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.     

Retinoic acid promotes neural conversion of mouse embryonic stem cells in adherent monoculture

Retinoic acid (RA) plays multiple roles in the nervous system, including induction of neural differentiation, axon outgrowth and neural patterning. Previously, RA for neural differentiation of embryonic stem (ES) cells always relies on embryoid bodies (EBs) formation. Here we report an in vitro adherent monoculture system to induce mouse ES cells into neural cells accompanied with RA. RA (1 μM) treatment, during initial 2 days of differentiation, can enhance the expression of neural markers, such as Nestin, Tuj1 and MAP2, and result in an earlier neural differentiation of ES cells. Furthermore, RA promotes a significant increase in neurite elongation of ES-derived neurons. Our study also implies that RA induced to express Wnt antagonist Dickkopf-1 (Dkk-1) for neural differentiation. However, the mechanisms of RA triggering neural induction remain to be determined. Our simple and efficient strategy is proposed to provide a basis for studying RA signaling pathways in neural differentiation in vitro.     

Potential role of heat shock proteins in neural differentiation of murine embryonal carcinoma stem cells (P19)

HSPs (heat shock proteins) have been recognized to maintain cellular homoeostasis during changes in microenvironment. The present study aimed to investigate the HSPs expression pattern in hierarchical neural differentiation stages from mouse embryonal carcinoma stem cells (P19) and its role in heat stressed exposed cells. For induction of HSPs, cells were heated at 42°C for 30 min and recovered at 37°C in different time points. For neural differentiation, EBs (embryoid bodies) were formed by plating P19 cells in bacterial dishes in the presence of 1 mM RA (retinoic acid) and 5% FBS (fetal bovine serum). Then, on the sixth day, EBs were trypsinized and plated in differentiation medium containing neurobasal medium, B27, N2 and 5% FBS and for an extra 4 days. The expression of HSPs and neural cell markers were evaluated by Western blot, flow cytometry and immunocytochemistry in different stages. Our results indicate that HSC (heat shock constant)70 and HSP60 expressions decreased following RA treatment, EB formation and in mature neural cells derived from heat-stressed single cells and not heat-treated EBs. While the level of HSP90 increased six times following maturation process, HSP25 was expressed constantly during neural differentiation; however, its level was enhanced with heat stress. Accordingly, heat shock 12 h before the initiation of differentiation did not affect the expression of neuroectodermal and neural markers, nestin and β-tubulin III, respectively. However, both markers increased when heat shock was induced after treatment and when EBs were formed. In conclusion, our results raise the possibility that HSPs could regulate cell differentiation and proliferation under both physiological and pathological conditions.     

Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.     

Vasculogeneic maturation of E14 embryonic stem cells with evidence of early vascular endothelial growth factor independency

Murine embryonic stem cells (ESC) provide a unique homogeneous cell system for studying early vasculogenic cell differentiation in vitro. In this report, we characterized endothelial development of cultured E14 ESCs and mapped the effects of vascular endothelial growth factor (VEGF) on these cells. After removal of leukemia inhibitory factor undifferentiated state ESCs were precultured for 6 days and then cultured for up to 30 days in differentiation culture medium, with or without supplemental VEGF. ELISA analysis was used to detect endogenous VEGF levels. Early vasculogenic development and expression of selected genes were characterized using flow cytometry for specific antigens and quantitative RT-PCR. ELISA analysis showed no endogenous VEGF after preculture and at day 2 in unsupplemented culture, therafter VEGF levels rise. Directly after preculture a high proportion (36%) of the ESCs showed positivity for endothelial CD31. We describe characteristic endothelial differentiation patterns in embryoid bodies (EB) kept in culture for up to 30 days. VEGF supplementation lead to qualitative changes in the EB vessels, specific activation of vasculogenesis-related genes (CD31, CD144, and ERG) and temporary down-regulation of the VEGF receptor gene flk-1. VEGF supplementation did not produce measurable changes in the endothelial cell fractions as judged by surface antigen presence. We conclude that early ESCs may undergo endothelial differentiation through VEGF-independent pathways, whereas endothelial cell patterns in EBs are cytokine dependent and fully stimulated by endogenous cytokine levels.     

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.     

Proliferation and Differentiation of Mouse Embryonic Stem Cells Modified by the Neural Growth Factor (NGF) Gene

The effect of the nerve growth factor (NGF) on the proliferation and differentiation of mouse embryonic stem cells (ESCs) during long-term culturing in vitro was studied using genetically modified cells with stable expression of ngf and gfp (green fluorescent protein) genes (clone R₁^NGF). It was shown that, under standard conditions in vitro with the addition of mouse cytokine LIF, R₁^NGF cells lose their adhesive properties and acquire the ability to form cellular conglomerates or embryoid bodies (EBs) spontaneously. It is noted that, after attachment to the surface of the culture on days 3–5, EBs differentiated towards the neuroectoderm, as evidenced by the appearance of markers of early (nestin) and late (vimentin) neural differentiation. During long-term cultivation up to 22 days, the production of endogenous NGF protein promotes predominant selection and survival of neuroectodermal derivatives.     

Identification of small signalling molecules promoting cardiac-specific differentiation of mouse embryonic stem cells.

Identification of signalling cascades involved in cardiomyogenesis is crucial for optimising the generation of cardiomyocytes from embryonic stem cells (ES cells) (in vitro). We used a transgenic ES cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-myosin heavy chain (alpha-MHC) promoter (palphaMHC-EGFP) to investigate the effects of 33 small molecules interfering with several signalling cascades on cardiomyogenesis. Interestingly, the L-Type Ca2+ channel blocker Verapamil as well as Cyclosporin, an inhibitor of the protein phosphatase 2B, exerted the most striking pro-cardiomyogenic effect. Forskolin (adenylate cyclase stimulator) exerted the most striking anti-cardiomyogenic effect. The cardiomyogenic effect of Cyclosporin and Verapamil correlated with an expression of early cardiac markers Nkx2.5 and GATA4.Compared to the effects on late developmental stage embryoid bodies (EBs) stimulation of early developmental stage EBs (1-day old) with Verapamil or Cyclosporin for 48 h resulted in a potent cardiomyogenic effect. Accordingly, enhanced expression of alpha-MHC mRNA and EGFP mRNA was observed after stimulation of the early developmental stage EBs for 48 h. No expression of alpha-smooth muscle actin or platelet endothelial cell adhesion molecule-1 (PECM-1) as well as of neuronal genes (Nestin, Neurofilament H) has been observed demonstrating a preferentially pro-cardiomyogenic effect by both molecules.     

Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.     

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.     

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.     

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.     

The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium

The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism.     

Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours

The intermediate filament protein nestin is predominantly expressed in some stem/progenitor cells and appears to be a useful molecular tool to characterise tumours originating from precursor cells of neuroectodermal and mesenchymal lineages. Leydig cells originate in the adult testis by differentiation from stem cells and express a variety of neural and neuroendocrine markers. The possible expression of the neural stem cell marker nestin in Leydig cells and testicular tumour cells was determined by analysing the patterns of nestin expression in normal and pathological human testes by Western blot and immunohistochemical methods. In normal testis, nestin was found in some vascular endothelial cells, a subset of peritubular spindle-shaped cells and some Leydig cells; spermatogenic and Sertoli cells were unstained. In normal Leydig cells, nestin was distributed in the perinuclear cytoplasm and accumulated in the crystalloids of Reinke with ageing. In non-tumour pathologies (cryptorchidism, impaired spermatogenesis), the seminiferous tubules were immunonegative, whereas hyperplastic Leydig cells showed cytoplasmic immunolabelling. In testicular malignancies, nestin was localised in the Sertoli cells of the seminiferous tubules affected with intratubular germ cell neoplasia, in the hyperplastic Leydig cells associated with these tumours and in some components (mesenchymal and neuroepithelial cells) of teratomas; spermatocytic and non-spermatocytic seminomas were unstained. Some vascular endothelial cells were immunolabelled in all tumour samples. Thus, nestin is expressed in a population of normal and hyperplastic Leydig cells and in Sertoli cells in the presence of intratubular germ-cell neoplasia. Nestin may be a good marker for identifying components of testicular teratomas.The two first authors participated equally in this workThis work was supported by a grant from the Fondo de Investigaciones Sanitarias (FIS 02/3003 to M.V.T. Lobo)     

VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.     

Down-regulation of nestin in mesenchymal stem cells derived from peripheral blood through blocking bone morphogenesis pathway

Different signaling pathways are implicated in proliferation and differentiation of stem cells. Bone Morphogenesis Pathway (BMP) signaling was known to display an important function in osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). In the present study, the authors investigated whether blocking BMP signaling was associated with down regulation of Nestin expression as neural stem cell marker in peripheral blood derived mesenchymal stem cells (PB-MSCs). At first, MSCs were isolated from peripheral blood by plastic adherent ability and flow cytometry analysis. After reaching the confluence, the cells were treated with medium containing Noggin as antagonist of BMP signaling upon 8 days. Real time PCR analysis indicated that the expression of Nestin was diminished in PB-MSCs by attenuating BMP signaling. The obtained results suggested that BMP signaling might have a regulatory function on the Nestin expression in mesenchymal stem cells.     

Directing endothelial differentiation of human embryonic stem cells via transduction with an adenoviral vector expressing the VEGF(165) gene

Endothelial progenitors derived from human embryonic stem cells (hESCs) hold much promise in clinical therapy. Conventionally, lineage-specific differentiation of hESCs is achieved through supplementation of various cytokines and chemical factors within the culture milieu. Nevertheless, this is a highly inefficient approach that is often limited by poor replicability. An alternative is through genetic modulation with recombinant DNA. Hence, this study investigated whether transduction of hESCs with an adenoviral vector expressing the human VEGF(165) gene (Ad-hVEGF(165)) can enhance endothelial-lineage differentiation. The hESCs were induced to form embryoid bodies (EBs) by culturing them within low-attachment plates for 7 days, and were subsequently trypsinized into single cells, prior to transduction with Ad-hVEGF(165). Optimal transduction efficiency with high cell viability was achieved by 4-h exposure of the differentiating hESCs to viral particles at a ratio of 1 : 500 for three consecutive days. ELISA results showed that Ad-hVEGF(165)-transduced cells secreted human vascular endothelial growth factor (hVEGF) for more than 30 days post-transduction, peaking on day 8, and the conditioned medium from the transduced cells stimulated extensive proliferation of HUVEC. Real-time PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF(165)-transduced cells. Additionally, flow cytometric analysis of CD133 cell surface marker revealed an approximately 5-fold increase in CD133 marker expression in Ad-hVEGF(165)-transduced cells compared to the non-transduced control. Hence, this study demonstrated that transduction of differentiating hESCs with Ad-hVEGF(165) facilitated expression of the VEGF transgene, which in turn significantly enhanced endothelial differentiation of hESCs.