Mesendoderm Differentiation Research Articles

Comparison of Potentials of Stem Cells Isolated from Tendon and Bone Marrow for Musculoskeletal Tissue Engineering

The use of tendon-derived stem cells (TDSCs) as a cell source for musculoskeletal tissue engineering has not been compared with that of bone marrow stromal cells (BMSC). This study compared the mesenchymal stem cell (MSC) and embryonic stem cells (ESC) markers, clonogenicity, proliferative capacity, and multilineage differentiation potential of rat TDSC and BMSC in vitro. The MSC and ESC marker profiles of paired TDSC and BMSC were compared using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Their clonogenicity and proliferative capacity were compared using colony-forming and 5-bromo-2'-deoxyuridine assays, respectively. The expression of tenogenic, osteogenic, and chondrogenic markers at basal state were examined using qRT-PCR. Their osteogenic, chondrogenic, and adipogenic differentiation potentials were compared using standard assays. TDSC and BMSC showed similar expression of CD90 and CD73. TDSC expressed higher levels of Oct4 than BMSC. TDSC exhibited higher clonogenicity, proliferated faster, and expressed higher tenomodulin, scleraxis, collagen 1 α 1 (Col1A1), decorin, alkaline phosphatase, Col2A1, and biglycan messenger RNA levels than BMSC. There was higher calcium nodule formation and osteogenic marker expression in TDSC than BMSC upon osteogenic induction. More chondrocyte-like cells and higher glycosaminoglycan deposition and chondrogenic marker expression were observed in TDSC than BMSC upon chondrogenic induction. There were more oil droplets and expression of an adipogenic marker in TDSC than BMSC upon adipogenic induction. TDSC expressed higher Oct4 levels, which was reported to positively regulate mesendodermal lineage differentiation, showed higher clonogenicity and proliferative capacity, and had greater tenogenic, osteogenic, chondrogenic, and adipogenic markers and differentiation potential than BMSC. TDSC might be a better cell source than BMSC for musculoskeletal tissue regeneration.

Id1 Maintains Embryonic Stem Cell Self-Renewal by Up-Regulation of Nanog and Repression of Brachyury Expression

Understanding the mechanism by which embryonic stem (ES) cells self-renew is crucial for the realization of their therapeutic potential. Earlier, overexpression of Id proteins was shown to be sufficient to maintain mouse ES cells in a self-renewing state even in the absence of serum. Here, we use ES cells derived from Id deficient mice to investigate the requirement for Id proteins in maintaining ES cell self-renewal. We find that Id1(-/-) ES cells have a defect in self-renewal and a propensity to differentiate. We observe that chronic or acute loss of Id1 leads to a down-regulation of Nanog, a critical regulator of self-renewal. In addition, in the absence of Id1, ES cells express elevated levels of Brachyury, a marker of mesendoderm differentiation. We find that loss of both Nanog and Id1 is required for the up-regulation of Brachyury, and ectopic Nanog expression in Id1(-/-) ES cells rescues the self-renewal defect, indicating that Nanog is the major downstream target of Id1. These results identify Id1 as a critical factor in the maintenance of ES cell self-renewal and suggest a plausible mechanism for its control of lineage commitment.

Distinct Developmental Ground States of Epiblast Stem Cell Lines Determine Different Pluripotency Features

Epiblast stem cells (EpiSCs) are pluripotent stem cells derived from mouse postimplantation embryos at embryonic day (E) 5.5-E7.5 at the onset of gastrulation, which makes them a valuable tool for studying mammalian postimplantation development in vitro. EpiSCs can also be reprogrammed into a mouse embryonic stem cell (mESC)-like state. Some reports have shown that the reversion of EpiSCs requires transcription factor overexpression, whereas others have suggested that use of stringent mESC culture conditions alone is sufficient for the reversion of EpiSCs. To clarify these discrepancies, we systematically compared a panel of independent EpiSC lines. We found that--regardless of the embryonic day of derivation--the different EpiSC lines shared a number of defining characteristics such as the ability to form teratomas. However, despite use of standard EpiSC culture conditions, some lines exhibited elevated expression of genes associated with mesendodermal differentiation. Pluripotency (Oct4) and mesodermal (Brachyury) marker genes were coexpressed in this subset of lines. Interestingly, the expression of mesendodermal marker genes was negatively correlated with the cells' ability to efficiently undergo neural induction. Moreover, these mesodermal marker gene-expressing cell lines could not be efficiently reverted to an mESC-like state by using stringent mESC culture conditions. Conversely, Brachyury overexpression diminished the reversion efficiency in otherwise Brachyury-negative lines. Overall, our data suggest that different EpiSC lines may undergo self-renewal into distinct developmental states, a finding with important implications for functional readouts such as reversion of EpiSCs to an mESC-like state as well as directed differentiation.

HEB and E2A function as SMAD/FOXH1 cofactors

Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We identified a new motif, termed SMAD complex-associated (SCA), that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation.

Dual Kinase/Phosphatase Regulating and Anchoring Protein Tribbles: Expression During Acquisition of Meiotic Competence and Meiosis in Mouse Oocytes.

Mammalian oocytes arrested at prophase of meiosis I undergo a growth phase and acquisition of meiotic competence prior to resumption of meiosis and progression to metaphase of meiosis II. Reversible phosphorylation, involving kinases and phosphatases, is involved in both of these intra-oocyte processes. However, current understandings of how numerous reversible phosphorylation events are orchestrated during acquisition of meiotic competence and maturation are significantly lacking. The Tribbles protein was originally described as a cell cycle regulator required for mesendodermal differentiation and oocyte specification during Drosophila embryogenesis. Drosophila Tribbles protein regulates cell proliferation by promoting degradation of CDC25 protein phosphatase. Mammalian somatic cells contain three Tribbles genes (Tribbles 1, 2, and 3) with more diverse functions, such as anchoring proteins that regulate protein kinase B, mitogen-activated protein kinases and CCAAT/enhancer-binding protein alpha. The objectives of this study were to determine if mammalian oocytes expressed Tribbles and compare Tribbles mRNA levels during acquisition of meiotic competence and maturation. Oocytes were collected from CF1 mice at postnatal d10-13 [growing / germinal vesicle-intact (GV-intact) / meiotically incompetent (Incomp)] and d19-21 following gonadotropin stimulation [GV-intact / meiotically competent (Comp)]. Denuded GV-intact oocytes were pooled and used for mRNA isolation or GV-intact/Comp oocytes were placed into culture and matured for 2, 7, and 16hrs to germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII), respectively, prior to mRNA isolation. Gene expression for Tribbles 1, 2, 3, and actin at each stage of oocyte development were quantified by real-time PCR, in triplicate per experiment, in three separate experiments. Expression ratios and/or fold changes were tested for significance by a pairwise fixed reallocation randomization test, and p<0.05 was considered significantly different. Mouse oocytes at all stages assessed expressed all three Tribbles genes. As oocytes gained meiotic competence gene expression of Tribbles 1 and 3 did not significantly change. However, during the transition from GV-intact Incomp to Comp Tribbles 2 gene expression significantly increased 15.7 fold; p<0.001. Tribbles 1 mRNA levels also did not significantly change during transitions from GV-intact to GVBD, to MI, or to MII. During the GV-intact to GVBD transition Tribbles 2 and Tribbles 3 mRNA levels significantly decreased (4.4 fold; p<0.01) and increased (5.7 fold; p<0.01), respectively. Finally, while no significant changes of any Tribbles mRNA occurred during the transition through MI, Tribbles 2 mRNA was found to significantly increase (2.1 fold; p<0.01) during the MI to MII transition. In conclusion, this is the first report of mammalian oocytes expressing all three Tribbles, which are known dual kinase/phosphatase regulatory and anchoring proteins. Tribbles 2 gene expression and mRNA levels change most significantly during acquisition of meiotic competence and meiotic maturation. Continued studies are required at the protein and functional levels and will help elucidate regulatory pathways of meiotic competence acquisition, meiotic maturation, and attainment of embryonic developmental competence. (poster)

An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafish

An evolutionarily conserved subcircuit (kernel) dedicated to a specific developmental function is found at the top of the gene regulatory networks (GRNs) hierarchy. Here we comprehensively demonstrate that a pan-deuterostome endoderm specification kernel exists in zebrafish. We analyzed interactions among gata5, gata6, otx2 and prdm1a using specific morpholino knockdowns and measured the gene expression profiles by quantitative real-time RT-PCR and in situ hybridization. The mRNA rescue experiment validated the specificity of the morpholino knockdown. We found that the interactions among gata5, gata6, otx2 and prdm1a determine the initial specification of the zebrafish endoderm. Although otx2 can activate both gata5 and gata6, and the prdm1a/krox homologue also activates some endoderm transcription factors, a feedback loop from Gata to otx2 and prdm1a is missing. Furthermore, we found the positive regulation between gata5 and gata6 to further lock-on the mesendoderm specification by the Gata family. Chromatin immunoprecipitation was used to further validate the recruitment of Otx2 to the gata5 and gata6 loci . Functional assays revealed that module B of gata6 and the basal promoter of gata5 drive the gene at the mesendoderm, and mutational analysis demonstrated that Otx2 and Gata5/6 contribute to reporter gene activation. This is the first direct evidence for evolutionarily conserved endoderm specification across echinoderms and vertebrates.

Pluripotency Factors in Embryonic Stem Cells Regulate Differentiation into Germ Layers

Cell fate decisions are fundamental for development, but we do not know how transcriptional networks reorganize during the transition from a pluripotent to a differentiated cell state. Here, we asked how mouse embryonic stem cells (ESCs) leave the pluripotent state and choose between germ layer fates. By analyzing the dynamics of the transcriptional circuit that maintains pluripotency, we found that Oct4 and Sox2, proteins that maintain ESC identity, also orchestrate germ layer fate selection. Oct4 suppresses neural ectodermal differentiation and promotes mesendodermal differentiation; Sox2 inhibits mesendodermal differentiation and promotes neural ectodermal differentiation. Differentiation signals continuously and asymmetrically modulate Oct4 and Sox2 protein levels, altering their binding pattern in the genome, and leading to cell fate choice. The same factors that maintain pluripotency thus also integrate external signals and control lineage selection. Our study provides a framework for understanding how complex transcription factor networks control cell fate decisions in progenitor cells.

Nodal-Dependent Mesendoderm Specification Requires the Combinatorial Activities of FoxH1 and Eomesodermin

Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA–binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals.

Smad-Interacting Protein-1 and MicroRNA 200 Family Define a Nitric Oxide–Dependent Molecular Circuitry Involved in Embryonic Stem Cell Mesendoderm Differentiation

Smad-interacting protein-1 (Sip1/ZEB2) is a transcriptional repressor of the telomerase reverse transcriptase catalytic subunit (Tert) and has recently been identified as a key regulator of embryonic cell fate with a phenotypic effect similar, in our opinion, to that reported for nitric oxide (NO). Remarkably, SIP1/ZEB2 is a known target of the microRNA 200 (miR-200) family. In this light, we postulated that Sip1/ZEB2 and the miR-200 family could play a role during the NO-dependent differentiation of mES. The results of the present study show that Sip1/ZEB2 expression is downregulated during the NO-dependent expression of mesendoderm and early cardiovascular precursor markers, including Flk1 and CXCR4 in mES. Coincidently, members of the miR-200 family, namely miR-429, -200a, -200b, and -200c, were transcriptionally induced in parallel to mouse Tert. This regulation occurred at the level of chromatin. Remarkably, miR-429/miR-200a overexpression or Sip1/ZEB2 knockdown by short hairpin RNA interference elicited a gene expression pattern similar to that of NO regardless of the presence of leukemia inhibitory factor. These results are the first demonstrating that the miR-200 family and Sip1/ZEB2 transcription factor are regulated by NO, indicating an unprecedented molecular circuitry important for telomerase regulation and early differentiation of mES.

A Mesoderm-Derived Precursor for Mesenchymal Stem and Endothelial Cells

Among the three embryonic germ layers, the mesoderm is a major source of the mesenchymal precursors giving rise to skeletal and connective tissues, but these precursors have not previously been identified and characterized. Using human embryonic stem cells directed toward mesendodermal differentiation, we show that mesenchymal stem/stromal cells (MSCs) originate from a population of mesodermal cells identified by expression of apelin receptor. In semisolid medium, these precursors form FGF2-dependent compact spheroid colonies containing mesenchymal cells with a transcriptional profile representative of mesoderm-derived embryonic mesenchyme. When transferred to adherent cultures, individual colonies give rise to MSC lines with chondro-, osteo-, and adipogenic differentiation potentials. Although the MSC lines lacked endothelial potential, endothelial cells could be derived from the mesenchymal colonies, suggesting that, similar to hematopoietic cells, MSCs arise from precursors with angiogenic potential. Together, these studies identified a common precursor of mesenchymal and endothelial cells, mesenchymoangioblast, as the source of mesoderm-derived MSCs.

Smad2 mediates Activin/Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells

Although Activin/Nodal signaling regulates pluripotency of human embryonic stem (ES) cells, how this signaling acts in mouse ES cells remains largely unclear. To investigate this, we confirmed that mouse ES cells possess active Smad2-mediated Activin/Nodal signaling and found that Smad2-mediated Activin/Nodal signaling is dispensable for self-renewal maintenance but is required for proper differentiation toward the mesendoderm lineage. To gain insights into the underlying mechanisms, Smad2-associated genes were identified by genome-wide chromatin immunoprecipitation-chip analysis. The results showed that there is a transcriptional correlation between Smad2 binding and Activin/Nodal signaling modulation, and that the development-related genes were enriched among the Smad2-bound targets. We further identified Tapbp as a key player in mesendoderm differentiation of mouse ES cells acting downstream of the Activin/Nodal-Smad2 pathway. Taken together, our findings suggest that Smad2-mediated Activin/Nodal signaling orchestrates mesendoderm lineage commitment of mouse ES cells through direct modulation of corresponding developmental regulator expression.

P26. Inhibition of phosphatidylinositol-3-kinase signalling promotes Activin A induced definitive endoderm differentiation of human embryonic stem cells

Efficient definitive endoderm (DE) differentiation is essential for the generation of hepatocytes from human embryonic stem cells (hESCs), which are in demand for drug development and clinical application. However, this has been hindered as signaling pathways regulating the differentiation remain unclear. Therefore, understanding the signaling pathways controlling DE formation is important for differentiation of hESCs to hepatic lineages. Generation of DE through the initial formation of mesendoderm is activin-dependent differentiation. Further induction by higher concentration of Activin A gives rise to DE, whereas lower concentration favors mesoderm lineage. On the other hand, phosphatidylinositol-3-kinase (PI3K) signaling, which is important for ESCs self-renew were shown to be potent inhibitors of the differentiation. It has been shown that supression of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then DE in conditioned medium. However, the differentiation efficiency and molecular mechanism underlying the mesendoderm and DE differentiation cannot be clearly addressed in this culture condition. Therefore, in this study, we have used chemically defined medium to assess differentiation potential by supression of PI3K in combination with Activin A. In this study, we demonstrate that suppression of PI3K signal in H1 hESCs causes downregulation of pluripotency marker (Oct4 and Sox2) and induces mesendoderm/endoderm gene expression (Brachyury, Gata 6 and FoxA2). Treatment of hESCs with combined Activin A (100 ng/ml) and PI3K inhibitor, Ly 294002 (20 μM) increased expression of mesendoderm and endoderm markers (Brachury, GSC, FOxA2, Sox17 and Wnt3a) several fold higher than that of Activin A (100 ng/ml) alone. As a result of improved DE formation, combined treatments also improved hepatocytes production, exhibited higher level of hepatic gene expression and increased Cyp3A4 activity (four-fold higher) than those derived from the treatment with activin alone. These results suggest that repression of PI3K is necessary for efficient DE differentiation and subsequently for the hepatocyte generation.

Differences in intracellular signaling patterns in HUVECs initially attaching to various ECM component combinations

Program/Abstract # 50Differences in intracellular signaling patterns in HUVECs initiallyattaching to various ECM component combinationsChristine Pauken, Michael CaplanSBHSE, Arizona State University, Tempe, AZ, USAWhen tissues are growing, injured, or in some pathological states,the vasculature must remodel itself in response to the new micro-environments. The new microenvironments include different compo-nents of the extracellular matrix (ECM) that the endothelial cells areexposed to and these cells must decipher and respond to the newcomponents. In this study, HUVECs were placed on 15 differentcombinationsoftwoECMcomponentsfor5–360 min.ThelevelsofErk1/2, Jnk, Akt, and NFKB phosphorylationwere measured at eight timepointsoverthisperiod.The480datapointswereanalyzedbyprincipalcomponent analysis.Thefirst principalcomponent correlateswithErk1/2signalingintheHUVECs,andthescoreplotshowshighcorrelationswithCol1/IV,ColIV/Lm,Vn/CSandVn/HS.FurtherstatisticalanalysisshowsthatdifferentErk1/2signalingpatternsoccurinHUVECsonCol1/Col IV, Col 1/Lm, Col IV/Lm, Col IV/HS, Vn/HS, and Vn/CS. OtherchangesinsignalingpatternsareseenintheJnkandAktphosphoryla-tionpatternswhentheHUVECsareplacedontheseECMcombinations.This data will enhance the knowledge of how HUVECs interpret theirmicroenvironment and allow for a better understanding of woundrepair, vascularization in tumors, and improved design of biomaterialsfor blood vessel repair and tissue engineering.doi:10.1016/j.ydbio.2010.05.083Program/Abstract # 51A role for Mxtx2 in mesendoderm inductionBenjamin Feldman, Sung-Kook Hong, Jamie L. BrownMedical Genetics Branch, National Human Genome Research Institute,NIH, Bethesda, MD 20892, USAA long-standing challenge to embryologists is deciphering theinstructions guiding germ layer differentiation at the onset ofgastrulation. In vertebrates, Nodal signaling is a core component ofmesoderm and endoderm (mesendoderm) differentiation and muchremains to be learned about how expression of the Nodal ligandsthemselves is activated. Motivated byevidence that the zebrafishyolkharbors Nodal- and mesendoderm-activating signals, we developed amicrodissection strategy for the genome-wide identification oftranscripts enriched in the early yolk. This analysis led us to reassessthe function of Mxtx2, a transcription factor that has been shown byothers to be expressed in the yolk and to be essential for normalepiboly. We find that Mxtx2 is a strong inducer of mesendoderm andits loss of function causes substantial reductions in the earlyexpression of the Nodal-related genes and several other mesendo-derm genes. We further find that Mxtx2 directly binds to the nodal-related 2 (cyclops)andsox32 loci, accounting for the Nodal-independent expression of these genes in the yolk. Thus, Mxtx2 is anew actor in zebrafish mesendoderm induction, with a role upstreamof Nodal-related genes.doi:10.1016/j.ydbio.2010.05.084Program/Abstract # 52Roles of cilia in Gli protein activationAimin Liu, Huiqing Zeng, Amber Hoover, Jinping JiaDept. of Biol., The Penn State Univ., University Park, PA, USAHedgehog (Hh) family of signaling molecules plays pivotal roles incell proliferation and cell fate determination during developmentthrough regulating the activities of the Gli-family proteins. Ourprevious genetic studies indicated that cilia are required forintracellular transduction of Hh signals to Gli proteins in developingmouse embryos. In the current study, we are investigating themolecular mechanisms underlying the roles of cilia in regulating Gliactivities. One unresolved issue in this field has been the roles of ciliain Gli1 activation. Previous studies showed that over-expression ofGli1 in cultured cells activates Hh signaling in the absence of cilia;however, the requirement for cilia in Gli1 activation in vivo has beendifficult to address due to diminished Gli1 transcription in ciliamutants. In the current study, we restore Gli1 transcription in ciliamutants through a knock-in approach and find that Gli1 protein failsto activate Hh pathway in vivo in the absence of cilia, suggesting thatGli1 activation in vivo requires cilia. Subsequently, we show that allthree mouse Gli-family members share the same molecular mechan-ism for their ciliary localization, which appears to be independent oftheir phosphorylation and ubiquitylation. Finally, we show thatSuppressor of Fused (Sufu), an essential negative regulator of Hhsignaling, exerts its repressive function on Gli proteins independentof cilia. Association between Sufu and Gli proteins is independent ofcilia and is critical for proper translocation of both proteins.doi:10.1016/j.ydbio.2010.05.085Program/Abstract # 53The signaling properties in MAPK cycle are modulated by itsdownstream substratesTsuyoshi Hirashima

Inhibition of ERK1/2 prevents neural and mesendodermal differentiation and promotes human embryonic stem cell self-renewal

Extracellular signal-regulated kinases (ERKs) have many important functions during embryogenesis. However, their role in embryonic stem (ES) cells is controversial. Previous studies reported that, in contrast to mouse ES cells, human ES cells differentiate if ERK1/2 is inhibited. We reexamined the role of ERK1/2 in human ES cells using a chemically defined culture system and found that when ERK1/2 is blocked with specific chemical inhibitors, neural and mesendodermal differentiation is prevented, but cells become sensitive to BMP-induced differentiation. Inhibition of ERK1/2 significantly reduced the clonogenicity of human ES cells by preventing cell adhesion and survival. When this negative effect was avoided, we were able to maintain human ES cell self-renewal for more than 3 months in the presence of ERK1/2 inhibitors in a chemically defined culture system containing FGF2 and activin A but no BMP4. Our results suggest that the functional outcome of FGF/ERK1/2 signaling in human ES cells is influenced by the relative levels of activin A/TGFβ and BMP activity. Moreover, activation of ERK1/2 in human ES cells is required for proper neural and mesendodermal differentiation. In contrast to mouse ES cells, a low level of BMP4 is sufficient to initiate extraembryonic differentiation when ERK1/2 is inhibited.

SIP1 Mediates Cell-Fate Decisions between Neuroectoderm and Mesendoderm in Human Pluripotent Stem Cells

Human embryonic stem cells (hESCs) rely on fibroblast growth factor and Activin-Nodal signaling to maintain their pluripotency. However, Activin-Nodal signaling is also known to induce mesendoderm differentiation. The mechanisms by which Activin-Nodal signaling can achieve these contradictory functions remain unknown. Here, we demonstrate that Smad-interacting protein 1 (SIP1) limits the mesendoderm-inducing effects of Activin-Nodal signaling without inhibiting the pluripotency-maintaining effects exerted by SMAD2/3. In turn, Activin-Nodal signaling cooperates with NANOG, OCT4, and SOX2 to control the expression of SIP1 in hESCs, thereby limiting the neuroectoderm-promoting effects of SIP1. Similar results were obtained with mouse epiblast stem cells, implying that these mechanisms are evolutionarily conserved and may operate in vivo during mammalian development. Overall, our results reveal the mechanisms by which Activin-Nodal signaling acts through SIP1 to regulate the cell-fate decision between neuroectoderm and mesendoderm in the progression from pluripotency to primary germ layer differentiation.

Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.

Zygotic VegT is required for Xenopus paraxial mesoderm formation and is regulated by Nodal signaling and Eomesodermin

The T-box gene VegT plays a crucial role during mesendoderm specification of the amphibian embryo. While the function of maternal VegT (mVegT) has been extensively investigated, little is known about the function and transcriptional regulation of zygotic VegT (zVegT). In the present study, we used comparative genomics and a knockdown experiment to demonstrate that zVegT is the orthologous gene of zebrafish Spadetail/Tbx16 and chick Tbx6L/Tbx6, and has an essential role in paraxial mesodermal formation. zVegT knockdown embryos show several defects in the patterning of trunk mesoderm, such as abnormal segmentation of somites, a reduction in muscle, and the formation of an abnormal mass of cells at the tail tip. We also identified the cis-regulatory elements of zVegT that are necessary and sufficient for mesoderm-specific expression. These cis-regulatory elements are located in two separate upstream regions of zVegT, corresponding to the first intron of mVegT. The results of in vitro binding and functional assays indicate that Forkhead box H1 (FoxH1) and Eomesodermin (Eomes) are the trans-acting factors required for zVegT expression. Our results highlight the essential role of zVegT in organization of paraxial mesoderm, and reveal that zVegT is regulated by a coherent feedforward loop of Nodal signaling via Eomes.

Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin

Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells. Differentiating mouse ES cells were treated with suramin (500 µM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis. Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells.

Substrate stiffness affects early differentiation events in embryonic stem cells

Embryonic stem cells (ESC) are both a potential source of cells for tissue replacement therapies and an accessible tool to model early embryonic development. Chemical factors such as soluble growth factors and insoluble components of the extracellular matrix are known to affect the differentiation of murine ESCs. However, there is also evidence to suggest that undifferentiated cells can both sense the mechanical properties of their environment and differentiate accordingly. By growing ESCs on flexible polydimethylsiloxane substrates with varying stiffness, we tested the hypothesis that substrate stiffness can influence ESC differentiation. While cell attachment was unaffected by the stiffness of the growth substrate, cell spreading and cell growth were all increased as a function of substrate stiffness. Similarly, several genes expressed in the primitive streak during gastrulation and implicated in early mesendoderm differentiation, such as Brachyury, Mixl1 and Eomes, were upregulated in cell cultures on stiffer compared to softer substrates. Finally, we demonstrated that osteogenic differentiation of ESCs was enhanced on stiff substrates compared to soft substrates, illustrating that the mechanical environment can play a role in both early and terminal ESC differentiation. Our results suggest a fundamental role for mechanosensing in mammalian development and illustrate that the mechanical environment should be taken into consideration when engineering implantable scaffolds or when producing therapeutically relevant cell populations in vitro.

Identification of a Novel Negative Regulator of Activin/Nodal Signaling in Mesendodermal Formation of Xenopus Embryos

Phosphotyrosine binding (PTB) domains, which are found in a large number of proteins, have been implicated in signal transduction mediated by growth factor receptors. However, the in vivo roles of these PTB-containing proteins remain to be investigated. Here, we show that Xdpcp (Xenopus dok-PTB containing protein) has a pivotal role in regulating mesendoderm formation in Xenopus, and negatively regulates the activin/nodal signaling pathway. We isolated cDNA for xdpcp and examined its potential role in Xenopus embryogenesis. We found that Xdpcp is strongly expressed in the animal hemisphere at the cleavage and blastula stages. The overexpression of xdpcp RNA affects activin/nodal signaling, which causes defects in mesendoderm formation. In addition, loss of Xdpcp function by injection of morpholino oligonucleotides leads to the expansion of the mesodermal territory. Moreover, we found that axis duplication by ventrally forced expression of activin is recovered by coexpression with Xdpcp. In addition, Xdpcp inhibits the phosphorylation and nuclear translocation of Smad2. Furthermore, we also found that Xdpcp interacts with Alk4, a type I activin receptor, and inhibits activin/nodal signaling by disturbing the interaction between Smad2 and Alk4. Taken together, these results indicate that Xdpcp regulates activin/nodal signaling that is essential for mesendoderm specification.