Hedgehog signaling is required for the differentiation of ES cells into neurectoderm

Mouse embryonic stem cells can differentiate in vitro into cells of the nervous system, neurons and glia. This differentiation mimics stages observed in vivo, including the generation of primitive ectoderm and neurectoderm in embryoid body culture. We demonstrate here that embryonic stem cell lines mutant for components of the Hedgehog signaling cascade are deficient at generating neurectoderm-containing embryoid bodies. The embryoid bodies derived from mutant cells are also unable to respond to retinoic acid treatment by producing nestin-positive neural stem cells, a response observed in cultures of heterozygous cells, and contain cores apparently arrested at the primitive ectoderm stage. The mutant cultures are also deficient in their capacity to differentiate into mature neurons and glia. These data are consistent with a role for Hedgehog signaling in generating neurectoderm capable of producing the appropriate neuronal and glial progenitors in ES cell culture.     

Trophoblast differentiation defect in human embryonic stem cells lacking PIG-A and GPI-anchored cell-surface proteins

Pluripotent human embryonic stem (hES) cells can differentiate into various cell types derived from the three embryonic germ layers and extraembryonic tissues such as trophoblasts. The mechanisms governing lineage choices of hES cells are largely unknown. Here, we report that we established two independent hES cell clones lacking a group of cell surface molecules, glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs). The GPI-AP deficiency in these two hES clones is due to the deficiency in the gene expression of PIG-A (phosphatidyl-inositol-glycan class A), which is required for the first step of GPI synthesis. GPI-AP-deficient hES cells were capable of forming embryoid bodies and initiating cell differentiation into the three embryonic germ layers. However, GPI-AP-deficient hES cells failed to form trophoblasts after differentiation induction by embryoid body formation or by adding exogenous BMP4. The defect in trophoblast formation was due to the lack of GPI-anchored BMP coreceptors, resulting in the impairment of full BMP4 signaling activation in the GPI-AP-deficient hES cells. These data reveal that GPI-AP-enhanced full activation of BMP signaling is required for human trophoblast formation.     

Protein kinases abundantly expressed in undifferentiated human ESC lines and derived embryoid bodies

The ability of human embryonic stem cells (ESCs) to unlimited proliferation and huge differentiation potential makes them very attractive tool both for basic research and biological medicine. There are still little known about mechanisms that govern their differentiation or keep them in a pluripotency state. A variety of signaling events determines gene expression profiles responsible for such mechanisms activation. Protein kinases are key components of the signaling cascades. The knowledge about protein kinases expression profile in undifferentiated ESCs and embryoid bodies (EBs) will allow to understand early differentiation events. We constructed cDNA libraries containing fragments of protein kinases catalytic domain that were expressed in undifferentiated cells or EB of hESM01, hESM02 cell lines. We detected high level of MAK-V expression using Northern-blot hybridization. Semi-quantitative RT-PCR was used to compare the level of abundantly expressed kinases MAK-V, A-RAF-1, MARK3, IGF1R, NEK3 and NEK7 in undifferentiated ESCs or derived EBs.     

Hedgehog is required for murine yolk sac angiogenesis.

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh(-/-) mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and alpha-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh(-/-) lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or SMO: Whereas Ihh(-/-) yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo(-/-) yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh(-/-) yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.     

In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.     

The weakest link: A new paradigm for stabilizing the integrin–actin connection

Human embryonic stem cells (hESCs) are to be considered as a valuable source for regenerative medicine because of their capacity to differentiate into all cell types. We have developed an efficient culture system to differentiate hECSs into endothelial cells without the formation of embryoid bodies Establishing appropriate culture conditions with a cocktail of growth factors allowed us to differentiate hESCs directly to endothelial primary culture with about 50% efficiency. CD31 immunomagnetic cell sorting was used to purify derived endothelium from the primary culture of hESCs. Isolated endothelial cells expressed immunological markers (vWF, CD105), specific genes (VE-cadherin, KDR, GATA-2, GATA-3, eNOS), and formed cord-like structures on collagen matrix and in Matrigel assay. During differentiation to endothelial lineage promoter regions of the genes involved in specific cell fate determination and homeostasis (GATA-2,-3, and eNOS) underwent intensive hypomethylation which correlated with the gene expression. Overall our data demonstrate that direct differentiation of hESCs leads to endothelial cells that acquire epigenetic patterning similar to the functional endothelial cells of the organism.     

Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies

Retinoid signaling has been implicated in embryonic stem cell differentiation. Here we present a systematic analysis of gene expression changes in mouse embryonic stem cells (mESCs), during their spontaneous differentiation into embryoid bodies and the effect of all-trans retinoic acid (ATRA) on this process. We show that retinoic acid is present in the serum and is sufficient to activate retinoid signaling at a basal level in undifferentiated mESCs. This signal disappears during embryoid body formation. However exogenously added ATRA resets the spontaneous differentiation programs in embryoid bodies and initiates a distinct genetic program. These data suggest that retinoid signaling not only promotes a particular pathway but also acts as a context dependent general coordinator of the differentiation states in embryonic stem cells.     

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.     

Neural differentiation of murine embryonic stem cells ES

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.     

Role of Sonic hedgehog signaling and the expression of its components in human embryonic stem cells

Human embryonic stem cells (hESC) are characterized by their ability to self-renew and differentiate into all cell types of the body, making them a valuable resource for regenerative medicine. Yet, the molecular mechanisms by which hESC retain their capacity for self-renewal and differentiation remain unclear. The Hedgehog signaling pathway plays a pivotal role in organogenesis and differentiation during development, and is also involved in the proliferation and cell-fate specification of neural stem cells and neural crest stem cells. As there has been no detailed study of the Sonic hedgehog (SHH) signaling pathway in hESC, this study examines the expression and functional role of SHH during hESC self-renewal and differentiation. Here, we show the gene and protein expression of key components of the SHH signaling pathway in hESC and differentiated embryoid bodies. Despite the presence of functioning pathway components, SHH plays a minimal role in maintaining pluripotency and regulating proliferation of undifferentiated hESC. However, during differentiation with retinoic acid, a GLI-responsive luciferase assay and target genes PTCH1 and GLI1 expression reveal that the SHH signaling pathway is highly activated. Besides, addition of exogenous SHH to hESC differentiated as embryoid bodies increases the expression of neuroectodermal markers Nestin, SOX1, MAP2, MSI1, and MSX1, suggesting that SHH signaling is important during hESC differentiation toward the neuroectodermal lineage. Our findings provide a new insight in understanding the SHH signaling in hESC and the further development of hESC differentiation for regenerative medicine.     

Protein kinases predominately expressed in human ES cell lines during differentiation

Capacity of human embryonic stem cells (ESC) for unlimited proliferation and differentiation make them an attractive object in fundamental science and medicine. Little is known about the mechanisms that direct cells to particular differentiation or sustain them in an undifferentiated state. Activation of these mechanisms is determined by gene expression mediated by cascades of signal transduction. Protein kinases are essential components of signal pathways. The study of protein kinases expression in ESC and embryoid bodies facilitates a better understanding of the processes underlying the differentiation stages. We isolated cDNA libraries with fragments of catalytic domains of protein kinases expressed in human ESC and embryoid bodies (EB) of hESM01 and hESM02 cell lines. Using Northern hybridization, we revealed a high level of protein kinases MAK-V in human ESC. Expressions of MAK-V, A-RAF-1, MAPK3, IGF1R, NEK3, and NEK7 in ESC and EB in hESM01 and hESM02 cell lines were compared by the semiquantitative method RT-PCR.     

Potentiating the Efficacy of Molecular Targeted Therapy for Hepatocellular Carcinoma by Inhibiting the Insulin-Like Growth Factor Pathway

Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.     

Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.     

Murine embryonic stem cell in vitro differentiation: applications to the study of vascular development

The present review summarizes knowledge accumulated during the last decade concerning in vitro endothelial differentiation from embryonic stem (ES) cells. There is now growing evidence that ES cells may provide a powerful model system to determine the cellular and molecular mechanisms of vascular development. ES cells differentiate into the endothelial lineage by successive maturation steps recapitulating in vivo events observed in the embryo. Further maturation of ES-derived embryoid bodies either in three dimensional gels or in confrontation cultures with tumor spheroids can also provide a model of physiological or tumoral angiogenesis. The data obtained from experimental in vitro differentiation of genetically modified mouse ES cells highlight the potential and the complementarity of this model system to in vivo gene knock out studies. We also consider and discuss some of the potential applications of ES cell technology in vascular biology for future directions in basic research and medicine, by manipulation of differentiation and the generation of cell populations for analysis and transplantation for therapeutic use.     

Building blood vessels--stem cell models in vascular biology

Spheroids of differentiating embryonic stem cells, denoted embryoid bodies, constitute a high-quality model for vascular development, particularly well suited for loss-of-function analysis of genes required for early embryogenesis. This review examines vasculogenesis and angiogenesis in murine embryoid bodies and discusses the promise of stem cell-based models for the study of human vascular development.     

The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF)

LIF (leukaemia inhibitory factor) is commonly used to maintain mouse embryonic stem cells in an undifferentiated state. These cells spontaneously differentiate when allowed to aggregate in the absence of LIF, forming embryoid bodies in which early embryonic cell lineages develop. Using embryoid bodies cultured in the presence and absence of LIF, we show that although LIF inhibited the development of visceral and parietal endodermal cells, it did not affect the differentiation of the primitive endodermal cell precursors of these extraembryonic cell lineages. Furthermore, deposition of the basement membrane produced by the primitive endodermal cells, which separates them from the remaining cells of the embryoid body, still occurred. The differentiation of primitive ectodermal cells and their progeny was inhibited by LIF, as evidenced by the lack of expression of FGF-5, muscle, and neuronal markers. However, cavitation of the embryoid body and maintenance of the cells in contact with the primitive endodermal basement membrane as an epiblast epithelium still occurred normally in the presence of LIF. These results indicate that cavitation and formation of the epiblast epithelium are regulated by mechanisms distinct from those controlling the differentiation of epiblast cell lineages. Furthermore, although epithelium formation and cavitation do not require the differentiation of visceral endodermal cells, the results are consistent with the hypothesis that the primitive endodermal basement membrane is sufficient to induce the epithelialization of undifferentiated embryonic stem cells necessary for cavitation.     

Hematopoietic differentiation from human ESCs as a model for developmental studies and future clinical translations. Invited review following the FEBS Anniversary Prize received on 5 July 2009 at the 34th FEBS Congress in Prague

Human embryonic stem cells (hESCs) and induced pluripotent stem cells are excellent models for the study of embryonic hematopoiesis in vitro, aiding the design of new differentiation models that may be applicable to cell-replacement therapies. Adult and fetal hematopoietic stem cells are currently being used in biomedical applications; however, the latest advances in regenerative medicine and stem cell biology suggest that hESC-derived hematopoietic stem cells are an outstanding tool for enhancing immunotherapy and treatments for blood disorders and cancer, for example. In this review, we compare various methods used for inducing in vitro hematopoietic differentiation from hESCs, based on co-culture with stromal cells or formation of embryoid bodies, and analyse their ability to give rise to hematopoietic precursors, with emphasis on their engraftment potential as a measure of their functionality in vivo.