protein, Rab11-FIP3. Thus, Slitrk5 acts as a TrkB co-receptor that mediates its BDNF-dependent trafficking and signaling., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Lung cancer is the leading cause of cancer related mortality worldwide, with non-small cell lung cancer (NSCLC) as the most prevalent form. Despite advances in treatment options including minimally invasive surgery, CT-guided radiation, novel chemotherapeutic regimens, and targeted therapeutics, prognosis remains dismal. Therefore, further molecular analysis of NSCLC is necessary to identify novel molecular targets that impact prognosis and the design of new-targeted therapies. In recent years, tumor "activated/reprogrammed" stromal cells that promote carcinogenesis have emerged as potential therapeutic targets. However, the contribution of stromal cells to NSCLC is poorly understood. Here, we show increased numbers of bone marrow (BM)-derived hematopoietic cells in the tumor parenchyma of NSCLC patients compared with matched adjacent non-neoplastic lung tissue. By sorting specific cellular fractions from lung cancer patients, we compared the transcriptomes of intratumoral myeloid compartments within the tumor bed with their counterparts within adjacent non-neoplastic tissue from NSCLC patients. The RNA sequencing of specific myeloid compartments (immature monocytic myeloid cells and polymorphonuclear neutrophils) identified differentially regulated genes and mRNA isoforms, which were inconspicuous in whole tumor analysis. Genes encoding secreted factors, including osteopontin (OPN), chemokine (C-C motif) ligand 7 (CCL7) and thrombospondin 1 (TSP1) were identified, which enhanced tumorigenic properties of lung cancer cells indicative of their potential as targets for therapy. This study demonstrates that analysis of homogeneous stromal populations isolated directly from fresh clinical specimens can detect important stromal genes of therapeutic value.

Hematopoietic stem cells (HSCs) first emerge during embryonic development within vessels such as the dorsal aorta of the aorta-gonad-mesonephros (AGM) region, suggesting that signals from the vascular microenvironment are critical for HSC development. Here, we demonstrated that AGM-derived endothelial cells (ECs) engineered to constitutively express AKT (AGM AKT-ECs) can provide an in vitro niche that recapitulates embryonic HSC specification and amplification. Specifically, nonengrafting embryonic precursors, including the VE-cadherin-expressing population that lacks hematopoietic surface markers, cocultured with AGM AKT-ECs specified into long-term, adult-engrafting HSCs, establishing that a vascular niche is sufficient to induce the endothelial-to-HSC transition in vitro. Subsequent to hematopoietic induction, coculture with AGM AKT-ECs also substantially increased the numbers of HSCs derived from VE-cadherin⁺CD45⁺ AGM hematopoietic cells, consistent with a role in supporting further HSC maturation and self-renewal. We also identified conditions that included NOTCH activation with an immobilized NOTCH ligand that were sufficient to amplify AGM-derived HSCs following their specification in the absence of AGM AKT-ECs. Together, these studies begin to define the critical niche components and resident signals required for HSC induction and self-renewal ex vivo, and thus provide insight for development of defined in vitro systems targeted toward HSC generation for therapeutic applications.

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC-derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina-induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain-null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood-derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence.

Emerging studies have begun to demonstrate that reprogrammed stromal cells play pivotal roles in tumor growth, metastasis, and resistance to therapy. However, the contribution of stromal cells to non-small-cell lung cancer (NSCLC) has remained underexplored. We used an orthotopic model of Kras-driven NSCLC to systematically dissect the contribution of specific hematopoietic stromal cells in lung cancer. RNA deep-sequencing analysis of individually sorted myeloid lineage and tumor epithelial cells revealed cell-type-specific differentially regulated genes, indicative of activated stroma. We developed a computational model for crosstalk signaling discovery based on ligand-receptor interactions and downstream signaling networks and identified known and novel tumor-stroma paracrine and tumor autocrine crosstalk-signaling pathways in NSCLC. We provide cellular and molecular insights into components of the lung cancer microenvironment that contribute to carcinogenesis. This study has the potential for development of therapeutic strategies that target tumor-stroma interactions and may complement conventional anti-cancer treatments., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC)-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP) self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The lung alveoli regenerate after surgical removal of the left lobe by pneumonectomy (PNX). How this alveolar regrowth/regeneration is initiated remains unknown. We found that platelets trigger lung regeneration by supplying stromal-cell-derived factor-1 (SDF-1, also known as CXCL12). After PNX, activated platelets stimulate SDF-1 receptors CXCR4 and CXCR7 on pulmonary capillary endothelial cells (PCECs) to deploy the angiocrine membrane-type metalloproteinase MMP14, stimulating alveolar epithelial cell (AEC) expansion and neo-alveolarization. In mice lacking platelets or platelet Sdf1, PNX-induced alveologenesis was diminished. Reciprocally, infusion of Sdf1(+/+) but not Sdf1-deficient platelets rescued lung regeneration in platelet-depleted mice. Endothelial-specific ablation of Cxcr4 and Cxcr7 in adult mice similarly impeded lung regeneration. Notably, mice with endothelial-specific Mmp14 deletion exhibited impaired expansion of AECs but not PCECs after PNX, which was not rescued by platelet infusion. Therefore, platelets prime PCECs to initiate lung regeneration, extending beyond their haemostatic contribution. Therapeutic targeting of this haemo-vascular niche could enable regenerative therapy for lung diseases.

Background: Endothelial cells (ECs) are responsible for creating a tumor vascular niche as well as producing angiocrine factors. ECs demonstrate functional and phenotypic heterogeneity when located under different microenvironments. Here, we describe a tumor-stimulated mesenchymal phenotype in ECs and investigate its impact on tumor growth, stemness, and invasiveness., Methods: Xenograft tumor assay in NOD/SCID mice and confocal imaging were conducted to show the acquisition of mesenchymal phenotype in tumor-associated ECs in vivo. Immunocytochemistry, qPCR and flow cytometry techniques showed the appearance of mesenchymal traits in ECs after contact with breast tumor cell lines MDA-MB231 or MCF-7. Cell proliferation, cell migration, and sphere formation assays were applied to display the functional advantages of mesenchymal ECs in tumor growth, invasiveness, and enrichment of tumor initiating cells. qPCR and western blotting were used to investigate the mechanisms underlying EC mesenchymal transition., Results: Our results showed that co-injection of ECs and tumor cells in NOD/SCID mice significantly enhanced tumor growth in vivo with tumor-associated ECs expressing mesenchymal markers while maintaining their intrinsic endothelial trait. We also showed that a mesenchymal phenotype is possibly detectable in human neoplastic breast biopsies as well as ECs pre-exposed to tumor cells (ECs(Mes)) in vitro. The ECs(Mes) acquired prolonged survival, increased migratory behavior and enhanced angiogenic properties. In return, ECs(Mes) were capable of enhancing tumor survival and invasiveness. The mesenchymal phenotypes in ECs(Mes) were the result of a contact-dependent transient phenomenon and reversed upon removal of the neoplastic contexture. We showed a synergistic role for TGFβ and notch pathways in this phenotypic change, as simultaneous inhibition of notch and TGFβ down-regulated Smad1/5 phosphorylation and Jag1(KD) tumor cells were unable to initiate the process., Conclusions: Overall, our data proposed a crosstalk mechanism between tumor and microenvironment where tumor-stimulated mesenchymal modulation of ECs enhanced the constitution of a transient mesenchymal/endothelial niche leading to significant increase in tumor proliferation, stemness, and invasiveness. The possible involvement of notch and TGFβ pathways in the initiation of mesenchymal phenotype may propose new stromal targets.

Malignant gliomas represent one of the most aggressive forms of cancer, displaying high mortality rates and limited treatment options. Specific subpopulations of cells residing in the tumor niche with stem-like characteristics have been postulated to initiate and maintain neoplasticity while resisting conventional therapies. The study presented here aims to define the role of glycogen synthase kinase 3 beta (GSK3b) in patient-derived glioblastoma (GBM) stem-like cell (GSC) proliferation, apoptosis and invasion. To evaluate the potential role of GSK3b in GBM, protein profiles from 68 GBM patients and 20 normal brain samples were analyzed for EGFR-mediated PI3kinase/Akt and GSK3b signaling molecules including protein phosphatase 2A (PP2A). To better understand the function of GSK3b in GBM, GSCs were isolated from GBM patient samples. Blocking GSK3b phosphorylation at Serine 9 attenuated cell proliferation while concomitantly stimulating apoptosis through activation of Caspase-3 in patient-derived GSCs. Increasing GSK3b protein content resulted in the inhibition of cell proliferation, colony formation and stimulated programmed cell death. Depleting GSK3b in GSCs down regulated PP2A. Furthermore, knocking down PP2A or blocking its activity by okadaic acid inactivated GSK3b by increasing GSK3b phosphorylation at Serine 9. Our data suggests that GSK3b may function as a regulator of apoptosis and tumorigenesis in GSCs. Therapeutic approaches targeting GSK3b in glioblastoma stem-like cells may be a useful addition to our current therapeutic armamentarium.

Ovarian cancer is the second leading cause of cancer-related death in women worldwide. Despite optimal cytoreduction and adequate adjuvant therapies, initial tumor response is often followed by relapse suggesting the existence of a tumor niche. Targeted therapies have been evaluated in ovarian cancer to overcome resistant disease. Among them, antiangiogenic therapies inhibit new blood vessel growth, induce endothelial cell apoptosis, and block the incorporation of hematopoietic and endothelial progenitor cells into new blood vessels. Despite in vitro and in vivo successes, antivascular therapy with bevacizumab targeting VEGF-A has limited efficacy in ovarian cancer. The precise molecular mechanisms underlying clinical resistance to anti-VEGF therapies are not yet well understood. Among them, tumor and stromal heterogeneity might determine the treatment outcomes. The present study investigates whether abnormalities in the tumor endothelium may contribute to treatment resistance to bevacizumab and promote a residual microscopic disease. Here, we showed that ovarian cancer cells activate Akt phosphorylation in endothelial cells inducing resistance to bevacizumab leading to an autocrine loop based on FGF2 secretion. Altogether, our results point out the role of an activated endothelium in the resistance to bevacizumab and in the constitution of a niche for a residual disease., (©2014 American Association for Cancer Research.)

Chromatin remodeling via incorporation of histone variants plays a key role in the regulation of embryonic development. The histone variant H3.3 has been associated with a number of early events including formation of the paternal pronucleus upon fertilization. The small number of amino acid differences between H3.3 and its canonical counterparts (H3.1 and H3.2) has limited studies of the developmental significance of H3.3 deposition into chromatin due to difficulties in distinguishing the H3 isoforms. To this end, we used zinc-finger nuclease (ZFN) mediated gene editing to introduce a small C-terminal hemagglutinin (HA) tag to the endogenous H3.3B locus in mouse embryonic stem cells (ESCs), along with an internal ribosome entry site (IRES) and a separately translated fluorescent reporter of expression. This system will allow detection of expression driven by the reporter in cells, animals, and embryos, and will facilitate investigation of differential roles of paternal and maternal H3.3 protein during embryogenesis that would not be possible using variant-specific antibodies. Further, the ability to monitor endogenous H3.3 protein in various cell lineages will enhance our understanding of the dynamics of this histone variant over the course of development., (© 2014 Wiley Periodicals, Inc.)

Treating metastasis has been challenging due to tumors complexity and heterogeneity. This complexity is partly related to the crosstalk between tumor and its microenvironment. Endothelial cells -the building blocks of tumor vasculature- have been shown to have additional roles in cancer progression than angiogenesis and supplying oxygen and nutrients. Here, we show an alternative role for endothelial cells in supporting breast cancer growth and spreading independent of their vascular functions. Using endothelial cells and breast cancer cell lines MDA-MB231 and MCF-7, we developed co-culture systems to study the influence of tumor endothelium on breast tumor development by both in vitro and in vivo approaches. Our results demonstrated that endothelial cells conferred survival advantage to tumor cells under complete starvation and enriched the CD44HighCD24Low/- stem cell population in tumor cells. Moreover, endothelial cells enhanced the pro-metastatic potential of breast cancer cells. The in vitro and in vivo results concordantly confirmed a role for endothelial Jagged1 to promote breast tumor through notch activation. Here, we propose a role for endothelial cells in enhancing breast cancer progression, stemness, and pro-metastatic traits through a perfusion-independent manner. Our findings may be beneficial in developing novel therapeutic approaches.

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.

Understanding the intricate cellular components of the bone marrow microenvironment can lead to the discovery of novel extrinsic factors that are responsible for the initiation and progression of leukemic disease. We have shown that endothelial cells (ECs) provide a fertile niche that allows for the propagation of primitive and aggressive leukemic clones. Activation of the ECs by vascular endothelial growth factor (VEGF)-A provides cues that enable leukemic cells to proliferate at higher rates and also increases the adhesion of leukemia to ECs. Vascular endothelial growth factor A-activated ECs decrease the efficacy of chemotherapeutic agents to target leukemic cells. Inhibiting VEGF-dependent activation of ECs by blocking their signaling through VEGF receptor 2 increases the susceptibility of leukemic cells to chemotherapy. Therefore, the development of drugs that target the activation state of the vascular niche could prove to be an effective adjuvant therapy in combination with chemotherapeutic agents., (Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

For future application of induced pluripotent stem cell (iPSC) technology, the ability to assess the overall quality of iPSC clones will be an important issue. Here we show that the histone variant H2A.X is a functional marker that can distinguish the developmental potentials of mouse iPSC lines. We found that H2A.X is specifically targeted to and negatively regulates extraembryonic lineage gene expression in embryonic stem cells (ESCs) and prevents trophectoderm lineage differentiation. ESC-specific H2A.X deposition patterns are faithfully recapitulated in iPSCs that support the development of "all-iPS" animals via tetraploid complementation, the most stringent test available of iPSC quality. In contrast, iPSCs that fail to support all-iPS embryonic development show aberrant H2A.X deposition, upregulation of extraembryonic lineage genes, and a predisposition to extraembryonic differentiation. Thus, our work has highlighted an epigenetic mechanism for maintaining cell lineage commitment in ESCs and iPSCs that can be used to distinguish the quality of iPSC lines., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Transfer of a somatic nucleus into an enucleated oocyte is the most efficient approach for somatic cell reprogramming. While this process is known to involve extensive chromatin remodeling of the donor nucleus, the maternal factors responsible and the underlying chromatin-based mechanisms remain largely unknown. Here we discuss our recent findings demonstrating that the histone variant H3.3 plays an essential role in reprogramming and is required for reactivation of key pluripotency genes in somatic cell nuclear transfer (SCNT) embryos. Maternal-derived H3.3 replaces H3 in the donor nucleus shortly after oocyte activation, with the amount of replacement directly related to the differentiation status of the donor nucleus in SCNT embryos. We provide additional evidence to suggest that de novo synthesized H3.3 replaces histone H3 carrying repressive modifications in the donor nuclei of SCNT embryos, and hypothesize that replacement may occur at specific loci that must be reprogrammed for gene reactivation.

The tumor stroma plays an essential role in tumor growth, resistance to therapy and occurrence of metastatic phenotype. Tumor vessels have been considered as passive conducts for nutrients but several studies have demonstrated secretion of pro-tumoral factors by endothelial cells. The failure of anti-angiogenic therapies to meet expectations raised by pre-clinical studies prompt us to better study the cross-talk between endothelial and cancer cells. Here, we hypothesized that tumor cells and the endothelium secrete bio-active microparticles (MPs) participating to a functional cross-talk. We characterized the cancer cells MPs, using breast and ovarian cancer cell lines (MCF7, MDA-MB231, SKOV3, OVCAR3 and a primary cell lines, APOCC). Our data show that MPs from mesenchymal-like cell lines (MDA-MB231, SKOV3 and APOCC) were able to promote an activation of endothelial cells through Akt phosphorylation, compared to MPs from epithelial-like cell lines (OVCAR3 and MCF7). The MPs from mesenchymal-like cells contained increased angiogenic molecules including PDGF, IL8 and angiogenin. The endothelial activation was associated to increased Arf6 expression and MPs secretion. Endothelial activation functionalized an MP dependent pro-tumoral vascular niche promoting cancer cells proliferation, invasiveness, stem cell phenotype and chemoresistance. MPs from cancer and endothelial cells displayed phenotypic heterogeneity, and participated to a functional cross-talk where endothelial activation by cancer MPs resulted in increased secretion of EC-MPs sustaining tumor cells. Such cross-talk may play a role in perfusion independent role of the endothelium.

Generating engraftable human haematopoietic cells from autologous tissues is a potential route to new therapies for blood diseases. However, directed differentiation of pluripotent stem cells yields haematopoietic cells that engraft poorly. Here, we have devised a method to phenocopy the vascular-niche microenvironment of haemogenic cells, thereby enabling reprogramming of human endothelial cells into engraftable haematopoietic cells without transition through a pluripotent intermediate. Highly purified non-haemogenic human umbilical vein endothelial cells or adult dermal microvascular endothelial cells were transduced with the transcription factors FOSB, GFI1, RUNX1 and SPI1 (hereafter referred to as FGRS), and then propagated on serum-free instructive vascular niche monolayers to induce outgrowth of haematopoietic colonies containing cells with functional and immunophenotypic features of multipotent progenitor cells (MPPs). These endothelial cells that have been reprogrammed into human MPPs (rEC-hMPPs) acquire colony-forming-cell potential and durably engraft into immune-deficient mice after primary and secondary transplantation, producing long-term rEC-hMPP-derived myeloid (granulocytic/monocytic, erythroid, megakaryocytic) and lymphoid (natural killer and B cell) progenies. Conditional expression of FGRS transgenes, combined with vascular induction, activates endogenous FGRS genes, endowing rEC-hMPPs with a transcriptional and functional profile similar to that of self-renewing MPPs. Our approach underscores the role of inductive cues from the vascular niche in coordinating and sustaining haematopoietic specification and may prove useful for engineering autologous haematopoietic grafts to treat inherited and acquired blood disorders.

During development, mammalian germ cells reprogram their epigenomes via a genome-wide erasure and de novo rewriting of DNA methylation marks. We know little of how methylation patterns are specifically determined. The piRNA pathway is thought to target the bulk of retrotransposon methylation. Here we show that most retrotransposon sequences are modified by default de novo methylation. However, potentially active retrotransposon copies evade this initial wave, likely mimicking features of protein-coding genes. These elements remain transcriptionally active and become targets of piRNA-mediated methylation. Thus, we posit that these two waves play essential roles in resetting germ cell epigenomes at each generation., (© 2014 Molaro et al.; Published by Cold Spring Harbor Laboratory Press.)

This review is intended to provide an overview of tissue engineering strategies using scaffold biomaterials to develop a vascularized tissue engineered construct for nano-pathophysiology. Two primary topics are discussed. The first is the biological or synthetic microenvironments that regulate cell behaviors in pathological conditions and tissue regeneration. Second is the use of scaffold biomaterials with angiogenic factors and/or cells to realize vascularized tissue engineered constructs for nano-pathophysiology. These topics are significantly overlapped in terms of three-dimensional (3-D) geometry of cells and blood vessels. Therefore, this review focuses on neovascularization of 3-D scaffold biomaterials induced by angiogenic factors and/or cells. The novel strategy of this approach in nano-pathophysiology is to utilize the vascularized tissue engineered construct as a tissue model to predict the distribution and subsequent therapeutic efficacy of a drug delivery system with different physicochemical and biological properties., (© 2013.)

Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA, into oocytes in somatic cell nuclear transfer embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity.

Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

Tumor endothelial cells (ECs) promote cancer progression in ways beyond their role as conduits supporting metabolism. However, it is unknown how vascular niche-derived paracrine factors, defined as angiocrine factors, provoke tumor aggressiveness. Here, we show that FGF4 produced by B cell lymphoma cells (LCs) through activating FGFR1 upregulates the Notch ligand Jagged1 (Jag1) on neighboring ECs. In turn, upregulation of Jag1 on ECs reciprocally induces Notch2-Hey1 in LCs. This crosstalk enforces aggressive CD44(+)IGF1R(+)CSF1R(+) LC phenotypes, including extranodal invasion and chemoresistance. Inducible EC-selective deletion of Fgfr1 or Jag1 in the Eμ-Myc lymphoma model or impairing Notch2 signaling in mouse and human LCs diminished lymphoma aggressiveness and prolonged mouse survival. Thus, targeting the angiocrine FGF4-FGFR1/Jag1-Notch2 loop inhibits LC aggressiveness and enhances chemosensitivity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Endothelial cells in the neointima undergo a transition to the mesenchymal phenotype, suggesting a therapeutic target for vein graft restenosis (Cooley et al., this issue).

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb (-/-)) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb(-/-) embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.

Chemical or traumatic damage to the liver is frequently associated with aberrant healing (fibrosis) that overrides liver regeneration. The mechanism by which hepatic niche cells differentially modulate regeneration and fibrosis during liver repair remains to be defined. Hepatic vascular niche predominantly represented by liver sinusoidal endothelial cells deploys paracrine trophogens, known as angiocrine factors, to stimulate regeneration. Nevertheless, it is not known how pro-regenerative angiocrine signals from liver sinusoidal endothelial cells is subverted to promote fibrosis. Here, by combining an inducible endothelial-cell-specific mouse gene deletion strategy and complementary models of acute and chronic liver injury, we show that divergent angiocrine signals from liver sinusoidal endothelial cells stimulate regeneration after immediate injury and provoke fibrosis after chronic insult. The pro-fibrotic transition of vascular niche results from differential expression of stromal-derived factor-1 receptors, CXCR7 and CXCR4 (refs 18, 19, 20, 21), in liver sinusoidal endothelial cells. After acute injury, CXCR7 upregulation in liver sinusoidal endothelial cells acts with CXCR4 to induce transcription factor Id1, deploying pro-regenerative angiocrine factors and triggering regeneration. Inducible deletion of Cxcr7 in sinusoidal endothelial cells (Cxcr7(iΔEC/iΔEC)) from the adult mouse liver impaired liver regeneration by diminishing Id1-mediated production of angiocrine factors. By contrast, after chronic injury inflicted by iterative hepatotoxin (carbon tetrachloride) injection and bile duct ligation, constitutive FGFR1 signalling in liver sinusoidal endothelial cells counterbalanced CXCR7-dependent pro-regenerative response and augmented CXCR4 expression. This predominance of CXCR4 over CXCR7 expression shifted angiocrine response of liver sinusoidal endothelial cells, stimulating proliferation of desmin(+) hepatic stellate-like cells and enforcing a pro-fibrotic vascular niche. Endothelial-cell-specific ablation of either Fgfr1 (Fgfr1(iΔEC/iΔEC)) or Cxcr4 (Cxcr4(iΔEC/iΔEC)) in mice restored the pro-regenerative pathway and prevented FGFR1-mediated maladaptive subversion of angiocrine factors. Similarly, selective CXCR7 activation in liver sinusoidal endothelial cells abrogated fibrogenesis. Thus, we demonstrate that in response to liver injury, differential recruitment of pro-regenerative CXCR7-Id1 versus pro-fibrotic FGFR1-CXCR4 angiocrine pathways in vascular niche balances regeneration and fibrosis. These results provide a therapeutic roadmap to achieve hepatic regeneration without provoking fibrosis.

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (ECs) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates., (© AlphaMed Press.)

Background: As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype., Methods: To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed., Results: We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process., Conclusion: Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem cells (HSCs). However, the number of HSCs in UCB is limited, and attempts to amplify them in vitro remain inefficient. Several publications have documented amplification of hematopoietic stem/progenitor cells (HSPCs) on endothelial or mesenchymal cells, but the lack of homogeneity in culture conditions and HSC definition impairs direct comparison of these results. We investigated the ability of different feeder layers, mesenchymal progenitors (MPs) and endothelial cells (ECs), to amplify hematopoietic stem/progenitor cells. Placental derived HSPCs (defined as Lin(-)CD45(-/dim)CD34(+)CD38(-)CD90(+)) were maintained on confluent feeder layers and the number of cells and their marker expression were monitored over 21 days. Although both types of feeder layers supported hematopoietic expansion, only endothelial cells triggered amplification of Lin(-)CD45(-/dim)CD34(+)CD38(-)CD90(+) cells, which peaked at 14 days. The amplified cells differentiated into all cell lineages, as attested by in vitro colony-forming assays, and were capable of engraftment and multi-lineage differentiation in sub-lethally irradiated mice. Mesenchymal progenitors promoted amplification of CD38(+) cells, previously defined as precursors with more limited differentiation potential. A competitive assay demonstrated that hematopoietic stem/progenitor cells had a preference for interacting with endothelial cells in vitro. Cytokine and transcriptomic analysis of both feeder cell types identified differences in gene expression that correlated with propensity of ECs and MPs to support hematopoietic cell amplification and differentiation respectively. Finally, we used RNA sequencing of endothelial cells and HSPCs to uncover relevant networks illustrating the complex interaction between endothelial cells and HSPCs leading to stem/progenitor cell expansion., (© 2013.)

Neovascular age-related macular degeneration is a leading cause of irreversible vision loss in the Western world. Cytokine-targeted therapies (such as anti-vascular endothelial growth factor) are effective in treating pathologic ocular angiogenesis, but have not led to a durable effect and often require indefinite treatment. Here, we show that Nutlin-3, a small molecule antagonist of the E3 ubiquitin protein ligase MDM2, inhibited angiogenesis in several model systems. We found that a functional p53 pathway was essential for Nutlin-3-mediated retinal antiangiogenesis and disruption of the p53 transcriptional network abolished the antiangiogenic activity of Nutlin-3. Nutlin-3 did not inhibit established, mature blood vessels in the adult mouse retina, suggesting that only proliferating retinal vessels are sensitive to Nutlin-3. Furthermore, Nutlin-3 inhibited angiogenesis in nonretinal models such as the hind limb ischemia model. Our work demonstrates that Nutlin-3 functions through an antiproliferative pathway with conceivable advantages over existing cytokine-targeted antiangiogenesis therapies.

Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila, polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. Here, we show that, in mouse embryonic stem cells (ESCs), H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones and reduced PRC2 occupancy. Further, we show H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. Our data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, our findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an "active" chromatin state., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The bone marrow (BM) microenvironment is composed of multiple niche cells that, by producing paracrine factors, maintain and regenerate the hematopoietic stem cell (HSC) pool (Morrison and Spradling, 2008). We have previously demonstrated that endothelial cells support the proper regeneration of the hematopoietic system following myeloablation (Butler et al., 2010; Hooper et al., 2009; Kobayashi et al., 2010). Here, we demonstrate that expression of the angiocrine factor Jagged-1, supplied by the BM vascular niche, regulates homeostatic and regenerative hematopoiesis through a Notch-dependent mechanism. Conditional deletion of Jagged-1 in endothelial cells (Jag1((ECKO)) mice) results in a profound decrease in hematopoiesis and premature exhaustion of the adult HSC pool, whereas quantification and functional assays demonstrate that loss of Jagged-1 does not perturb vascular or mesenchymal compartments. Taken together, these data demonstrate that the instructive function of endothelial-specific Jagged-1 is required to support the self-renewal and regenerative capacity of HSCs in the adult BM vascular niche., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Microvascular endothelial cells (ECs) within different tissues are endowed with distinct but as yet unrecognized structural, phenotypic, and functional attributes. We devised EC purification, cultivation, profiling, and transplantation models that establish tissue-specific molecular libraries of ECs devoid of lymphatic ECs or parenchymal cells. These libraries identify attributes that confer ECs with their organotypic features. We show that clusters of transcription factors, angiocrine growth factors, adhesion molecules, and chemokines are expressed in unique combinations by ECs of each organ. Furthermore, ECs respond distinctly in tissue regeneration models, hepatectomy, and myeloablation. To test the data set, we developed a transplantation model that employs generic ECs differentiated from embryonic stem cells. Transplanted generic ECs engraft into regenerating tissues and acquire features of organotypic ECs. Collectively, we demonstrate the utility of informational databases of ECs toward uncovering the extravascular and intrinsic signals that define EC heterogeneity. These factors could be exploited therapeutically to engineer tissue-specific ECs for regeneration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Our vision of cancer has changed during the past decades. Indeed tumors are now perceived as complex entities where tumoral and stromal components interact closely. Among the different elements of tumor stroma the cellular component play a primordial role. Bone Marrow derived mesenchymal cells (MSCs) are attracted to tumor sites and support tumor growth. Endothelial cells (ECs) play a major role in angiogenesis. While the literature documents many aspects of the cross talk between stromal and cancer cells, the role of direct hetero-cellular contact is not clearly established. Recently, Tunneling nanotubes (TnTs) have been shown to support cell-to-cell transfers of plasma membrane components, cytosolic molecules and organelles within cell lines. Herein, we have investigated the formation of heterocellular TnTs between stromal (MSCs and ECs) and cancer cells. We demonstrate that TnTs occur between different cancer cells, stromal cells and cancer-stromal cell lines. We showed that TnTs-like structure occurred in 3D anchorage independent spheroids and also in tumor explant cultures. In our culture condition, TnTs formation occurred after large membrane adhesion. We showed that intercellular transfers of cytoplasmic content occurred similarly between cancer cells and MSCs or ECs, but we highlighted that the exchange of mitochondria occurred preferentially between endothelial cells and cancer cells. We illustrated that the cancer cells acquiring mitochondria displayed chemoresistance. Our results illustrate the perfusion-independent role of the endothelium by showing a direct endothelial to cancer cell mitochondrial exchange associated to phenotypic modulation. This supports another role of the endothelium in the constitution of the metastatic niche.

Myelosuppression is a life-threatening complication of antineoplastic therapy, but treatment is restricted to a few cytokines with unilineage hematopoietic activity. Although hematopoietic stem cells (HSCs) are predominantly quiescent during homeostasis, they are rapidly recruited into cell cycle by stresses, including myelosuppressive chemotherapy. Factors that induce HSCs to proliferate during stress have been characterized, but it is not known how HSC quiescence is then reestablished. In this study, we show that TGFβ signaling is transiently activated in hematopoietic stem and progenitor cells (HSPCs) during hematopoietic regeneration. Blockade of TGFβ signaling after chemotherapy accelerates hematopoietic reconstitution and delays the return of cycling HSCs to quiescence. In contrast, TGFβ blockade during homeostasis fails to induce cycling of HSPCs. We identified the cyclin-dependent kinase inhibitor Cdkn1c (p57) as a key downstream mediator of TGFβ during regeneration because the recovery of chimeric mice, incapable of expressing p57 in HSPCs, phenocopies blockade of TGFβ signaling after chemotherapy. This study demonstrates that context-dependent activation of TGFβ signaling is central to an unrecognized counterregulatory mechanism that promotes homeostasis once hematopoiesis has sufficiently recovered from myelosuppressive chemotherapy. These results open the door to new, potentially superior, approaches to promote multilineage hematopoietic recovery by blocking the TGFβ signaling that dampens regeneration.

Unlabelled: Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation., Key Points: Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.

Remarkable advancements have been made in understanding the pathophysiology of hypertrophic cardiomyopathy (HCM), since the first implantable cardioverter defibrillator (ICD) was placed in a human, more than 25 years ago to prevent sudden cardiac death (SCD). ICD has become the cornerstone in the management of HCM, with an ability to change the natural course of this complex disease. American College of Cardiology/American Heart Association guidelines have been charted out to help risk stratify asymptomatic or minimally symptomatic adults with HCM, in order to prevent SCD in such individuals. Our patient with left ventricular outflow tract gradient <30 mm Hg, left ventricular wall thickness < 30 mm, negative medical history of syncope and no family history of SCD, would have been at low risk for SCD.

Background: Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment., Methods: Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed., Results: In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro., Conclusion: EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Recent work has highlighted the multitude of biological functions of sphingosine 1-phosphate (S1P), which include roles in hematopoietic cell trafficking, organization of immune organs, vascular development, and neuroinflammation. Indeed, a functional antagonist of S1P(1) receptor, FTY720/Gilenya, has entered the clinic as a novel therapeutic for multiple sclerosis. In this issue of the JEM, Zhang et al. highlight yet another function of this lipid mediator: thrombopoiesis. The S1P(1) receptor is required for the growth of proplatelet strings in the bloodstream and the shedding of platelets into the circulation. Notably, the sharp gradient of S1P between blood and the interstitial fluids seems to be essential to ensure the production of platelets, and S1P appears to cooperate with the CXCL12-CXCR4 axis. Pharmacologic modulation of the S1P(1) receptor altered circulating platelet numbers acutely, suggesting a potential therapeutic strategy for controlling thrombocytopenic states. However, the S1P(4) receptor may also regulate thrombopoiesis during stress-induced accelerated platelet production. This work reveals a novel physiological action of the S1P/S1P(1) duet that could potentially be harnessed for clinical translation.

ETS transcription factors ETV2, FLI1, and ERG1 specify pluripotent stem cells into induced vascular endothelial cells (iVECs). However, iVECs are unstable and drift toward nonvascular cells. We show that human midgestation c-Kit(-) lineage-committed amniotic cells (ACs) can be reprogrammed into vascular endothelial cells (rAC-VECs) without transitioning through a pluripotent state. Transient ETV2 expression in ACs generates immature rAC-VECs, whereas coexpression with FLI1/ERG1 endows rAC-VECs with a vascular repertoire and morphology matching mature endothelial cells (ECs). Brief TGFβ-inhibition functionalizes VEGFR2 signaling, augmenting specification of ACs into rAC-VECs. Genome-wide transcriptional analyses showed that rAC-VECs are similar to adult ECs in which vascular-specific genes are expressed and nonvascular genes are silenced. Functionally, rAC-VECs form stable vasculature in Matrigel plugs and regenerating livers. Therefore, short-term ETV2 expression and TGFβ inhibition with constitutive ERG1/FLI1 coexpression reprogram mature ACs into durable rAC-VECs with clinical-scale expansion potential. Banking of HLA-typed rAC-VECs establishes a vascular inventory for treatment of diverse disorders., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In this issue of Blood, Pitchford et al demonstrate that intravenous administration of VEGF-A promotes megakaryocyte (Mk) maturation and localization to bone marrow sinusoidal endothelial cells (BMECs), stimulating thrombopoiesis.

During angiogenesis, nascent vascular sprouts fuse to form vascular networks, enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S1P receptor-1 (S1P1) stabilizes the primary vascular network. A gradient of S1P1 expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S1P1, adherens junctions are destabilized, barrier function is breached, and flow is perturbed, resulting in abnormal vascular hypersprouting. Interestingly, S1P1 responds to S1P as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in vitro and in vivo. These data demonstrate that blood flow and circulating S1P activate endothelial S1P1 to stabilize blood vessels in development and homeostasis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.

The human airway epithelium is a pseudostratified heterogenous layer comprised of ciliated, secretory, intermediate, and basal cells. As the stem/progenitor population of the airway epithelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the three major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2-dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell-secreted VEGFA activated endothelium to express mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA-mediated cross-talk between airway basal cells and endothelium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

Background: Public hospitals can privatize management activities by contracting with a private organization or person to perform the work. Management contract is a method which uses private sector for major government projects like hospitals. This study evaluates contract management in health care institutes of developing countries., Methods: Information has been collected by reviewing the management contract condition of selected countries. Different forms of public private partnership for private participation in hospitals were surveyed., Results: The effects of management contract is expanding market opportunities to include public sector clients, capturing a market to be protected from competitors and providing a reliable and timely source of revenue., Conclusion: Contracting with non-governmental entities will provide better results than government provision of the same services. Contracting initiatives must be regulated and monitored at the highest level of government by experienced and astute policy makers, economists and operational personnel.