Embryonic Stem Cell Differentiation System Research Articles

Expression of RUNX1-ETO Rapidly Alters the Chromatin Landscape and Growth of Early Human Myeloid Precursor Cells

SummaryAcute myeloid leukemia (AML) is a hematopoietic malignancy caused by recurrent mutations in genes encoding transcriptional, chromatin, and/or signaling regulators. The t(8;21) translocation generates the aberrant transcription factor RUNX1-ETO (RUNX1-RUNX1T1), which by itself is insufficient to cause disease. t(8;21) AML patients show extensive chromatin reprogramming and have acquired additional mutations. Therefore, the genomic and developmental effects directly and solely attributable to RUNX1-ETO expression are unclear. To address this, we employ a human embryonic stem cell differentiation system capable of forming definitive myeloid progenitor cells to express RUNX1-ETO in an inducible fashion. Induction of RUNX1-ETO causes extensive chromatin reprogramming by interfering with RUNX1 binding, blocks differentiation, and arrests cellular growth, whereby growth arrest is reversible following RUNX1-ETO removal. Single-cell gene expression analyses show that RUNX1-ETO induction alters the differentiation of early myeloid progenitors, but not of other progenitor types, indicating that oncoprotein-mediated transcriptional reprogramming is highly target cell specific.

Effect of Wnt/β-catenin signal regulating EMT level on the differentiation of mouse ESC to liver tissue

Objective: To study embryonic stem cell (ESC) differentiation into liver tissue structure from the perspective of epithelial mesenchymal transition (EMT). Methods: ESC of Balb/c mice was selected to induced into hepatic cell using hepatocyte growth factor (HGF), fibroblast growth factor (FGF) in vitro, and at the time points of metaphase (13 d) and maturity (17 d) of differentiation, dynamic inhibition of Wnt/β-catenin signal was made to reduce the level of EMT. Finally, three-dimensional organization structure growth of the differentiation cells was observed in the differentiation system.Expressions of the liver cells vascular markers[albumin (Alb) and vascular endothelial growth factor (VEGFR)]were detected. Results: During the differentiation of ESC, the level of early EMT in the experimental group and the control group was not significantly different. The level of mid-late EMT in the experiment group was significantly lower than the control group. On the day 18 and 20 of differentiation, the relative mRNA expression level of E-cadherin was 0.61±0.15 and 0.47±0.05 in the experimental group, and 0.07±0.05 and 0 in the control group, respectively.The expression level of ALB/AFP/CK8/CK19 in the experimental group was generally higher than that of the control group in the same period, while CD31/VEGFR1 markers in the experimental group decreased more slowly in the late period of differentiation compared with the control group. In the supernatant of ESC culture, the Alb of the experimental group could be detected onday 7, and the concentration was (0.32±0.02) mg/L, while Alb in the control group was (0.19±0.05) mg/L. Urea in the experimental group could be detected on the day 13, and the concentration was (8.7 ±1.0) μmol/L, and the urea concentration of the control group was (3.1±1.2) μmol/L. The concentration of Alb and urea in the culture supernatant of ESC differentiation system increased significantly with the prolongation of the differentiation time, and the Alb and urea concentrations in the experimental group were significantly higher than those in the control group at the same time period. In addition, the differentiated cells in the experimental group could maintain the growth of three-dimensional tissue, while the differentiated cells in the control group eventually showed a single cell state. The expression of hepatic and vascular cell markers could be detected in the experimental group. Immunofluorescence results showed that the hepatocytes and vascular structures were tightly arranged. HE staining showed the formation of hepatic lobular structure, while the control group had no vascular component markers. Conclusion: The differentiation of ESC into liver tissue can be effectively promoted by decreasing the level of EMT at the mid-late stage of ESC differentiation.

Regulation of RUNX1 dosage is crucial for efficient blood formation from hemogenic endothelium

ABSTRACTDuring ontogeny, hematopoietic stem and progenitor cells arise from hemogenic endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in hemogenic endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects hemogenic endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFβ allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in hemogenic endothelium maturation and the establishment of the hematopoietic system.

Activation of the TGFβ pathway impairs endothelial to haematopoietic transition.

The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFβ signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFβ activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFβ pathway. Quantitative proteomics analysis showed that TGFβ treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFβ blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFβ signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFβ signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFβ pathway does not enhance EHT as we hypothesised but instead impairs it.

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients. To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFβ, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFβ, and FLI-1. RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

Wnt/β-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse

Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse β-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of β-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for β-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of β-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/β-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/β-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.

Embryonic stem cell differentiation system for evaluating gene functions involved in physiological megakaryocytic differentiation

Megakaryocytic differentiation is accompanied by marked morphological changes induced by endomitosis and proplatelet formation. Molecular mechanisms underlying this unique cell differentiation process have been investigated by gain/loss-of-function studies using leukemic cell lines. However, these cell lines cannot completely mimic physiological megakaryocytic differentiation, including the morphological changes, and sometimes lead to contradictory results between cell lines. The goal of this study was to establish a novel cell differentiation system that completely mimics physiological megakaryocytic differentiation for analyzing gene function. To that end, we used homologous recombination to prepare an embryonic stem (ES) cell line containing a GFP-transgene driven by the PF4 promoter at the Hprt locus. Differentiation of these cells resulted in megakaryocytes and proplatelets, suggesting physiological megakaryocytic differentiation. However, the number of GFP-expressing cells was low (1.7% GFP+ cells among CD41+ cells). Insertion of full-length or small core β-globin insulators on either side of the transgene significantly increased the number of GFP-expressing cells (∼60% GFP+ cells among CD41+ cells), and GFP-expression was specifically observed in megakaryocytic cells. Similar results were obtained with other ES cells containing a GPIIb-GFP transgene. Altogether, we have succeeded in efficiently expressing exogenous genes specifically in differentiating megakaryocytes and in establishing a novel ES cell differentiation system for analyzing gene function involved in physiological megakaryocytic differentiation.

ES cell differentiation system recapitulates the establishment of imprinted gene expression in a cell-type-specific manner

Genomic imprinting is a phenomenon whereby monoallelic gene expression occurs in a parent-of-origin-specific manner. A subset of imprinted genes acquires a tissue-specific imprinted status during the course of tissue development, and this process can be analyzed by means of an in vitro differentiation system utilizing embryonic stem (ES) cells. In neurons, the gene Ube3a is expressed from the maternal allele only, and a paternally expressed non-coding, antisense RNA has been implicated in the imprinting process in mice and humans. Here, to study the genomic imprinting mechanism, we established F1 hybrid ES cells derived from two sub-species of Mus musculus and established an in vitro neuronal differentiation system in which neuron-specific imprinting of Ube3a was recapitulated. With this system, we revealed that the switch from biallelic expression to maternal, monoallelic expression of Ube3a occurs late in neuronal development, during the neurite outgrowth period, and that the expression of endogenous antisense transcript from the Ube3a locus is up-regulated several hundred-fold during the same period. Our results suggest that evaluation of the quality of ES cells by studying their differentiation in vitro should include evaluation of epigenetic aspects, such as a comparison with the genomic imprinting status found in tissues in vivo, in addition to the evaluation of differentiation gene markers and morphology. Our F1 hybrid ES cells and in vitro differentiation system will allow researchers to investigate complex end-points such as neuron-specific genomic imprinting, and our F1 hybrid ES cells are a useful resource for other tissue-specific genomic imprinting and epigenetic analyses.

ES & iPS Cell Research for Cardiovascular Regeneration

We have been investigating cardiovascular cell differentiation and regeneration using embryonic stem (ES) and induced pluripotent stem (iPS) cells. Previously, we established an ES cell differentiation system for cardiovascular cells using Flk1+ cells as common progenitors (Nature, 2000). We recently found an efficient expansion method for cardimyocytes and cardiac progenitors from ES cells. That is, an immunosuppressant, cyclosporin-A (CSA), showed a novel effect specifically acting on mesoderm cells to drastically increase cardiac progenitors as well as cardiomyocytes (Biochem Biophys Res Commun, 2009). Recently, novel pluripotent stem cell lines, iPS cells, were established from mouse and human somatic cells. We applied our ES cell system to mouse iPS cells, and succeeded in systematically inducing cardiovascular cells from iPS cells (Circulation, 2008). Combining our technologies in ES cells and iPS cells, we recently revealed that cardiac cells were efficiently induced from mouse and human iPS cells by CSA treatment (PLoS One, 2011). Now we are exploring cell transplantation methods using the cell sheets technology (Shimizu, Curr Pharm Des, 2009) and small molecules for cardiac regeneration. These studies would largely contribute to cardiovascular regeneration with iPS cells.

DifferentialHoxExpression in Murine Embryonic Stem Cell Models of Normal and Malignant Hematopoiesis

The Hox family are master transcriptional regulators of developmental processes, including hematopoiesis. The Hox regulators, caudal homeobox factors (Cdx1-4), and Meis1, along with several individual Hox proteins, are implicated in stem cell expansion during embryonic development, with gene dosage playing a significant role in the overall function of the integrated Hox network. To investigate the role of this network in normal and aberrant, early hematopoiesis, we employed an in vitro embryonic stem cell differentiation system, which recapitulates mouse developmental hematopoiesis. Expression profiles of Hox, Pbx1, and Meis1 genes were quantified at distinct stages during the hematopoietic differentiation process and compared with the effects of expressing the leukemic oncogene Tel/PDGFRβ. During normal differentiation the Hoxa cluster, Pbx1 and Meis1 predominated, with a marked reduction in the majority of Hox genes (27/39) and Meis1 occurring during hematopoietic commitment. Only the posterior Hoxa cluster genes (a9, a10, a11, and a13) maintained or increased expression at the hematopoietic colony stage. Cdx4, Meis1, and a subset of Hox genes, including a7 and a9, were differentially expressed after short-term oncogenic (Tel/PDGFRβ) induction. Whereas Hoxa4-10, b1, b2, b4, and b9 were upregulated during oncogenic driven myelomonocytic differentiation. Heterodimers between Hoxa7/Hoxa9, Meis1, and Pbx have previously been implicated in regulating target genes involved in hematopoietic stem cell (HSC) expansion and leukemic progression. These results provide direct evidence that transcriptional flux through the Hox network occurs at very early stages during hematopoietic differentiation and validates embryonic stem cell models for gaining insights into the genetic regulation of normal and malignant hematopoiesis.

Roles of Cyclic Adenosine Monophosphate Signaling in Endothelial Cell Differentiation and Arterial-Venous Specification During Vascular Development

Cyclic adenosine monophosphate (cAMP) is an important second messenger mediating physiological functions, including metabolism, gene expression, cell growth and differentiation. Recently, we demonstrated novel roles of cAMP pathway in endothelial cell (EC) differentiation and arterial-venous specification using an embryonic stem cell differentiation system. These studies offered a concept that vascular formation is accomplished by a 2-layered mechanism: (1) a basal mechanism for common EC differentiation, whereby vascular endothelial growth factor (VEGF) signaling plays a central role in the basal mechanism, and (2) a vascular diversification mechanism working on the basis of common EC differentiation. Vascular diversification, such as artery and vein formation, can be only achieved by enacting specific machineries in the presence of the basal EC machinery. cAMP/protein kinase A signaling contributes to common EC differentiation through upregulation of the VEGF-A receptors, Flk1 and neuropilin1. On the other hand, cAMP can activate phosphatidylinositol-3 kinase, which induces an arterial fate in vascular progenitors via dual activation of Notch and β-catenin signaling as an arterial-specific machinery. cAMP signaling thus plays a pivotal role in both the basal and diversification machinery during vascular development.

Convergence of Notch and β-catenin signaling induces arterial fate in vascular progenitors

Molecular mechanisms controlling arterial–venous specification have not been fully elucidated. Previously, we established an embryonic stem cell differentiation system and demonstrated that activation of cAMP signaling together with VEGF induces arterial endothelial cells (ECs) from Flk1+ vascular progenitor cells. Here, we show novel arterial specification machinery regulated by Notch and β-catenin signaling. Notch and GSK3β-mediated β-catenin signaling were activated downstream of cAMP through phosphatidylinositol-3 kinase. Forced activation of Notch and β-catenin with VEGF completely reconstituted cAMP-elicited arterial EC induction, and synergistically enhanced target gene promoter activity in vitro and arterial gene expression during in vivo angiogenesis. A protein complex with RBP-J, the intracellular domain of Notch, and β-catenin was formed on RBP-J binding sites of arterial genes in arterial, but not venous ECs. This molecular machinery for arterial specification leads to an integrated and more comprehensive understanding of vascular signaling.

Transcriptional Activation by the Mixl1 Homeodomain Protein in Differentiating Mouse Embryonic Stem Cells

Members of the Mix/Bix family of paired class homeobox genes play important roles in the development of vertebrate mesoderm and endoderm. The single Mix/Bix family member identified in the mouse, Mix-like 1 (Mixl1), is required for mesendoderm patterning during gastrulation and promotes mesoderm formation and hematopoiesis in embryonic stem cell (ESC)-derived embryoid bodies. Despite its crucial functions the transcriptional activity and targets of Mixl1 have not been well described. To investigate the molecular mechanisms of Mixl1-mediated transcriptional regulation, we have characterized the DNA-binding specificity and transcriptional properties of this homeodomain protein in differentiating ESCs. Mixl1 binds preferentially as a dimer to an 11-base pair (bp) Mixl1 binding sequence (MBS) that contains two inverted repeats separated by a 3-bp spacer. The MBS mediates transcriptional activation by Mixl1 in both NIH 3T3 cells and in a new application of an inducible ESC differentiation system. Consistent with our previous observation that early induction of Mixl1 expression in ESCs results in premature activation of Goosecoid (Gsc), we have found that Mixl1 occupies two variant MBSs within and activates transcription from the Gsc promoter in vitro and in vivo. These results strongly suggest that Gsc is a direct target gene of Mixl1 during embryogenesis. STEM CELLS 2009;27:2884-2895.

Estimation of embryotoxic effect of fluoxetine using embryonic stem cell differentiation system

Aims Fluoxetine is an antidepressant drug of the selective serotonin reuptake inhibitor (SSRI) class, which is commonly prescribed to treat a wide spectrum of mood disorders including depression during pregnancy and lactation. Recent studies have proposed a possible association between an increase in major malformations and the maternal use of SSRI drugs during pregnancy. Here, we assess the effects of fluoxetine using a mouse ES cell differentiation system to clarify the possible association. Main methods Using a mouse embryonic stem (ES) cell differentiation system, we evaluated cell viability and differentiation affected by fluoxetine. Key findings Fluoxetine adversely affected cell viability and differentiation from undifferentiated ES cells to cardiomyocytes in a dose-dependent manner. The IC 50 values of fluoxetine for ES cells and NIH-3T3 fibroblasts were 1.79 μM and 4.67 μM, respectively, and the ID 50 value for ES cells was 3.79 μM. These results indicate that fluoxetine has strong toxicity evaluated by a mouse embryonic stem cell test (EST). Analysis of tissue-specific markers revealed that fluoxetine potently inhibits mesodermal development, although it promotes ectodermal differentiation in a lineage-specific manner. Significance These results using the in vitro ES cell assay system suggest a possible relationship between the teratogenicity of fluoxetine and its molecular mechanism.

Abstract 3944: Converged Signaling of β-Catenin and Notch Induces Differentiation of Arterial Endothelial Cells from VEGFR2+ Vascular Progenitors

We have been investigating molecular mechanisms of vascular development and diversification using embryonic stem (ES) cells. Previously, we established an ES cell differentiation system that reproduces early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors (Nature, 2000). Endothelial cells (ECs) were induced from VEGFR2 + progenitor cells with VEGF. VEGF alone mainly induced venous ECs, which are negative for arterial EC markers, ephrinB2 and CXCR4. Interestingly, addition of 8bromo-cAMP with VEGF activated Notch signaling in ECs and substantially induced arterial ECs. Nevertheless, activation of Notch using notch intracellular domain (NICD)-estrogen receptor fusion protein failed to induce arterial ECs (Arterioscler Thromb Vasc Biol, 2006). These results indicate that Notch activation was not sufficient to induce arterial ECs from vascular progenitors. Then we further investigated the downstream of cAMP, which would suffice signals for constructive induction of arterial ECs. We found that phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, completely inhibited cAMP-induced arterial EC differentiation as well as Notch activation. GSK3β inhibitor, Bio, partially reversed the inhibitory effects of LY294002. Whereas activation of β-catenin signaling alone using Doxycycline-induced constitutive active form of β-catenin expression showed only a weak effect on arterial EC induction, simultaneous activation of β-catenin and Notch signaling completely restored arterial EC induction even in the absence of cAMP. Thus, cAMP-PI3K-GSK3β-β-catenin pathway plays a critical role in arterial EC differentiation. Furthermore, chromatin immunoprecipitation assay on the two RBP-J binding sites of mouse EphrinB2 revealed that RBP-J, NICD, and β-catenin formed a protein complex on these two sites only in arterial ECs, but not in venous ECs. These findings suggest that Notch and β-catenin signaling converged to form a novel arterial EC-specific protein complex to induce arterial ECs. This study would provide novel understandings of cellular and molecular mechanisms for arterial-venous specification and novel molecular targets for drug discovery.

Numb mediates the interaction between Wnt and Notch to modulate primitive erythropoietic specification from the hemangioblast

During embryonic development, the establishment of the primitive erythroid lineage in the yolk sac is a temporally and spatially restricted program that defines the onset of hematopoiesis. In this report, we have used the embryonic stem cell differentiation system to investigate the regulation of primitive erythroid development at the level of the hemangioblast. We show that the combination of Wnt signaling with inhibition of the Notch pathway is required for the development of this lineage. Inhibition of Notch signaling at this stage appears to be mediated by the transient expression of Numb in the hemangioblast-derived blast cell colonies. Activation of the Notch pathway was found to inhibit primitive erythropoiesis efficiently through the upregulation of inhibitors of the Wnt pathway. Together, these findings demonstrate that specification of the primitive erythroid lineage is controlled, in part, by the coordinated interaction of the Wnt and Notch pathways, and position Numb as a key mediator of this process.

Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity

Embryonic stem (ES) cells have the potential to differentiate into all cell types of the adult body, and could allow regeneration of damaged tissues. The challenge is to alter differentiation toward functional cell types or tissues by directing ES cells to a specific fate. Efforts have been made to understand the molecular mechanisms that are required for the formation of the different germ layers and tissues from ES cells, and these mechanisms appear to be very similar in the mouse embryo. Differentiation toward mesoderm and mesoderm derivatives such as cardiac tissue or hemangioblasts has been demonstrated; however, the roles of Activin A/Nodal, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) signaling in the early patterning of ES cell-derived pan-mesoderm and anterior visceral endoderm (aVE) have not been reported yet. We therefore analyzed the roles of Activin A/Nodal, BMP, and FGF signaling in the patterning of ES cell-derived mesoderm as well as specification of the aVE by using a dual ES cell differentiation system combining a loss-of-function with a gain-of-function approach. We found that Activin A or Nodal directed the nascent mesoderm toward axial mesoderm and mesendoderm, while Bmp4 was inducing posterior and extraembryonic mesoderm at the expense of anterior primitive streak cells. FGF signaling appeared to have an important role in mesoderm differentiation by allowing an epithelial-to-mesenchymal transition of the newly formed mesoderm cells that would lead to their further patterning. Moreover, inhibition of FGF signaling resulted in increased expression of axial mesoderm markers. Additionally, we revealed that the formation of aVE cells from ES cells requires FGF-dependent Activin A/Nodal signaling and the attenuation of Bmp4 signaling.

Wnt, Activin, and BMP Signaling Regulate Distinct Stages in the Developmental Pathway from Embryonic Stem Cells to Blood

The embryonic stem cell differentiation system was used to define the roles of the Activin/Nodal, BMP, and canonical Wnt signaling pathways at three distinct developmental stages during hematopoietic ontogeny: induction of a primitive streak-like population, formation of Flk1(+) mesoderm, and induction of hematopoietic progenitors. Activin/Nodal and Wnt, but not BMP, signaling are required for the induction of the primitive streak. Although BMP is not required for primitive streak induction, it displays a strong posteriorizing effect on this population. All three signaling pathways regulate induction of Flk1(+) mesoderm. The specification of Flk1(+) mesoderm to the hematopoietic lineages requires VEGF and Wnt, but not BMP or Activin/Nodal signaling. Specifically, Wnt signaling is essential for commitment of the primitive erythroid, but not the definitive lineages. These findings highlight dynamic changes in signaling requirements during blood cell development and identify a role for Wnt signaling in the establishment of the primitive erythroid lineage.

Regulated Nodal signaling promotes differentiation of the definitive endoderm and mesoderm from ES cells

Nodal signaling induces the formation of the endoderm and mesoderm during gastrulation. Nodal expression persists until the definitive endoderm progenitor has completely formed, and disappears thereafter. A tightly regulated Nodal expression system is essential for the differentiation of embryonic stem (ES) cells into distinct tissue lineages. On this basis, we established an ES cell differentiation system with the tetracycline-regulated expression of Nodal. The upregulated Nodal signaling pathway and its downstream transcriptional targets induced the specification of ES cells into definitive endoderm and mesoderm derivatives, and the subsequent downregulation of Nodal signaling promoted further maturation of the gut tube both in vitro and in vivo. Sustained expression of the Nodal gene inhibited the maturation of the definitive endoderm owing to persistent Oct3 and/or Oct4 expression and teratoma formation. Furthermore, quantitative single cell analysis by flow cytometry using CXCR4, VEGFR2 and PDGFR-alpha indicated that this protocol for definitive endoderm and mesoderm differentiation is superior to any other available protocol. Our findings also indicated that the Nodal or Nodal-related molecules secreted from Nodal-expressing ES cells could cause genetically unmanipulated ES cells to induce the expression of the Nodal signaling pathway and its downstream targets, which consequently leads to the differentiation of the ES cells into definitive endoderm and mesoderm. Our differentiation system, using tightly regulated Nodal expression, enabled us to investigate the mechanism of ES cell differentiation into definitive endoderm or mesoderm derivatives. Our findings also demonstrate that Nodal-expressing ES cells might be a source of highly active proteins that could be used for developing endoderm or mesoderm tissues in regenerative medicine.

Characterization of Developmental Pathway of Natural Killer Cells from Embryonic Stem Cells In Vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34+ EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34+ EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34+ EB cells, NK and T cell potentials were detected in a CD45− subset, whereas CD45+ EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34+CD45− EB cells showed that CD45+Mac-1−Ter119− ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45−CD34+ EB cells have lymphoid potential, and they differentiate into more mature CD45+Lin− hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122− and they rapidly acquire CD122 as they differentiate along the NK lineage.