RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells.

During angiogenesis, VEGF acts as an attractive cue for endothelial cells (ECs), while Sema3E mediates repulsive cues. Here, we show that the small GTPase RhoJ integrates these opposing signals in directional EC migration. In the GTP-bound state, RhoJ interacts with the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ released from PlexinD1 induces cell contraction. PlexinD1-bound RhoJ further facilitates Sema3E-induced PlexinD1-VEGFR2 association, VEGFR2 transphosphorylation at Y1214, and p38 MAPK activation, leading to reverse EC migration. Upon VEGF stimulation, RhoJ is required for the formation of the holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, thereby preventing degradation of internalized VEGFR2, prolonging downstream signal transductions via PLCγ, Erk, and Akt, and promoting forward EC migration. After conversion to the GDP-bound state, RhoJ shifts from PlexinD1 to VEGFR2, which then terminates the VEGFR2 signals. RhoJ deficiency in ECs efficiently suppressed aberrant angiogenesis in ischemic retina. These findings suggest that distinct Rho GTPases may act as context-dependent integrators of chemotactic cues in directional cell migration and may serve as candidate therapeutic targets to manipulate cell motility in disease or tissue regeneration.

Decoding the regulatory network of early blood development from single-cell gene expression measurements.

Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.

Circulation-independent differentiation pathway from extraembryonic mesoderm toward hematopoietic stem cells via hemogenic angioblasts.

A large gap exists in our understanding of the course of differentiation from mesoderm to definitive hematopoietic stem cells (HSCs). Previously, we reported that Runx1(+) cells in embryonic day 7.5 (E7.5) embryos contribute to the hemogenic endothelium in the E10.5 aorta-gonad-mesonephros (AGM) region and HSCs in the adult bone marrow. Here, we show that two Runx1(+) populations subdivided by Gata1 expression exist in E7.5 embryos. The hemogenic endothelium and the HSCs are derived only from the Runx1(+)Gata1(-) population. A subset of this population moves from the extra- to intraembryonic region during E7.5-E8.0, where it contributes to the hemogenic endothelium of the dorsal aorta (DA). Migration occurs before the heartbeat is initiated, and it is independent of circulation. This suggests a developmental trajectory from Runx1(+) cells in the E7.5 extraembryonic region to definitive HSCs via the hemogenic endothelium.

A simple and highly effective method for slow-freezing human pluripotent stem cells using dimethyl sulfoxide, hydroxyethyl starch and ethylene glycol.

Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional -80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application.

Ectopic cerebellar cell migration causes maldevelopment of Purkinje cells and abnormal motor behaviour in Cxcr4 null mice.

SDF-1/CXCR4 signalling plays an important role in neuronal cell migration and brain development. However, the impact of CXCR4 deficiency in the postnatal mouse brain is still poorly understood. Here, we demonstrate the importance of CXCR4 on cerebellar development and motor behaviour by conditional inactivation of Cxcr4 in the central nervous system. We found CXCR4 plays a key role in cerebellar development. Its loss leads to defects in Purkinje cell dentritogenesis and axonal projection in vivo but not in cell culture. Transcriptome analysis revealed the most significantly affected pathways in the Cxcr4 deficient developing cerebellum are involved in extra cellular matrix receptor interactions and focal adhesion. Consistent with functional impairment of the cerebellum, Cxcr4 knockout mice have poor coordination and balance performance in skilled motor tests. Together, these results suggest ectopic the migration of granule cells impairs development of Purkinje cells, causes gross cerebellar anatomical disruption and leads to behavioural motor defects in Cxcr4 null mice.

Vascular RhoJ is an effective and selective target for tumor angiogenesis and vascular disruption.

Current antiangiogenic therapy is limited by its cytostatic nature and systemic side effects. To address these limitations, we have unveiled the role of RhoJ, an endothelial-enriched Rho GTPase, during tumor progression. RhoJ blockade provides a double assault on tumor vessels by both inhibiting tumor angiogenesis and disrupting the preformed tumor vessels through the activation of the RhoA-ROCK (Rho kinase) signaling pathway in tumor endothelial cells, consequently resulting in a functional failure of tumor vasculatures. Moreover, enhanced anticancer effects were observed when RhoJ blockade was employed in concert with a cytotoxic chemotherapeutic agent, angiogenesis-inhibiting agent, or vascular-disrupting agent. These results identify RhoJ blockade as a selective and effective therapeutic strategy for targeting tumor vasculature with minimal side effects.

Prolonged treatment with DNMT inhibitors induces distinct effects in promoters and gene-bodies.

Treatment with the demethylating drugs 5-azacytidine (AZA) and decitabine (DAC) is now recognised as an effective therapy for patients with Myelodysplastic Syndromes (MDS), a range of disorders arising in clones of hematopoietic progenitor cells. A variety of cell models have been used to study the effect of these drugs on the methylation of promoter regions of tumour suppressor genes, with recent efforts focusing on the ability of these drugs to inhibit DNA methylation at low doses. However, it is still not clear how nano-molar drug treatment exerts its effects on the methylome. In this study, we have characterised changes in DNA methylation caused by prolonged low-dose treatment in a leukemic cell model (SKM-1), and present a genome-wide analysis of the effects of AZA and DAC. At nano-molar dosages, a one-month continuous treatment halved the total number of hypermethylated probes in leukemic cells and our analysis identified 803 candidate regions with significant demethylation after treatment. Demethylated regions were enriched in promoter sequences whereas gene-body CGIs were more resistant to the demethylation process. CGI methylation in promoters was strongly correlated with gene expression but this correlation was lost after treatment. Our results indicate that CGI demethylation occurs preferentially at promoters, but that it is not generally sufficient to modify expression patterns, and emphasises the roles of other means of maintaining cell state.

Role of Etv2-positive cells in the remodeling morphogenesis during vascular development.

Etv2 is a critical determinant for the commitment of endothelial (EC) and hematopoietic (HPC) cells from mesoderm. Etv2 is assumed to be transiently required for EC commitment but dispensable after most ECs differentiate around E9.5. To confirm the time window of Etv2 requirement, Etv2 was ablated at different time points using ROSA26CreER mice. Unexpectedly, Etv2 ablation at E9.5 caused vascular remodeling defects in cranial and yolk sac vasculature. Immunostaining showed that Etv2+/VE-cadherin (VECAD)- cells were present around forming vasculature, mostly co-expressing Flk-1 with a small number of Etv2+/VECAD+ cells, indicating that Etv2+/Flk-1+/VECAD- cells are the major Etv2+ population promoting vascular remodeling around E9.5. Gene expression analysis showed up-regulation of Fgf proteins, Il-6, Glypican-3 and matrix metalloproteases in Etv2+/VEDAC- cells over Etv2-/VECAD+ mature ECs. Blockade of those factors caused reduced EC sprouting in ex vivo explant culture from E9.5 embryos, suggesting the functional significance of environmental factors derived from Etv2+ cells. Altogether, we propose that Etv2+/VEDAC- cells around E9.5-E10.5 provide extracellular factors to complete vascular morphogenesis in addition to becoming differentiated ECs incorporated into vessels. This insight for the new role of Ets protein in perivascular Flk-1+/VECAD-/(Etv2+) cells to induce expression of angiogenic factors may provide another strategy to control angiogenesis.

High-level expression and efficient one-step chromatographic purification of a soluble human leukemia inhibitory factor (LIF) in Escherichia coli.

Leukemia inhibitor factor (LIF) is a three disulfide bridge-containing cytokine with numerous regulatory effects. In this report, we present the high level expression of a soluble recombinant human LIF (rhLIF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hLIF cDNA was cloned into pET3b vector, and transformed into E. coli OrigamiB(DE3) harboring the bacterial thioredoxin coexpression vector. By using an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™), the yield of soluble proteins was significantly improved in comparison to commonly-used 2 × YT media. The recombinant protein was purified via a single chromatographic step using an affinity tag-based protein purification system that processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Soluble rhLIF yield was estimated to be approximately 1mg/g of wet weight cells, with above 98% purity. The rhLIF protein specifically inhibited the proliferation of the murine myeloblastic leukemia M1 cell in a dose-dependent manner, and induced Stat3 phosphorylation in mouse ES cells. This novel expression and purification protocol for the production of recombinant hLIF is a simple, suitable, and effective method.

EphrinB2-EphB4 signals regulate formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries.

PURPOSE: To elucidate the role of signals mediated by EphB4 receptor tyrosine kinase and its transmembrane ephrinB2 ligand in corneal lymphatic capillaries. METHODS: To detect expression of ephrinB2 and EphB4 in mouse corneas, immunohistochemistry of flat-mount corneas from 6- to 10-week-old wild-type, Efnb2-lacZ, and Ephb4-lacZ mice on a C57BL/6 background was performed. To induce formation of new blood vessels and lymphatic vessels, mouse corneal epithelia were swabbed with 0.1 M sodium hydroxide. To antagonize endogenous receptor-ligand interactions in corneal lymphatic vessels, recombinant EphB4/Fc proteins were injected into the subconjunctival spaces. To visualize the corneal lymphatic flow, FITC-dextran was injected subconjunctivally. RESULTS: In lymphatic capillaries of adult mouse corneas, EphB4 was intensively expressed in lymphatic endothelial cells (LECs) of funnel-shaped valves, which were segregated from ephrinB2-expressing LECs. The number of corneal lymphatic valves was significantly decreased by Efnb2 haploinsufficiency, and subconjunctival EphB4/Fc injections resulted in the deformation of preexisting valves of corneal lymphatic capillaries. In alkali-burn corneas, ephrinB2 and EphB4 were highly expressed in LECs of valve-forming areas. Subconjunctival EphB4/Fc injections perturbed the morphologic maturation of new lymphatic valves, leading to reflux of FITC-dextran to peripheral lymphatic branches. CONCLUSIONS: The results demonstrate a pivotal role of ephrinB2-EphB4 signals in the formation and maintenance of funnel-shaped valves in corneal lymphatic capillaries, and further suggest the potential of ephrinB2-EphB4 signals as a target to therapeutically manipulate corneal lymphangiogenesis.

Peyer's patch inducer cells play a leading role in the formation of B and T cell zone architecture.

Peripheral lymphoid tissues, such as lymph nodes and Peyer's patches (PPs), are organs required for mounting highly efficient immune responses to small quantities of Ag. The compartmentalization of the cellular components involved in the immune response into distinct zones supports the function of these tissues; however, little is known about how this compartmentalization is achieved. In this study, we analyzed neonatal PP development and present evidence that the CD3(-)IL-7Rα(+) PP inducer cells that initially play a pivotal role in the formation of the PP anlagen are involved in the formation of B and T cell zones in neonatal mice. PP inducer cells migrate between these zones by undergoing chemokine receptor switching.

Region-specific Etv2 ablation revealed the critical origin of hemogenic capacity from Hox6-positive caudal-lateral primitive mesoderm.

Hematopoietic cells (HPCs) develop from hemogenic endothelial cells (ECs), a specialized type of ECs undergoing hematopoietic transition. However, the mesoderm origin for hemogenic ECs or HPCs has not been clarified. To examine the origin for hemogenic mesoderm, we inactivated Etv2, a master regulator for EC/HPC commitment, in specific regions. Region-specific Etv2 ablation in early mesoderm caused local EC differentiation block, resulting in the loss of specific vascular beds without compensatory migration of residual ECs into avascular area. This feature of local EC/HPC differentiation block was correlated to the hemogenic potential of each mesoderm subset. We found that caudal-lateral mesoderm of E7.5-8.5 embryos represent the pre-committed population critical for generating hemogenic ECs. Etv2 ablation in caudal-lateral mesoderm by Hoxb6 Cre or Hoxb6CreER transgene affected vitelline plexus formation and intra-aortic hematopoietic clusters. In differentiated embryonic stem cells, this mesoderm subset marked by Hoxb6-lateral mesoderm promoter showed enriched T lymphopoietic potential among Flk-1(+) cells, which could be regarded as a characteristic for definitive HPCs. These findings indicate that critical mesoderm precursors possibly for definitive type hemogenic ECs are regionally specified in primitive mesoderm, suggesting that Hoxb6(+) caudal-lateral mesoderm represents the critical source of HPCs, which are potentially useful to enrich definitive HPCs from embryonic stem cells.

PDGF receptor alpha+ mesoderm contributes to endothelial and hematopoietic cells in mice.

BACKGROUND: Early mesoderm can be classified into Flk-1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk-1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato-endothelial lineages has not been proved in mouse embryos. RESULTS: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin-Kit+Sca-1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk-1+ population that exists until E8.5 may be the source for hemato-endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin-Kit+Sca-1+ (KSL) cells, respectively. CONCLUSIONS: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos.

Arhgef15 promotes retinal angiogenesis by mediating VEGF-induced Cdc42 activation and potentiating RhoJ inactivation in endothelial cells.

BACKGROUND: Drugs inhibiting vascular endothelial growth factor (VEGF) signaling are globally administered to suppress deregulated angiogenesis in a variety of eye diseases. However, anti-VEGF therapy potentially affects the normal functions of retinal neurons and glias which constitutively express VEGF receptor 2. Thus, it is desirable to identify novel drug targets which are exclusively expressed in endothelial cells (ECs). Here we attempted to identify an EC-specific Rho guanine nucleotide exchange factor (GEF) and evaluate its role in retinal angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By exploiting fluorescence-activated cell sorting and microarray analyses in conjunction with in silico bioinformatics analyses, we comprehensively identified endothelial genes in angiogenic retinal vessels of postnatal mice. Of 9 RhoGEFs which were highly expressed in retinal ECs, we show that Arhgef15 acted as an EC-specific GEF to mediate VEGF-induced Cdc42 activation and potentiated RhoJ inactivation, thereby promoting actin polymerization and cell motility. Disruption of the Arhgef15 gene led to delayed extension of vascular networks and subsequent reduction of total vessel areas in postnatal mouse retinas. CONCLUSIONS/SIGNIFICANCE: Our study provides information useful to the development of new means of selectively manipulating angiogenesis without affecting homeostasis in un-targeted tissues; not only in eyes but also in various disease settings such as cancer.

Protective effects of GLP-1 on glomerular endothelium and its inhibition by PKCß activation in diabetes.

To characterize glucagon-like peptide (GLP)-1 signaling and its effect on renal endothelial dysfunction and glomerulopathy. We studied the expression and signaling of GLP-1 receptor (GLP-1R) on glomerular endothelial cells and the novel finding of protein kinase A-dependent phosphorylation of c-Raf at Ser259 and its inhibition of angiotensin II (Ang II) phospho-c-Raf(Ser338) and Erk1/2 phosphorylation. Mice overexpressing protein kinase C (PKC)ß2 in endothelial cells (EC-PKCß2Tg) were established. Ang II and GLP-1 actions in glomerular endothelial cells were analyzed with small interfering RNA of GLP-1R. PKCß isoform activation induced by diabetes decreased GLP-1R expression and protective action on the renal endothelium by increasing its degradation via ubiquitination and enhancing phospho-c-Raf(Ser338) and Ang II activation of phospho-Erk1/2. EC-PKCß2Tg mice exhibited decreased GLP-1R expression and increased phospho-c-Raf(Ser338), leading to enhanced effects of Ang II. Diabetic EC-PKCß2Tg mice exhibited greater loss of endothelial GLP-1R expression and exendin-4-protective actions and exhibited more albuminuria and mesangial expansion than diabetic controls. These results showed that the renal protective effects of GLP-1 were mediated via the inhibition of Ang II actions on cRaf(Ser259) and diminished by diabetes because of PKCß activation and the increased degradation of GLP-1R in the glomerular endothelial cells.

Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice.

Etv2 is a master gene for the commitment of hematopoietic/endothelial cells and is a potent inducer of endothelial/hematopoietic cells from embryonic stem cells. Etv2 is highly expressed in endothelial/hematopoietic precursors but is downregulated when they are differentiated, indicating that Etv2 should have transient but not constitutive function. However, relatively little attention has been paid to the importance of transient Etv2 expression. To determine whether transient Etv2 expression is essential to normal development and cell differentiation, we generated mice that constitutively express Etv2 from a Cre-activatable ROSA26 locus in endothelial/hematopoietic, somite, or neuronal lineages. Constitutive Etv2 expression caused profound phenotypes in hematopoietic/endothelial cells, with little effect on somite or neuronal lineages. In hematopoietic/endothelial lineages, constitutive Etv2 expression induced by Tie-2 Cre transgene caused abnormal yolk sac vasculature. Prolonged vascular endothelial cadherin expression and decreased B lymphopoiesis were observed in Etv2 expressing vascular endothelial cadherin(+)/CD45(+) cells, indicating that Etv2 forces endothelial program on hematopoietic cells. Etv2 expression in adult hematopoietic cells by Vav-iCre transgene also conferred an endothelial phenotype on hematopoietic stem cells and suppressed hematopoiesis, with erythropoiesis severely affected. We conclude that constitutive Etv2 expression perturbs vascular development and hematopoiesis. While promoting hematopoiesis/vasculogenesis, Etv2 expression should be tightly regulated to achieve normal vascular development and hematopoiesis.

Phf14, a novel regulator of mesenchyme growth via platelet-derived growth factor (PDGF) receptor-α.

The regulation of mesenchymal cell growth by signaling molecules plays an important role in maintaining tissue functions. Aberrant mesenchymal cell proliferation caused by disruption of this regulatory process leads to pathogenetic events such as fibrosis. In the current study we have identified a novel nuclear factor, Phf14, which controls the proliferation of mesenchymal cells by regulating PDGFRα expression. Phf14-null mice died just after birth due to respiratory failure. Histological analyses of the lungs of these mice showed interstitial hyperplasia with an increased number of PDGFRα(+) mesenchymal cells. PDGFRα expression was elevated in Phf14-null mesenchymal fibroblasts, resulting in increased proliferation. We demonstrated that Phf14 acts as a transcription factor that directly represses PDGFRα expression. Based on these results, we used an antibody against PDGFRα to successfully treat mouse lung fibrosis. This study shows that Phf14 acts as a negative regulator of PDGFRα expression in mesenchymal cells undergoing normal and abnormal proliferation, and is a potential target for new treatments of lung fibrosis.

Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.

Early ontogenic origin of the hematopoietic stem cell lineage.

Several lines of evidence suggest that the adult hematopoietic system has multiple developmental origins, but the ontogenic relationship between nascent hematopoietic populations under this scheme is poorly understood. In an alternative theory, the earliest definitive blood precursors arise from a single anatomical location, which constitutes the cellular source for subsequent hematopoietic populations. To deconvolute hematopoietic ontogeny, we designed an embryo-rescue system in which the key hematopoietic factor Runx1 is reactivated in Runx1-null conceptuses at specific developmental stages. Using complementary in vivo and ex vivo approaches, we provide evidence that definitive hematopoiesis and adult-type hematopoietic stem cells originate predominantly in the nascent extraembryonic mesoderm. Our data also suggest that other anatomical sites that have been proposed to be sources of the definitive hematopoietic hierarchy are unlikely to play a substantial role in de novo blood generation.