The pro-inflammatory signature of lipopolysaccharide in spontaneous contracting embryoid bodies differentiated from mouse embryonic stem cells.

Embryonic stem (ES) cells differentiate towards all three germ layers, including cardiac cells and leukocytes, and may be therefore suitable to model inflammatory reactions in vitro. In the present study, embryoid bodies differentiated from mouse ES cells were treated with increasing doses of lipopolysaccharide (LPS) to mimic infection with gram-negative bacteria. LPS treatment dose-dependent increased contraction frequency of cardiac cell areas and calcium spikes and increased protein expression of α-actinin. LPS treatment increased the expression of the macrophage marker CD68 and CD69, which is upregulated after activation on T cells, B cells and NK cells. LPS dose-dependent increased protein expression of toll-like receptor 4 (TLR4). Moreover, upregulation of NLR family pyrin domain containing 3 (NLRP3), IL-1ß and cleaved caspase 1 was observed, indicating activation of inflammasome. In parallel, generation of reactive oxygen species (ROS), nitric oxide (NO), and expression of NOX1, NOX2, NOX4 and eNOS occurred. ROS generation, NOX2 expression and NO generation were downregulated by the TLR4 receptor antagonist TAK-242 which abolished the LPS-induced positive chronotropic effect of LPS. In conclusion, our data demonstrate that LPS induced a pro-inflammatory cellular immune response in tissues derived from ES cells, recommending the in vitro model of embryoid bodies for inflammation research.

The nicotinamide phosphoribosyltransferase antagonist FK866 inhibits growth of prostate tumour spheroids and increases doxorubicin retention without changes in drug transporter and cancer stem cell protein expression.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD) synthesis and is involved in cancer cell proliferation through regulation of energy production pathways. Therefore, NAMPT inhibitors are promising drugs for cancer therapy by limiting energy supply of tumours. Herein, we demonstrated that the NAMPT inhibitor FK866 ((E)-N-(4-(1-Benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) dose-dependently inhibited growth and cell motility of DU-145 prostate tumour spheroids and decreased the intracellular ATP concentration. The apoptosis marker cleaved caspase-3 remained unchanged, but the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) was upregulated. Growth inhibition was reversed upon co-administration of NAD to the cell culture medium. FK866 decreased calcein as well as pheophorbide A efflux from tumour spheroids and increased doxorubicin toxicity, indicating interference with function of drug efflux transporters. DU-145 multicellular tumour spheroids expressed the stem cell associated markers CD133, CD44, Oct4, Nanog, Sox2, and drug transporters ABCB1, ABCG2, and ABCC1 which are associated with stem cell properties in cancer cells. The ABCB1 inhibitor zosuquidar, the ABCG2 inhibitor Ko143, and the ABCC1 inhibitor MK571 increased calcein retention. Neither protein expression of stem cell markers, nor drug transporters was significantly changed upon FK866 treatment. In conclusion, our data suggest that FK866 inhibits prostate cancer cell proliferation by interference with the energy metabolism, and function of drug efflux transporters.

Omega-3 and Omega-6 polyunsaturated fatty acids stimulate vascular differentiation of mouse embryonic stem cells.

Polyunsaturated fatty acids (PUFAs) and their metabolites may influence cell fate regulation. Herein, we investigated the effects of linoleic acid (LA) as ω-6 PUFA, eicosapentaenoic acid (EPA) as ω-3 PUFA and palmitic acid (PA) on vasculogenesis of embryonic stem (ES) cells. LA and EPA increased vascular structure formation and protein expression of the endothelial-specific markers fetal liver kinase-1, CD31 as well as VE-cadherin, whereas PA was without effect. LA and EPA increased reactive oxygen species (ROS) and nitric oxide (NO), activated endothelial NO synthase (eNOS) and raised intracellular calcium. The calcium response was inhibited by the intracellular calcium chelator BAPTA, sulfo-N-succinimidyl oleate which is an antagonist of CD36, the scavenger receptor for fatty acid uptake as well as by a CD36 blocking antibody. Prevention of ROS generation by radical scavengers or the NADPH oxidase inhibitor VAS2870 and inhibition of eNOS by L-NAME blunted vasculogenesis. PUFAs stimulated AMP activated protein kinase-α (AMPK-α) as well as peroxisome proliferator-activated receptor-α (PPAR-α). AMPK activation was abolished by calcium chelation as well as inhibition of ROS and NO generation. Moreover, PUFA-induced vasculogenesis was blunted by the PPAR-α inhibitor GW6471. In conclusion, ω-3 and ω-6 PUFAs stimulate vascular differentiation of ES cells via mechanisms involving calcium, ROS and NO, which regulate function of the energy sensors AMPK and PPAR-α and determine the metabolic signature of vascular cell differentiation.

Longitudinal metabolic profiling of cardiomyocytes derived from human-induced pluripotent stem cells.

Human-induced pluripotent stem cells (h-iPSCs) are a unique in vitro model for cardiovascular research. To realize the potential applications of h-iPSCs-derived cardiomyocytes (CMs) for drug testing or regenerative medicine and disease modeling, characterization of the metabolic features is critical. Here, we show the transcriptional profile during stages of cardiomyogenesis of h-iPSCs-derived CMs. CM differentiation was not only characterized by the expression of mature structural components (MLC2v, MYH7) but also accompanied by a significant increase in mature metabolic gene expression and activity. Our data revealed a distinct substrate switch from glucose to fatty acids utilization for ATP production. Basal respiration and respiratory capacity in 9 days h-iPSCs-derived CMs were glycolysis-dependent with a shift towards a more oxidative metabolic phenotype at 14 and 28 day old CMs. Furthermore, mitochondrial analysis characterized the early and mature forms of mitochondria during cardiomyogenesis. These results suggest that changes in cellular metabolic phenotype are accompanied by increased O2 consumption and ATP synthesis to fulfill the metabolic needs of mature CMs activity. To further determine functionality, the physiological response of h-iPSCs-derived CMs to ß-adrenergic stimulation was tested. These data provide a unique in vitro human heart model for the understanding of CM physiology and metabolic function which may provide useful insight into metabolic diseases as well as novel therapeutic options.

The milk thistle (Silybum marianum) compound Silibinin stimulates leukopoiesis from mouse embryonic stem cells.

The milk thistle compound Silibinin (i.e., a 1:1 mixture of Silybin A and Silybin B) stimulates vasculogenesis of mouse embryonic stem (ES) cells. Because vasculogenesis and leukopoiesis are interrelated, the effect of Silibinin on leukopoiesis of ES cells was investigated. Treatment of differentiating ES cells with hydrosoluble Silibinin-C-2',3-dihydrogen succinate dose-dependent increased the number of CD18+ , CD45+ , and CD68+ cells, indicating leukocyte/macrophage differentiation. Silibinin treatment activated phosphoinositide 3-kinase (PI3K), AKT (protein kinase B), signal transducer and activator of transcription 3 (STAT3), stimulated hypoxia-induced factor-1α (HIF-1α), and vascular endothelial growth factor receptor 2 (VEGFR2) expression and raised intracellular nitric oxide (NO). Western blot experiments showed that upon coincubation with either the PI3K inhibitor LY294002, the STAT3 inhibitor Stattic, the AKT antagonist AKT inhibitor VIII, or the NO inhibitor L-NAME, the Silibinin-induced expression of CD18, CD45, and CD68 was abolished. Moreover, the stimulation of HIF-1α and VEGFR2 expression was blunted upon STAT3 and PI3K/AKT inhibition. Treatment of differentiating ES cells with L-NAME abolished the stimulation of VEGFR2 and VE-cadherin expression achieved with Silibinin, indicating that NO is involved in vasculogenesis and leukocyte differentiation pathways. In summary, the data of the present study demonstrate that Silibinin stimulates leukocyte differentiation of ES cells, which is associated to vasculogenesis and regulated by PI3K/AKT-, STAT3-, and NO-mediated signaling.

Silibinin from Silybum marianum Stimulates Embryonic Stem Cell Vascular Differentiation via the STAT3/PI3-K/AKT Axis and Nitric Oxide.

Silibinin, the bioactive compound of milk thistle (Silybum marianum), exerts tissue protective and regenerative effects that may include stem cell differentiation toward vascular cells. The purpose of the present study was to investigate whether silibinin stimulates blood vessel formation from mouse embryonic stem (ES) cells and to unravel the underlying signaling cascade. Vascular branching points were assessed by confocal laser scanning microscopy and computer-assisted image analysis of CD31-positive cell structures. Protein expression of vascular markers and activation of protein kinases were determined by western blot. Nitric oxide (NO) generation was investigated by use of the fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. Silibinin dose-dependently increased CD31-positive vascular branching points in embryoid bodies cultivated from ES cells. This was paralleled by increase of protein expression levels for the endothelial-specific markers vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor 2, and hypoxia-inducible factor-1α. Moreover, silibinin increased activation of endothelial nitric oxide synthase (eNOS), which boosted generation of NO in embryoid bodies and enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphoinositide 3-kinase (PI3-K) and AKT. Vasculogenesis, VE-cadherin expression, STAT3 and AKT phosphorylation, NO generation, and eNOS phosphorylation were inhibited by the small molecule STAT3 inhibitor Stattic, AKT inhibitor VIII, the PI3-K inhibitor LY294002, or the NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride. In conclusion, our findings indicate that silibinin induces vasculogenesis of ES cells via activation of STAT3, PI3-K, and AKT, which regulate NO generation by eNOS.

Mechanical strain stimulates vasculogenesis and expression of angiogenesis guidance molecules of embryonic stem cells through elevation of intracellular calcium, reactive oxygen species and nitric oxide generation.

OBJECTIVES: Differentiation of embryonic stem (ES) cells may be regulated by mechanical strain. Herein, signaling molecules underlying mechanical stimulation of vasculogenesis and expression of angiogenesis guidance cues were investigated in ES cell-derived embryoid bodies. METHODS AND RESULTS: Treatment of embryoid bodies with 10% static mechanical strain using a Flexercell strain system significantly increased CD31-positive vascular structures and the angiogenesis guidance molecules plexinB1, ephrin B2, neuropilin1 (NRP1), semaphorin 4D (sem4D) and robo4 as well as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) as evaluated by Western blot and real time RT-PCR. In contrast ephrin type 4 receptor B (EphB4) expression was down-regulated upon mechanical strain, indicating an arterial-type differentiation. Robo1 protein expression was modestly increased with no change in mRNA expression. Mechanical strain increased intracellular calcium as well as reactive oxygen species (ROS) and nitric oxide (NO). Mechanical strain-induced vasculogenesis was abolished by the NOS inhibitor L-NAME, the NADPH oxidase inhibitor VAS2870, upon chelation of intracellular calcium by BAPTA as well as upon siRNA inactivation of ephrin B2, NRP1 and robo4. BAPTA blunted the strain-induced expression of angiogenic growth factors, the increase in NO and ROS as well as the expression of NRP1, sem4D and plexinB1, whereas ephrin B2, EphB4 as well as robo1 and robo4 expression were not impaired. CONCLUSIONS: Mechanical strain stimulates vasculogenesis of ES cells by the intracellular messengers ROS, NO and calcium as well as by upregulation of angiogenesis guidance molecules and the angiogenic growth factors VEGF, FGF-2 and PDGF-BB.

Embryonic Stem Cells for Tissue Biocompatibility, Angiogenesis, and Inflammation Testing.

AIM: To introduce embryoid bodies derived from mouse embryonic stem (ES) cells, which differentiate blood vessel-like structures and leukocytes, as a novel in vitro model system for biocompatibility, inflammation, and angiogenesis studies. METHODOLOGY/RESULTS: Punched spherical discs of bioabsorbable polymers (ε-caprolactone and L-lactide in different compositions) with a diameter of 2 mm and a thickness of 0.2 mm were inoculated with embryoid bodies for cocultivation. As reference material for biocompatible, nonbioabsorbable, and bioincompatible materials, polymer punched discs of petriPERM (PP) membrane (polytetrafluoroethylene) as well as polyvinylchloride (PVC) were used. Tissue outgrowth on the polymer discs decreased and cell toxicity increased upon confrontation on bioabsorbable biomaterials and PVC. Bioabsorbable polymers as well as PVC decreased the branching points and total tube length of CD31-positive vascular structures in embryoid bodies. With the exception of PP, all applied materials increased the differentiation of CD68-positive macrophages and the generation of reactive oxygen species, which is indicative of proinflammatory processes upon contact of tissue with biomaterials. Consequently, cocultivation with polymers increased secretion of the cytokines interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α. CONCLUSION: Three-dimensional tissues cultivated from ES cells are well-suited for testing the biocompatibility, the vascular response, and the inflammatory reaction towards bioabsorbable and nonbioabsorbable polymers.

Involvement of phosphoinositide 3-kinase class IA (PI3K 110α) and NADPH oxidase 1 (NOX1) in regulation of vascular differentiation induced by vascular endothelial growth factor (VEGF) in mouse embryonic stem cells.

The impact of reactive oxygen species and phosphoinositide 3-kinase (PI3K) in differentiating embryonic stem (ES) cells is largely unknown. Here, we show that the silencing of the PI3K catalytic subunit p110α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) by short hairpin RNA or pharmacological inhibition of NOX and ras-related C3 botulinum toxin substrate 1 (Rac1) abolishes superoxide production by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES-cell-derived fetal liver kinase-1(+) (Flk-1(+)) vascular progenitor cells, whereas the mitochondrial complex I inhibitor rotenone does not have an effect. Silencing p110α or inhibiting Rac1 arrests vasculogenesis at initial stages in embryoid bodies, even under VEGF treatment, as indicated by platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive areas and branching points. In the absence of p110α, tube-like structure formation on matrigel and cell migration of Flk-1(+) cells in scratch migration assays are totally impaired. Silencing NOX1 causes a reduction in PECAM-1-positive areas, branching points, cell migration and tube length upon VEGF treatment, despite the expression of vascular differentiation markers. Interestingly, silencing p110α but not NOX1 inhibits the activation of Rac1, Ras homologue gene family member A (RhoA) and Akt leading to the abrogation of VEGF-induced lamellipodia structure formation. Thus, our data demonstrate that the PI3K p110α-Akt/Rac1 and NOX1 signalling pathways play a pivotal role in VEGF-induced vascular differentiation and cell migration. Rac1, RhoA and Akt phosphorylation occur downstream of PI3K and upstream of NOX1 underscoring a role of PI3K p110α in the regulation of cell polarity and migration.

Impact of Arachidonic Acid and the Leukotriene Signaling Pathway on Vasculogenesis of Mouse Embryonic Stem Cells.

Embryonic stem (ES) cells can differentiate into various kinds of cells, such as endothelial and hematopoietic cells. In addition, some evidence suggests that inflammatory mediators such as leukotrienes (LTs), which include the 5-lipoxygenase (LOX) family, can regulate endothelial cell differentiation. In the present study, the eicosanoid precursor arachidonic acid (AA) stimulated vasculogenesis of ES cells by increasing the number of fetal liver kinase-1+ vascular progenitor cells as well as vascular structures positive for platelet endothelial cell adhesion protein-1 and vascular endothelial cadherin. The stimulation of vasculogenesis and expression of the rate-limiting enzyme in the LT signaling pathway, 5-LOX-activating protein (FLAP), was blunted upon treatment with the FLAP inhibitors AM643 and REV5901. Vasculogenesis was significantly restored upon exogenous addition of LTs. Downstream of FLAP, the LTB4 receptor (BLT1) blocker U75302, the BLT2 receptor blocker LY255283 as well as the cysteinyl LT blocker BAY-u9773 inhibited vasculogenesis of ES cells. AA treatment of differentiating ES cells increased reactive oxygen species (ROS) generation, which was not affected upon either FLAP or cyclooxygenase-2 inhibition. Prevention of ROS generation by either the free radical scavengers vitamin E and N-(2-mercaptopropionyl)glycine or the NADPH oxidase inhibitor VAS2870 downregulated vasculogenesis of ES cells and blunted the provasculogenic effect of AA. In summary, our data demonstrate that proinflammatory AA stimulates vasculogenesis of ES cells via the LT pathway by mechanisms involving ROS generation.

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP.

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists ß,γ-methylenadenosine 5'-triphosphate (ß,γ-MetATP) and 8-bromoadenosine 5'-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca(2+)](i)) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca(2+)](i) transients induced by ADP/ATP were abolished by the phospholipase C-ß (PLC-ß) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca(2+)](i) transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca(2+)](i) response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca(2+)](i) transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

ß-Adrenergic receptor antagonists inhibit vasculogenesis of embryonic stem cells by downregulation of nitric oxide generation and interference with VEGF signalling.

The ß-adrenoceptor antagonist Propranolol has been successfully used to treat infantile hemangioma. However, its mechanism of action is so far unknown. The hypothesis of this research was that ß-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific ß-adrenergic receptor (ß-adrenoceptor) antagonist Propranolol, the ß1-adrenoceptor-specific antagonist Atenolol and the ß2-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three ß-blockers dose-dependently downregulated formation of capillary structures in ES cell-derived embryoid bodies and decreased the expression of the vascular cell markers CD31 and VE-cadherin. Furthermore, ß-blockers downregulated the expression of fibroblast growth factor-2 (FGF-2), hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor 165 (VEGF165), VEGF receptor 2 (VEGF-R2) and phospho VEGF-R2, as well as neuropilin 1 (NRP1) and plexin-B1 which are essential modulators of embryonic angiogenesis with additional roles in vessel remodelling and arteriogenesis. Under conditions of ß-adrenoceptor inhibition, the endogenous generation of nitric oxide (NO) as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) was decreased in embryoid bodies, whereas an increase in NO generation was observed with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Consequently, vasculogenesis of ES cells was restored upon treatment of differentiating ES cells with ß-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that ß-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.

VEGF-mediated PI3K class IA and PKC signaling in cardiomyogenesis and vasculogenesis of mouse embryonic stem cells.

VEGF-, phosphoinositide 3-kinase (PI3K)- and protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in mouse ES cells and in ES cell-derived Flk-1⁺ cardiovascular progenitor cells. Inhibition of PI3K by wortmannin and LY294002, disruption of PI3K catalytic subunits p110α and p110δ using short hairpin RNA (shRNA), or inhibition of p110α with compound 15e and of p110δ with IC-87114 impaired cardiac and vascular differentiation. By contrast, TGX-221, an inhibitor of p110ß, and shRNA knockdown of p110ß were without significant effects. Antagonists of the PKC family, i.e. bisindolylmaleimide-1 (BIM-1), GÖ 6976 (targeting PKCα/ßII) and rottlerin (targeting PKCδ) abolished vasculogenesis, but not cardiomyogenesis. Inhibition of Akt blunted cardiac as well as vascular differentiation. VEGF induced phosphorylation of PKCα/ßII and PKCδ but not PKCζ. This was abolished by PI3K inhibitors and the VEGFR-2 antagonist SU5614. Furthermore, phosphorylation of Akt and phosphoinositide-dependent kinase-1 (PDK1) was blunted upon inhibition of PI3K, but not upon inhibition of PKC by BIM-1, suggesting that activation of Akt and PDK1 by VEGF required PI3K but not PKC. In summary, we demonstrate that PI3K catalytic subunits p110α and p110δ are central to cardiovasculogenesis of ES cells. Akt downstream of PI3K is involved in both cardiomyogenesis and vasculogenesis, whereas PKC is involved only in vasculogenesis.

Generation of a Syngeneic Heterozygous ACVRL1(wt/mut) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis.

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

Induction of Stem-Cell-Derived Cardiomyogenesis by Fibroblast Growth Factor 10 (FGF10) and Its Interplay with Cardiotrophin-1 (CT-1).

For heart regeneration purposes, embryonic stem cell (ES)-based strategies have been developed to induce the proliferation of cardiac progenitor cells towards cardiomyocytes. Fibroblast growth factor 10 (FGF10) contributes to cardiac development and induces cardiomyocyte differentiation in vitro. Yet, among pro-cardiogenic factors, including cardiotrophin-1 (CT-1), the hyperplastic function of FGF10 in cardiomyocyte turnover remains to be further characterized. We investigated the proliferative effects of FGF10 on ES-derived cardiac progenitor cells in the intermediate developmental stage and examined the putative interplay between FGF10 and CT-1 in cardiomyocyte proliferation. Mouse ES cells were treated with FGF10 and/or CT-1. Differential expression of cardiomyocyte-specific gene markers was analyzed at transcript and protein levels. Substantial upregulation of sarcomeric α-actinin was detected by qPCR, flow cytometry, Western blot and immunocytochemistry. FGF10 enhanced the expression of other structural proteins (MLC-2a, MLC-2v and TNNT2), transcriptional factors (NKX2-5 and GATA4), and proliferation markers (Aurora B and YAP-1). FGF10/CT-1 co-administration led to an upregulation of proliferation markers, suggesting the synergistic potential of FGF10 + CT-1 on cardiomyogenesis. In summary, we provided evidence that FGF10 and CT-1 induce cardiomyocyte structural proteins, associated transcription factors, and cardiac cell proliferation, which could be applicable in therapies to replenish damaged cardiomyocytes.

NOS inhibition synchronizes calcium oscillations in human adipose tissue-derived mesenchymal stem cells by increasing gap-junctional coupling.

Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising stem cell source for cell transplantation. We demonstrate that undifferentiated ASCs display robust oscillations of intracellular calcium [Ca(2+) ](i) which may be associated with stem cell maintenance since oscillations were absent in endothelial cell differentiation medium supplemented with FGF-2. [Ca(2+) ](i) oscillations were dependent on extracellular Ca(2+) and Ca(2+) release from intracellular stores since they were abolished in Ca(2+) -free medium and in the presence of the store-depleting agent thapsigargin. They were inhibited by the phospholipase C antagonist U73,122, the inositol 1,4,5-trisphosphate (InsP(3) ) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as by the gap-junction uncouplers 1-heptanol and carbenoxolone, indicating regulation by the InsP(3) pathway and dependence on gap-junctional coupling. Cells endogenously generated nitric oxide (NO), expressed NO synthase 1 (NOS 1) and connexin 43 (Cx 43). The nitric oxide NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), 2-ethyl-2-thiopseudourea, and diphenylene iodonium as well as si-RNA-mediated down-regulation of NOS 1 synchronized [Ca(2+) ](i) oscillations between individual cells, whereas the NO-donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) as well as the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were without effects. The synchronization of [Ca(2+) ](i) oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) revealed increased reflow of fluorescent calcein into the bleached area in the presence of the NOS inhibitors DPI and L-NAME. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca(2+) ](i) oscillations in undifferentiated ASCs by controlling gap-junctional coupling.

Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells.

BACKGROUND: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. METHODS AND RESULTS: We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects. CONCLUSIONS: SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

Mimicry of a constitutively active pre-B cell receptor in acute lymphoblastic leukemia cells.

Pre-B cells undergo apoptosis unless they are rescued by pre-B cell receptor-dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre-B lymphoblastic leukemia bypasses selection for pre-B cell receptor-dependent survival signals. Investigating possible interference of BCR-ABL1 with pre-B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre-B cell receptor engagement. Instead, Bruton's tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre-B cell receptor, including activation of PLCgamma1, autonomous Ca2+ signaling, STAT5-phosphorylation, and up-regulation of BCLX(L). Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1+ leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1-dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre-B cell receptor.

Imaging herpes simplex virus type 1 amplicon vector-mediated gene expression in human glioma spheroids.

Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector-mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP). After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector-mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 µm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of < 150 µm. Guided vector injection into the spheroids showed transduction efficiencies ranging between < 10 and > 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application-injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.

The role of ceramide accumulation in human induced pluripotent stem cell-derived cardiomyocytes on mitochondrial oxidative stress and mitophagy.

Oversupply of fatty acids (FAs) to cardiomyocytes (CMs) is associated with increased ceramide content and elevated the risk of lipotoxic cardiomyopathy. Here we investigate the role of ceramide accumulation on mitochondrial function and mitophagy in cardiac lipotoxicity using CMs derived from human induced pluripotent stem cell (hiPSC). Mature CMs derived from hiPSC exposed to the diabetic-like environment or transfected with plasmids overexpressing serine-palmitoyltransferase long chain base subunit 1 (SPTLC1), a subunit of the serine-palmitoyltransferase (SPT) complex, resulted in increased intracellular ceramide levels. Accumulation of ceramides impaired insulin-dependent phosphorylation of Akt through activating protein phosphatase 2A (PP2A) and disturbed gene and protein levels of key metabolic enzymes including GLUT4, AMPK, PGC-1α, PPARα, CD36, PDK4, and PPARγ compared to controls. Analysis of CMs oxidative metabolism using a Seahorse analyzer showed a significant reduction in ATP synthesis-related O2 consumption, mitochondrial ß-oxidation and respiratory capacity, indicating an impaired mitochondrial function under diabetic-like conditions or SPTLC1-overexpression. Further, ceramide accumulation increased mitochondrial fission regulators such as dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF) as well as auto/mitophagic proteins LC3B and PINK-1 compared to control. Incubation of CMs with the specific SPT inhibitor (myriocin) showed a significant increase in mitochondrial fusion regulators the mitofusin 2 (MFN2) and optic atrophy 1 (OPA1) as well as p-Akt, PGC-1 α, GLUT-4, and ATP production. In addition, a significant decrease in auto/mitophagy and apoptosis was found in CMs treated with myriocin. Our results suggest that ceramide accumulation has important implications in driving insulin resistance, oxidative stress, increased auto/mitophagy, and mitochondrial dysfunction in the setting of lipotoxic cardiomyopathy. Therefore, modulation of the de novo ceramide synthesis pathway may serve as a novel therapeutic target to treat metabolic cardiomyopathy.