Expression of estrogen receptor-alpha and -beta, glucocorticoid receptor, and progesterone receptor genes in human embryonic stem cells and embryoid bodies

Human embryonic stem cells (hESCs) have the potential to differentiate into various cell types, and the three germ layers in vivo and in vitro. They are therefore useful in transplantation and tissue engineering. Here, we describe the expression patterns of selected steroid receptor mRNAs - estrogen receptor-alpha (ER-alpha), ER-beta, glucocorticoid receptor (GR), and progesterone receptor (PR) - in undifferentiated hESCs and embryoid bodies (EBs) cultured for 2, 4, and 6 d, as assessed by real-time PCR, in order to define the possible influence of steroid hormones on the differentiation of hESCs. These receptor mRNAs were expressed in undifferentiated hESCs and EBs. The expression of PR mRNA only decreased during the differentiation of EBs but not of hESCs. Immunohistochemical analysis gave strong staining of ER-alpha, ER-beta, and GR proteins in the nuclei of hESCs and EBs, whereas PR was not detected. We also examined the potential of these steroid hormones to direct the differentiation of hESCs in vitro. The expression of 11 cell-specific markers representing 3 germ layers and 5 tissue types was used to assess the differentiation of hESCs. We found that certain endodermal marker genes were either only expressed in the estrogen-treated group or their expression was stimulated in that group, suggesting that steroid hormones can control the differentiation of hESCs into various cell types.     

Electrical stimulation of human embryonic stem cells: Cardiac differentiation and the generation of reactive oxygen species

Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride-coated titanium, titanium), length of stimulus (1 and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s and for 4-day-old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H₂O₂. Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of troponin T and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS.     

The SHB adapter protein is required for normal maturation of mesoderm during in vitro differentiation of embryonic stem cells

Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.     

Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies

The mammalian reproductive tract is known to contain 1.5–5.3% oxygen (O₂), but human embryonic stem cells (hESCs) derived from preimplantation embryos are typically cultured under 21% O₂ tension. The aim of this study was to investigate the effects of O₂ tension on the long-term culture of hESCs and on cell-fate determination during early differentiation. hESCs and embryoid bodies (EBs) were grown under different O₂ tensions (3, 12, and 21% O₂). The expression of markers associated with pluripotency, embryonic germ layers, and hypoxia was analyzed using RTPCR, immunostaining, and Western blotting. Proliferation, apoptosis, and chromosomal aberrations were examined using BrdU incorporation, caspase-3 immunostaining, and karyotype analysis, respectively. Structural and morphological changes of EBs under different O₂ tensions were comparatively examined using azan- and hematoxylineosin staining, and scanning and transmission electron microscopy. Mild hypoxia (12% O₂) increased the number of cells expressing Oct4/Nanog and reduced BrdU incorporation and aneuploidy. The percentage of cells positive for active caspase-3, which was high during normoxia (21% O₂), gradually decreased when hESCs were continuously cultured under mild hypoxia. EBs subjected to hypoxia (3% O₂) exhibited well-differentiated microvilli on their surface, secreted high levels of collagen, and showed enhanced differentiation into primitive endoderm. These changes were associated with increased expression of Foxa2, Sox17, AFP, and GATA4 on the EB periphery. Our data suggest that mild hypoxia facilitates the slow mitotic division of hESCs in long-term culture and reduces the frequency of chromosomal abnormalities and apoptosis. In addition, hypoxia promotes the differentiation of EBs into extraembryonic endoderm.     

Basic fibroblast growth factor and its receptors in human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent stem cells with long-lasting capacity to self-renew and differentiate into various cell types of endodermal, ectodermal or mesodermal origin. Unlike mouse ESCs (mESCs), which can be maintained in an undifferentiated state simply by adding leukemia inhibitory factor (LIF) into the culture medium, hESCs are notorious for the sustained willingness to differentiate and not yet clearly defined signaling pathways that are crucial for their "stemness". Presently, our knowledge involves only limited number of growth factor signaling pathways that appear to be biologically relevant for stem cell functions in vitro. These include BMP, TGFbeta, Wnt, and FGF signaling pathway. The purpose of this review is to summarize recent data on the expression of FGFs and their receptors in hESCs, and critically evaluate the potential effects of FGF signals for their undifferentiated growth and/or differentiation in context with our current understanding of FGF/FGFR biology.     

Regulatory roles of ganglioside GQ1b in neuronal cell differentiation of mouse embryonic stem cells

Gangliosides play an important role in neuronal differentiation processes. The regulation of ganglioside levels is related to the induction of neuronal cell differentiation. In this study, the ST8Sia5 gene was transfected into mESCs and then differentiated into neuronal cells. Interestingly, ST8Sia5 gene transfected mESCs expressed GQ1b by HPTLC and immunofluorescence analysis. To investigate the effects of GQ1b over-expression in neurogenesis, neuronal cells were differentiated from GQ1b expressing mESCs in the presence of retinoic acid. In GQ1b expressing mESCs, increased EBs formation was observed. After 4 days, EBs were co-localized with GQ1b and nestin, and GFAP. Moreover, GQ1b co-localized with MAP-2 expressing cells in GQ1b expressing mESCs in 7-day-old EBs. Furthermore, GQ1b expressing mESCs increased the ERK1/2 MAP kinase pathway. These results suggest that the ST8Sia5 gene increases ganglioside GQ1b and improves neuronal differentiation via the ERK1/2 MAP kinase pathway.     

Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm

Nodal, a member of the TGF-β family of signaling molecules, has been implicated in pluripotency in human embryonic stem cells (hESCs) [Vallier, L., Reynolds, D., Pedersen, R.A., 2004a. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev. Biol. 275, 403-421], a finding that seems paradoxical given Nodal's central role in mesoderm/endoderm specification during gastrulation. In this study, we sought to clarify the role of Nodal signaling during hESC differentiation by constitutive overexpression of the endogenous Nodal inhibitors Lefty2 (Lefty) and truncated Cerberus (Cerb-S) and by pharmacological interference using the Nodal receptor antagonist SB431542. Compared to wildtype (WT) controls, embryoid bodies (EBs) derived from either Lefty or Cerb-S overexpressing hESCs showed increased expression of neuroectoderm markers Sox1, Sox3, and Nestin. Conversely, they were negative for a definitive endoderm marker (Sox17) and did not generate beating cardiomyocyte structures in conditions that allowed mesendoderm differentiation from WT hESCs. EBs derived from either Lefty or Cerb-S expressing hESCs also contained a greater abundance of neural rosette structures as compared to controls. Differentiating EBs derived from Lefty expressing hESCs generated a dense network of β-tubulin III positive neurites, and when Lefty expressing hESCs were grown as a monolayer and allowed to differentiate, they generated significantly higher numbers of β-tubulin positive neurons as compared to wildtype hESCs. SB431542 treatments reproduced the neuralising effects of Lefty overexpression in hESCs. These results show that inhibition of Nodal signaling promotes neuronal specification, indicating a role for this pathway in controlling early neural development of pluripotent cells.     

Differentiation of mouse embryonic stem cells into dental epithelial-like cells induced by ameloblasts serum-free conditioned medium

Embryonic stem cells (ESCs) possess an intrinsic self-renewal ability and can differentiate into numerous types of functional tissue cells; however, whether ESCs can differentiate toward the odontogenic lineage is still unknown. In this study, we developed an efficient culture strategy to induce the differentiation of murine ESCs (mESCs) into dental epithelial cells. By culturing mESCs in ameloblasts serum-free conditioned medium (ASF-CM), we could induce their differentiation toward dental epithelial cell lineages; however, similar experiments with the tooth germ cell-conditioned medium (TGC-CM) did not yield effective results. After culturing the cells for 14 days in the differentiation-inducing media, the expression of ameloblast-specific proteins such as cytokeratin (CK)14, ameloblastin (AMBN), and amelogenin (AMGN) was markedly higher in mESCs obtained with embryoid body (EB) formation than in mESCs obtained without EB formation. We observed that immunocompromised mice implanted with induced murine EBs (mEBs) showed tissue regenerative capacity and produced odontogenic epithelial-like structures, whereas those implanted with mSCE monolayer cells mainly formed connective tissues. Thus, for the first time, we report that ASF-CM provides a suitable microenvironment for inducing mESC differentiation along the odontogenic epithelial cell lineage. This result has important implications for tooth tissue engineering.