Embryonic Stem Cell-derived Embryoid Bodies Research Articles

Recreating Tissue Structures Representative of Teratomas In Vitro Using a Combination of 3D Cell Culture Technology and Human Embryonic Stem Cells.

In vitro studies using human embryonic stem cells (hESCs) are a valuable method to study aspects of embryogenesis, avoiding ethical issues when using embryonic materials and species dissimilarities. The xenograft teratoma assay is often traditionally used to establish pluripotency in putative PSC populations, but also has additional applications, including the study of tissue differentiation. The stem cell field has long sought an alternative due to various well-established issues with the in vivo technique, including significant protocol variability and animal usage. We have established a two-step culture method which combines PSC-derived embryoid bodies (EBs) with porous scaffolds to enhance their viability, prolonging the time these structures can be maintained, and therefore, permitting more complex, mature differentiation. Here, we have utilised human embryonic stem cell-derived EBs, demonstrating the formation of tissue rudiments of increasing complexity over time and the ability to manipulate their differentiation through the application of exogenous morphogens to achieve specific lineages. Crucially, these EB-derived tissues are highly reminiscent of xenograft teratoma samples derived from the same cell line. We believe this in vitro approach represents a reproducible, animal-free alternative to the teratoma assay, which can be used to study human tissue development.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.

Spatial protein analysis in developing tissues: a sampling-based image processing approach.

Advances in fluorescence microscopy approaches have made it relatively easy to generate multi-dimensional image volumes and have highlighted the need for flexible image analysis tools for the extraction of quantitative information from such data. Here we demonstrate that by focusing on simplified feature-based nuclear segmentation and probabilistic cytoplasmic detection we can create a tool that is able to extract geometry-based information from diverse mammalian tissue images. Our open-source image analysis platform, called ‘SilentMark’, can cope with three-dimensional noisy images and with crowded fields of cells to quantify signal intensity in different cellular compartments. Additionally, it provides tissue geometry related information, which allows one to quantify protein distribution with respect to marked regions of interest. The lightweight SilentMark algorithms have the advantage of not requiring multiple processors, graphics cards or training datasets and can be run even with just several hundred megabytes of memory. This makes it possible to use the method as a Web application, effectively eliminating setup hurdles and compatibility issues with operating systems. We test this platform on mouse pre-implantation embryos, embryonic stem cell-derived embryoid bodies and mouse embryonic heart, and relate protein localization to tissue geometry.This article is part of a discussion meeting issue ‘Contemporary morphogenesis’.

Second-phase validation study of an alternative developmental toxicity test using mouse embryonic stem cell-derived embryoid bodies.

The embryoid body test (EBT) is a developmental toxicity test method that measures the size of embryoid bodies (EBs) and the viability of mouse embryonic stem cells (mESCs) and fibroblasts (3T3 cells). The previous pre-validation study confirmed the high accuracy (above 80%) of EBT using 26 coded test chemicals. This second-phase validation study assessed the inter-laboratory reproducibility (5 chemicals in common) and predictive capacity (10 chemicals in each laboratory) test using the coded test chemicals at three laboratories. For the prediction model, the accuracy is increased when more data is accumulated. Therefore, we updated the prediction model and analyzed the results of the second year with the newly created-prediction model. Statistical analysis of the inter-laboratory reproducibility test results indicated that accuracy, sensitivity, and specificity were 87%, 78%, and 100%, respectively. The results of the statistical analysis of the predictive capacity test showed an accuracy of 80%, sensitivity of 78%, and specificity of 81%. In conclusion, the EBT can accurately classify various embryotoxicants within a short period and with relatively little effort. Therefore, EBT can be used as a good way to test developmental toxicity.

Pre-validation study of alternative developmental toxicity test using mouse embryonic stem cell-derived embryoid bodies

The embryoid body test (EBT) is a developmental toxicity test method that assesses the half inhibitory concentrations of substances in the area of embryoid bodies (EBs), and in the viability of mouse embryonic stem cells (ESCs) and fibroblasts (3T3 cells) following chemical exposure for three and four days, respectively. In the previous study, the EBT showed more advanced than the embryonic stem cell test (EST) from the European Centre for the Validation of Alternative Methods (ECVAM) applying cardiac differentiation of mouse ESCs, because the EBT greatly reduced the exposure time, labor, and amount of materials required, and misclassification of embryotoxic potential. This pre-validation study evaluated the predictive accuracy of the EBT using 26 coded test substances by two steps: intra-laboratory and inter-laboratory reproducibility tests. Since some substances have different embryotoxic potentials at different pregnancy periods, in this study, a new prediction model consisting of non-toxic and toxic classes was used, instead of the existing prediction model assessing embryotoxicants in four classes. The results of the intra- and inter-laboratory tests were highly accurate (above 80%) when substances were classified using the predictive model. In conclusion, EBT can accurately classify various embryotoxicants in a short time with less effort and greater validation.

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

ObjectivesDifferentiation 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 resultsTreatment 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. ConclusionsMechanical 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.

Differential effects of high and low strength magnetic fields on mouse embryonic development and vasculogenesis of embryonic stem cells.

Man-made magnetic fields (MFs) may exert adverse effects on mammalian embryonic development. Herein, we analysed the effect of 10mT 50Hz sinusoidal (AC) or static (DC) MFs versus 1mT MFs on embryonic development of mice. Exposure for 20days during gestation to 10mT MFs increased resorptions and dead fetuses, decreased crown-rump length and fresh weight, reduced blood vessel differentiation and caused histological changes, accompanied with diminished vascular endothelial growth factor (VEGF) protein expression in several organs. In embryonic stem (ES) cell-derived embryoid bodies exposure towards 10mT MFs increased reactive oxygen species (ROS), decreased vascular marker as well as VEGF expression and enhanced apoptosis. In conclusion, our combined data from in vivo and in vitro experiments identified VEGF as an important mediator during embryonic development that can be influenced by high strength MFs, which in consequence leads to severe abnormalities in fetus organs and blood vessel formation.

Fibronectin modified expression of Sonic hedgehog in ATRA-mediated neuronal differentiation.

In this study, the effect of fibronectin on the neurite outgrowth from embryoid bodies (EBs) in neurodifferentiated embryonal carcinoma P19 cells was examined. The neurite outgrowth on fibronectin was maintained for a longer time in comparison with those on collagen or laminin. Quantitative RT-PCR revealed that mRNA level corresponding to sonic hedgehog (Shh) in neurodifferentiated P19 cells was upregulated on fibronectin, whereas collagen or laminin did not affect. Further knockdown of integrin αv subunit in P19 cells demonstrated that expression of Shh was mediated through interaction between fibronectin and integrin. Additionally, exogenous Shh agonist accelerated neurite outgrowth from embryonic stem cell-derived EBs without large change of neuronal phenotype expression. Taken together, fibronectin could maintain neurite outgrowth via increased Shh expression.

PTEN induces apoptosis and cavitation via HIF-2-dependent Bnip3 upregulation during epithelial lumen formation.

The tumor suppressor phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and antagonizes the prosurvival PI3K-Akt pathway. Targeted deletion of PTEN in mice led to early embryonic lethality. To elucidate its role in embryonic epithelial morphogenesis and the underlying mechanisms, we used embryonic stem cell-derived embryoid body (EB), an epithelial cyst structurally similar to the periimplantation embryo. PTEN is upregulated during EB morphogenesis in parallel with apoptosis of core cells, which mediates EB cavitation. Genetic ablation of PTEN causes Akt overactivation, apoptosis resistance and cavitation blockade. However, rescue experiments using mutant PTEN and pharmacological inhibition of Akt suggest that the phosphatase activity of PTEN and Akt are not involved in apoptosis-mediated cavitation. Instead, hypoxia-induced upregulation of Bnip3, a proapoptotic BH3-only protein, mediates PTEN-dependent apoptosis and cavitation. PTEN inactivation inhibits hypoxia- and reactive oxygen species-induced Bnip3 elevation. Overexpression of Bnip3 in PTEN-null EBs rescues apoptosis of the core cells. Mechanistically, suppression of Bnip3 following PTEN loss is likely due to reduction of hypoxia-inducible factor-2α (HIF-2α) because forced expression of an oxygen-stable HIF-2α mutant rescues Bnip3 expression and apoptosis. Lastly, we show that HIF-2α is upregulated by PTEN at both transcriptional and posttranscriptional levels. Ablation of prolyl hydroxylase domain-containing protein 2 (PHD2) in normal EBs or inhibition of PHD activities in PTEN-null EBs stabilizes HIF-2α and induces Bnip3 and caspase-3 activation. Altogether, these results suggest that PTEN is required for apoptosis-mediated cavitation during epithelial morphogenesis by regulating the expression of HIF-2α and Bnip3.

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

The β-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 β-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific β-adrenergic receptor (β-adrenoceptor) antagonist Propranolol, the β1-adrenoceptor-specific antagonist Atenolol and the β2-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three β-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, β-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 β-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 β-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that β-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.

Bone marrow derived cells with defective c-Myb activity protect against neointimal remodeling following injury

Purpose: We previously showed that the transcription factor c-myb regulates the differentiation of contractile vascular smooth muscle cells in embryonic stem cell-derived embryoid bodies. While we also showed that c-Myb regulates the expansion of a specific embryonic hemangiogenic progenitor, it is unclear if this population has a role in the adult animal. However, the role of c-Myb in the proliferation and potential (trans)differentiation of bone marrow (BM)-derived vs. vessel-resident cells in arterial remodelling in the adult has eluded genetic examination due to the embryonic lethality of c-myb-/- mice from hematopoietic failure. To examine the role of c-myb in BM-derived vs. vessel-resident cells, we have used a mouse harboring a non-lethal point mutation resulting in a hypomorphic (h) allele of c-myb with defective c-Myb activity. Methods and results: Histology of common carotid arteries of 12 wk-old male c-mybh/h and c-mybwt mice was performed 14 days after carotid artery wire denudation injury. Uninjured vessels from wt and h/h mice did not differ in lumen, intima, and media morphometry or I/M ratio. Following wire injury, h/h mice showed reduced intima formation (16,723±2,550 vs. 31,402±49,890 μm2; p<0.05; N=6-7/group) and I/M ratio (0.313±0.044 vs. 0.565±0.089; p<0.01) than wt controls. To determine the relative contribution of BM-derived vs. vessel cells in arterial remodeling, reciprocal BM transplants were performed in 5wk old wt and h/h mice, which were allowed to reconstitute their BM for 7 wks before wire injury. Compared to wt→wt mice, injured arteries from h/h→wt mice had decreased intima formation (19,222±2,622 vs. 8,080±1,337 μm2; p<0.0001; N= 7/group) and I/M ratio (0.491±0.068 vs. 0.217±0.01; p<0.0001). However, injured arteries from wt→h/h mice had no differences in intima formation as compared to h/h→h/h mice (14,519±486 vs. 13,437±1,175 μm2; p=NS; N= 7/group), with no differences observed in uninjured BM-transplanted control arteries. Conclusions: The blunted arterial remodelling response observed in h/h→wt mice is due to a c-Myb dependent defect in BM-derived cell populations that participate in the pathogenesis of neointimal proliferation following arterial injury.

Activation of the epidermal growth factor receptor promotes lymphangiogenesis in the skin

BackgroundThe lymphatic vascular system regulates tissue fluid homeostasis and plays important roles in immune surveillance, inflammation and cancer metastasis. However, the molecular mechanisms involved in the regulation of lymphangiogenesis remain incompletely characterized. ObjectiveWe aimed to identify new pathways involved in the promotion of skin lymphangiogenesis. MethodsWe used a mouse embryonic stem cell-derived embryoid body vascular differentiation assay to investigate the effects of a selection of pharmacological agents with the potential to inhibit blood and/or lymphatic vessel formation. We also used a subcutaneous Matrigel assay to study candidate lymphangiogenesis factors as well as skin-specific transgenic mice. ResultsWe found that compounds inhibiting the epidermal growth factor (EGF) receptor (EGFR) led to an impaired formation of lymphatic vessel-like structures. In vitro studies with human dermal lymphatic endothelial cells (LECs), that were found to express EGFR, revealed that EGF promotes lymphatic vessel formation. This effect was inhibited by EGFR-blocking antibodies and by low molecular weight inhibitors of the EGFR associated tyrosine kinase. Incorporation of EGF into a mouse matrigel plug assay showed that EGF promotes enlargement of lymphatic vessels in the skin in vivo. Moreover, transgenic mice with skin-specific overexpression of amphiregulin, another agonistic ligand of the EGFR, displayed an enhanced size and density of lymphatic vessels in the skin. ConclusionThese findings reveal that EGFR activation is involved in lymphatic remodeling and suggest that specific EGFR antagonists might be used to inhibit pathological lymphangiogenesis.

α2-Macroglobulin Enhances Vasculogenesis/Angiogenesis of Mouse Embryonic Stem Cells by Stimulation of Nitric Oxide Generation and Induction of Fibroblast Growth Factor-2 Expression

α2-macroglobulin (α2M) is an acute-phase protein released upon challenges like cardiac hypertrophy and infarction. α2M signals via the low density lipoprotein receptor-related protein (LRP-1) and may induce stem cell activation. In the present study, the effects of α2M on vasculogenesis/angiogenesis and underlying signaling cascades were investigated in mouse embryonic stem (ES) cells. LRP-1 was expressed in ES cells and upregulated during differentiation. α2M dose dependently increased CD31-positive vascular structures in ES cell-derived embryoid bodies, the early cardiovascular markers isl-1, Nkx-2.5, and flk-1 as well as numbers of VE-cadherin and flk-1-positive cells, but downregulated α-smooth muscle actin. Enhancement of vasculogenesis/angiogenesis by α2M was abolished by the LRP-1 antagonist receptor-associated protein (RAP) and LRP-1 blocking antibody. Notably, α2M stimulated vascular growth in the chicken chorioallantois membrane assay, but not in a human umbilical vein endothelial cell spheroid model. α2M increased fibroblast growth factor-2 (FGF-2) protein expression, which was abolished by RAP, induced nitric oxide (NO) generation as determined by 4,5-diaminofluorescein diacetate microfluorometry, and activated nitric oxide synthase-3 (NOS-3) as well as extracellular-regulated kinase 1,2 (ERK1/2) and phosphatidyl inositol 3-kinase (PI3K). NO generation, the increase in FGF-2 expression, and the stimulation of vasculogenesis/angiogenesis by α2M were blunted by the NO synthase inhibitor L-NAME, the ERK1/2 inhibitor PD98059, and the PI3K inhibitor LY294002. Furthermore, vasculogenesis/angiogenesis by α2M was inhibited in the presence of the FGF receptor 1 antagonist SU5402. In conclusion, α2M stimulates endothelial and early cardiac, but not smooth muscle differentiation of ES cells through generation of NO, activation of ERK1/2 as well as PI3K, and induction of FGF-2 expression.

Simvastatin modulates mouse embryonic stem cell-derived chondrogenesis in vitro

It has been studied in detail that cellular differentiation during chondrogenesis can be recapitulated in vitro by differentiation of embryonic stem (ES) cells as embryoid bodies (EBs). We here used this model system of cartilage development to analyze the effect of simvastatin, a potentially embryotoxic substance. Statins are a group of drugs used to treat hypercholesterolaemia. We found that simvastatin activated cartilage nodule formation during EB differentiation. Extended application of simvastatin resulted in enhanced expression of cartilage marker molecules and prolonged persistence of cartilage nodules. Expression of collagen type II was upregulated during simvastatin-induced chondrogenic ES cell differentiation as demonstrated by quantitative real time PCR. However, immunostaining for cartilage marker molecules revealed that cartilage nodules within simvastatin-treated EBs were defective, bearing cavities of cell loss. Furthermore, caspase activity was reduced in comparison to untreated controls indicating reduced apoptosis. Taken together, we may speculate that simvastatin prolongs survival of chondrocytes and disrupts cellular integrity of cartilage nodules during EB development by affecting apoptotic mechanisms. The study underlines that ES cell-derived EBs are a useful in vitro model to screen substances for their embryotoxic and teratogenic potential.

Synergistic effects of hypoxia and extracellular matrix cues in cardiomyogenesis

Limited characterization of how the stem cell niche evolves has hindered our ability to mimic the physiological environment. In this paper, we hypothesized that hypoxia-induced extracellular matrix (ECM) cues may facilitate cardiomyogenesis. We evaluated the expression of four ECM proteins – fibronectin, collagen I, collagen IV, and laminin - over a period of 20 days in H1 and H9 human embryonic stem cell-derived embryoid bodies (EBs) under hypoxic (5% oxygen) and normoxic (21% oxygen) conditions. Hypoxic EBs exhibited increased collagen I, collagen IV and fibronectin expression relative to normoxic EBs between days 9–13, which coincided with increased expression of mesoderm genes. The effect of ECM cues was confirmed by plating day 9 EBs on collagen IV, gelatin, and fibronectin-rich substrates for 11 days. Hypoxia/gelatin cultures synergistically increased the cardiomyocyte yield by 1.7 and 5.5 fold relative to normoxia/gelatin and normoxia/collagen IV cultures, respectively. Current differentiation protocols may underestimate the contribution of hypoxia and ECM cues that evolve during EB maturation.

Abstract 3334: Antiangiogenic activity of honokiol through inhibition of vascular vessel formation in the mouse embryonic stem cell-derived endothelial cells

Abstract Embryonic stem (ES) cells, which are characterized by pluripotency and self-renewal, have recently been highlighted in several aspects of drug discovery. In particular, the potential of ES cells to differentiate into specific-cell types make them an extremely useful tool in the evaluation of the biological activity of test compounds. In the present study, we employed mouse embryonic stem (mES) cell-derived embryoid bodies (EBs) to evaluate the antiangiogenic activity of natural compounds. Honokiol, a major neolignan derived from the bark of Magnolia obovata Thunberg (Magnoliaceae), was capable of inhibiting differentiation and vascular vessel formation in mES cell models. EBs were formed using hanging drop cultures. The induction of vascular formation was carried out on gelatin-coated plates in EGM-2 medium. Cell proliferation was measured by MTT assay. RT-PCR, immunocytochemistry, and Western blotting were used to measure differentiation of EBs into endothelial cells and vessel formation. The growth inhibition of honokiol in EB-derived endothelial cells was found that honokiol is more sensitive in the differentiated cells (on day 8) compared to the undifferentiated EB-derived endothelial cells (on day 1) with the IC50 values of 6.0 and 17.8 μM, respectively. Honokiol also dramatically decreased the expressions of an endothelial biomarker PECAM in the differentiated EB-derived endothelial cells (on day 11). Honokiol also suppressed the activation of p38 MAPK, Akt, ERK1/2 and SAPK/JNK in EB-derived endothelial cells. The antiangiogenic activity of honokiol is associated with the suppression of PECAM and the MAPK pathway in EB-derived endothelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3334. doi:1538-7445.AM2012-3334

Static magnetic fields increase cardiomyocyte differentiation of Flk-1+ cells derived from mouse embryonic stem cells via Ca2+ influx and ROS production

AimsTo investigate the effects of static magnetic fields (MFs) on cardiomyogenesis of mouse embryonic stem (ES) cell-derived embryoid bodies and Flk-1+ cardiac progenitor cells and to assess the impact of cytosolic calcium [Ca2+]c and reactive oxygen species (ROS). Methods and resultsEmbryoid bodies and ES cell-derived Flk-1+ cardiovascular progenitor cells were exposed to static MFs. The expression of cardiac genes was evaluated by RT-PCR; sarcomeric structures were assessed by immunohistochemistry; intracellular ROS and [Ca2+]c of ES cells were examined by H2DCF-DA- and fluo-4-based microfluorometry. Treatment of embryoid bodies with MFs dose-dependent increased the number of contracting foci and cardiac areas as well as mRNA expression of the cardiac genes MLC2a, MLC2v, α-MHC and β-MHC. In Flk-1+ cells MFs (1mT) elevated both [Ca2+]c and ROS, increased expression of the cardiogenic transcription factors Nkx-2.5 and GATA-4 as well as cardiac genes. This effect was due to Ca2+ influx, since extracellular Ca2+ chelation abrogated ROS production and MF-induced cardiomyogenesis. Furthermore absence of extracellular calcium impaired sarcomere structures. Neither the phospholipase C inhibitor U73122 nor thapsigargin inhibited MF-induced increase in [Ca2+]c excluding involvement of intracellular calcium stores. ROS were generated through NAD(P)H oxidase, since NOX-4 but not NOX-1 and NOX-2 mRNA was upregulated upon MF exposure. Ablation of NOX-4 by sh-RNA and treatment with the NAD(P)H oxidase inhibitor diphenylen iodonium (DPI) totally abolished MF-induced cardiomyogenesis. ConclusionThe ability of static MFs to enhance cardiomyocyte differentiation of ES cells allows high throughput generation of cardiomyocytes without pharmacological or genetic modification.

Promotion of hematopoietic differentiation from mouse induced pluripotent stem cells by transient HoxB4 transduction

Ectopic expression of HoxB4 in embryonic stem (ES) cells leads to an efficient production of hematopoietic cells, including hematopoietic stem/progenitor cells. Previous studies have utilized a constitutive HoxB4 expression system or tetracycline-regulated HoxB4 expression system to induce hematopoietic cells from ES cells. However, these methods cannot be applied therapeutically due to the risk of transgenes being integrated into the host genome. Here, we report the promotion of hematopoietic differentiation from mouse ES cells and induced pluripotent stem (iPS) cells by transient HoxB4 expression using an adenovirus (Ad) vector. Ad vector could mediate efficient HoxB4 expression in ES cell-derived embryoid bodies (ES-EBs) and iPS-EBs, and its expression was decreased during cultivation, showing that Ad vector transduction was transient. A colony-forming assay revealed that the number of hematopoietic progenitor cells with colony-forming potential in HoxB4-transduced cells was significantly increased in comparison with that in non-transduced cells or LacZ-transduced cells. HoxB4-transduced cells also showed more efficient generation of CD41-, CD45-, or Sca-1-positive cells than control cells. These results indicate that transient, but not constitutive, HoxB4 expression is sufficient to augment the hematopoietic differentiation of ES and iPS cells, and that our method would be useful for clinical applications, such as cell transplantation therapy.

A Role for All-Trans-Retinoic Acid in the Early Steps of Lymphatic Vasculature Development

The molecular mechanisms that regulate the earliest steps of lymphatic vascular system development are unknown. To identify regulators of lymphatic competence and commitment, we used an in vitro vascular assay with mouse embryonic stem cell-derived embryoid bodies (EBs). We found that incubation with retinoic acid (RA) and, more potently, with RA in combination with cAMP, induced the expression of the lymphatic competence marker LYVE-1 in the vascular structures of the EBs. This effect was dependent on RA receptor (RAR)-α and protein kinase A signaling. RA-cAMP incubation also promoted the development of CD31+/LYVE-1+/Prox1+ cell clusters. In situ studies revealed that RAR-α is expressed by endothelial cells of the cardinal vein in ED 9.5–11.5 mouse embryos. Timed exposure of mouse and Xenopus embryos to excess of RA upregulated LYVE-1 and VEGFR-3 on embryonic veins and increased formation of Prox1-positive lymphatic progenitors. These findings indicate that RA signaling mediates the earliest steps of lymphatic vasculature development.