Endodermal Gene Expression Research Articles

Differential responses to Wnt and PCP disruption predict expression and developmental function of conserved and novel genes in a cnidarian.

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at “oral” and “aboral” poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.

Long noncoding RNAs are spatially correlated with transcription factors and regulate lung development.

Long noncoding RNAs (lncRNAs) are thought to play important roles in regulating gene transcription, but few have well-defined expression patterns or known biological functions during mammalian development. Using a conservative pipeline to identify lncRNAs that have important biological functions, we identified 363 lncRNAs in the lung and foregut endoderm. Importantly, we show that these lncRNAs are spatially correlated with transcription factors across the genome. In-depth expression analyses of lncRNAs with genomic loci adjacent to the critical transcription factors Nkx2.1, Gata6, Foxa2 (forkhead box a2), and Foxf1 mimic the expression patterns of their protein-coding neighbor. Loss-of-function analysis demonstrates that two lncRNAs, LL18/NANCI (Nkx2.1-associated noncoding intergenic RNA) and LL34, play distinct roles in endoderm development by controlling expression of critical developmental transcription factors and pathways, including retinoic acid signaling. In particular, we show that LL18/NANCI acts upstream of Nkx2.1 and downstream from Wnt signaling to regulate lung endoderm gene expression. These studies reveal that lncRNAs play an important role in foregut and lung endoderm development by regulating multiple aspects of gene transcription, often through regulation of transcription factor expression.

Effects of Fluid Dynamic Forces Created by Rotary Orbital Suspension Culture on Cardiomyogenic Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the potential to become an effective resource for regenerative medicine in heart disease. The efficiency of cardiomyocyte differentiation changes drastically depending on the quality of hESC aggregates known as embryoid bodies (EBs). This study compares the production of EBs in rotary orbital suspension culture at various speeds with that in a simple static suspension culture. None of the outgrowths of EBs formed in static suspension culture developed beating. In contrast, outgrowths from EBs formed at 100 rpm had the highest rate of beating, 70%, and increased the expression of Nkx2.5, a master gene of cardiomyocyte differentiation. Outgrowths from EBs developed at slower rotational speeds showed more endoderm gene expression. At faster speeds, the expression of ectoderm markers increased. A computational hydrodynamic simulation showed that the liquid medium shear stress on the bottom was restricted to the periphery at 30 and 55 rpm and the center at 120 rpm. The analysis showed that at 100 rpm, the shear stress was uniformly distributed in the dish. Our results suggest that shear stress by fluid dynamic forces induces the differentiation of specific cell phenotypes from hESCs depending on rotational speed. For cardiomyocyte differentiation, 100 rpm was the most effective speed.

Valproic acid promotes differentiation of hepatocyte-like cells from whole human umbilical cord-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are mesoderm-derived cells that are considered a good source of somatic cells for treatment of many degenerative diseases. Previous studies have reported the differentiation of mesodermal MSCs into endodermal and ectodermal cell types beyond their embryonic lineages, including hepatocytes and neurons. However, the molecular pathways responsible for the direct or indirect cell type conversion and the functional ability of the differentiated cells remain unclear and need further research. In the present study, we demonstrated that valproic acid (VPA), which is a histone deacetylase inhibitor, induced an increase in the expression of endodermal genes including CXCR4, SOX17, FOXA1, FOXA2, GSC, c-MET, EOMES, and HNF-1β in human umbilical cord derived MSCs (hUCMSCs). In addition, we found that VPA is able to increase these endodermal genes in hUCMSCs by activating signal transduction of AKT and ERK. VPA pretreatment increased hepatic differentiation at the expense of adipogenic differentiation. The effects of VPA on modulating hUCMSCs fate were diminished by blocking AKT and ERK activation using specific signaling inhibitors. Together, our results suggest that VPA contributes to the lineage conversion of hUCMSCs to hepatic cell fate by upregulating the expression of endodermal genes through AKT and ERK activation.

Oct4 switches partnering from Sox2 to Sox17 to reinterpret the enhancer code and specify endoderm

How regulatory information is encoded in the genome is poorly understood and poses a challenge when studying biological processes. We demonstrate here that genomic redistribution of Oct4 by alternative partnering with Sox2 and Sox17 is a fundamental regulatory event of endodermal specification. We show that Sox17 partners with Oct4 and binds to a unique 'compressed' Sox/Oct motif that earmarks endodermal genes. This is in contrast to the pluripotent state where Oct4 selectively partners with Sox2 at 'canonical' binding sites. The distinct selection of binding sites by alternative Sox/Oct partnering is underscored by our demonstration that rationally point-mutated Sox17 partners with Oct4 on pluripotency genes earmarked by the canonical Sox/Oct motif. In an endodermal differentiation assay, we demonstrate that the compressed motif is required for proper expression of endodermal genes. Evidently, Oct4 drives alternative developmental programs by switching Sox partners that affects enhancer selection, leading to either an endodermal or pluripotent cell fate. This work provides insights in understanding cell fate transcriptional regulation by highlighting the direct link between the DNA sequence of an enhancer and a developmental outcome.

Inositol-requiring enzyme 1α is required for gut development inXenopus laviesembryos

To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos. Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH. One and half nanogram of IRE1α, 1 ng of IRE1α-GR mRNA, 1 ng of IRE1αΔC-GR mRNA, and 50 ng of IRE1α morpholino oligonucleotide (MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis. To rescue the effect of IRE1α MO, 1 ng of IRE1α-GR mRNA was co-injected with 50 ng of MO. For the activation of the GR-fusion proteins, dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1α, IRE1β, Xbra and Xsox17α. IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting. In the whole-mount in situ hybridization analysis, xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1α was observed in the pancreas, and significant transcription of IRE1β was found in the liver. IRE1α protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1α-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages. We did not observe a significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage. IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.

Enhancement of osteogenic and chondrogenic differentiation of human embryonic stem cells by mesodermal lineage induction with BMP-4 and FGF2 treatment

Recently, it was reported that bone morphogenetic protein 4 (BMP4) alone or BMP4 combined with fibroblast growth factor 2 (FGF2) treatment enhanced mesodermal differentiation of human embryonic stem cells (hESCs) that were cultured feeder-free on Matrigel. In this study, we show that mesodermal lineage-induced embryoid bodies (EBs) generate greater numbers of osteogenic and chondrogenic lineage cells. To induce the mesodermal lineage, hESCs were treated with BMP4 and FGF2 during the EB state. Quantitative real-time reverse transcription-polymerase chain reaction analysis showed that the treatment decreased endodermal and ectodermal lineage gene expression and increased mesodermal lineage gene expression. Importantly, the mesodermal lineage-induced EBs underwent enhanced osteogenic and chondrogenic differentiation after differentiation induction. This method could be useful to enhance the osteogenic or chondrogenic differentiation of hESCs.

The Histone Methyltransferase Inhibitor BIX01294 Enhances the Cardiac Potential of Bone Marrow Cells

Bone marrow (BM) has long been considered a potential stem cell source for cardiac repair due to its abundance and accessibility. Although previous investigations have generated cardiomyocytes from BM, yields have been low, and far less than produced from ES or induced pluripotent stem cells (iPSCs). Since differentiation of pluripotent cells is difficult to control, we investigated whether BM cardiac competency could be enhanced without making cells pluripotent. From screens of various molecules that have been shown to assist iPSC production or maintain the ES cell phenotype, we identified the G9a histone methyltransferase inhibitor BIX01294 as a potential reprogramming agent for converting BM cells to a cardiac-competent phenotype. BM cells exposed to BIX01294 displayed significantly elevated expression of brachyury, Mesp1, and islet1, which are genes associated with embryonic cardiac progenitors. In contrast, BIX01294 treatment minimally affected ectodermal, endodermal, and pluripotency gene expression by BM cells. Expression of cardiac-associated genes Nkx2.5, GATA4, Hand1, Hand2, Tbx5, myocardin, and titin was enhanced 114, 76, 276, 46, 635, 123, and 5-fold in response to the cardiogenic stimulator Wnt11 when BM cells were pretreated with BIX01294. Immunofluorescent analysis demonstrated that BIX01294 exposure allowed for the subsequent display of various muscle proteins within the cells. The effect of BIX01294 on the BM cell phenotype and differentiation potential corresponded to an overall decrease in methylation of histone H3 at lysine9, which is the primary target of G9a histone methyltransferase. In summary, these data suggest that BIX01294 inhibition of chromatin methylation reprograms BM cells to a cardiac-competent progenitor phenotype.

Effect of Hypoxia on Neural Induction in Colonies of Human Parthenogenetic Stem Cells

We studied neural induction and generation of neuroectoderm in the colonies of human parthenogenetic SC cultured in the presence of 5 and 19±2% oxygen. We found that neuroectoderm was more actively generated at high oxygen content. At the same time, the transcription of stem cell pluripotency genes was not completely suppressed during neural induction at low oxygen content, while the expression of endoderm and mesodermal marker genes attested to the absence of specific differentiation. These findings demonstrate more efficient neuroectoderm generation induced in the colonies of pluripotent stem cells under conditions of normoxia.

Hepatic differentiation of human embryonic stem cells on growth factor-containing surfaces

Embryonic stem cells (ESCs) hold considerable promise in tissue engineering and regenerative medicine as a source of tissue-specific cells. Hepatocytes derived from ESCs will be useful for therapies, bioartificial liver assistance devices and drug discovery. In traditional stem cell cultivation/differentiation experiments, growth factors (GFs) are added in soluble form in order to provide signals for tissue-specific differentiation. In contrast, we investigated differentiation of hESCs cultured on top of GFs. In these experiments, glass substrates were imprinted with a mixture of ECM and GF molecules to form 500 µm diameter spots. hESCs were cultured onto these GF-containing ECM spots for up to 12 days to induce differentiation towards the hepatic lineage. The dynamics of differentiation were examined by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and immunocytochemistry. Stem cells cultured on GF-containing surfaces stained positive for the endoderm markers SOX17 and FOXA2, as well as early liver markers such as α-fetoprotein and albumin. qRT-PCR confirmed that pluripotency, endoderm and liver gene expression of hESCs cultured on GF-containing surfaces was consistent with hepatic differentiation. In comparison, hESCs cultured on ECM spots without GFs showed less pronounced loss of pluripotency and lower levels of liver gene expression. In summary, our study demonstrates that hESCs receive differentiation-inducing signals from GF-containing surfaces and may be pushed along the hepatic lineage when cultured on such surfaces. Compared to traditional approaches, the advantages of GF immobilization include reduction in the cost of experiments, stronger and longer stimulation and the possibility of screening GF-stem cell interactions in a multiplexed manner.

XenopusNanos1 is required to prevent endoderm gene expression and apoptosis in primordial germ cells

Nanos is expressed in multipotent cells, stem cells and primordial germ cells (PGCs) of organisms as diverse as jellyfish and humans. It functions together with Pumilio to translationally repress targeted mRNAs. Here we show by loss-of-function experiments that Xenopus Nanos1 is required to preserve PGC fate. Morpholino knockdown of maternal Nanos1 resulted in a striking decrease in PGCs and a loss of germ cells from the gonads. Lineage tracing and TUNEL staining reveal that Nanos1-deficient PGCs fail to migrate out of the endoderm. They appear to undergo apoptosis rather than convert to normal endoderm. Whereas normal PGCs do not become transcriptionally active until neurula, Nanos1-depleted PGCs prematurely exhibit a hyperphosphorylated RNA polymerase II C-terminal domain at the midblastula transition. Furthermore, they inappropriately express somatic genes characteristic of endoderm regulated by maternal VegT, including Xsox17α, Bix4, Mixer, GATA4 and Edd. We further demonstrate that Pumilio specifically binds VegT RNA in vitro and represses, along with Nanos1, VegT translation within PGCs. Repressed VegT RNA in wild-type PGCs is significantly less stable than VegT in Nanos1-depleted PGCs. Our data indicate that maternal VegT RNA is an authentic target of Nanos1/Pumilio translational repression. We propose that Nanos1 functions to translationally repress RNAs that normally specify endoderm and promote apoptosis, thus preserving the germline.

Functional analyses of vertebrate TCF proteins in C. elegans embryos

In the canonical Wnt pathway, signaling results in the stabilization and increased levels of β-catenin in responding cells. β-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in β-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognizes specific promoter sequences upstream of endoderm genes and interacts with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans β-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.

The zebrafish maternal-effect gene mission impossible encodes the DEAH-box helicase Dhx16 and is essential for the expression of downstream endodermal genes

Early animal embryonic development requires maternal products that drive developmental processes prior to the activation of the zygotic genome at the mid-blastula transition. During and after this transition, maternal products may continue to act within incipient zygotic developmental programs. Mechanisms that control maternally-inherited products to spatially and temporally restrict developmental responses remain poorly understood, but necessarily depend on posttranscriptional regulation. We report the functional analysis and molecular identification of the zebrafish maternal-effect gene mission impossible (mis). Our studies suggest requirements for maternally-derived mis function in events that occur during gastrulation, including cell movement and the activation of some endodermal target genes. Cell transplantation experiments show that the cell movement defect is cell autonomous. Within the endoderm induction pathway, mis is not required for the activation of early zygotic genes, but is essential to implement nodal activity downstream of casanova/sox 32 but upstream of sox17 expression. Activation of nodal signaling in blastoderm explants shows that the requirement for mis function in endoderm gene induction is independent of the underlying yolk cell. Positional cloning of mis, including genetic rescue and complementation analysis, shows that it encodes the DEAH-box RNA helicase Dhx16, shown in other systems to act in RNA regulatory processes such as splicing and translational control. Analysis of a previously identified insertional dhx16 mutation shows that the zygotic component of this gene is also essential for embryonic viability. Our studies provide a striking example of the interweaving of maternal and zygotic genetic functions during the egg-to-embryo transition. Maternal RNA helicases have long been known to be involved in the development of the animal germ line, but our findings add to growing evidence that these factors may also control specific gene expression programs in somatic tissues.

Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Maternal and Zygotic aldh1a2 Activity Is Required for Pancreas Development in Zebrafish

We have isolated and characterized a novel zebrafish pancreas mutant. Mutant embryos lack expression of isl1 and sst in the endocrine pancreas, but retain isl1 expression in the CNS. Non-endocrine endodermal gene expression is less affected in the mutant, with varying degrees of residual expression observed for pdx1, carbA, hhex, prox1, sid4, transferrin and ifabp. In addition, mutant embryos display a swollen pericardium and lack fin buds. Genetic mapping revealed a mutation resulting in a glycine to arginine change in the catalytic domain of the aldh1a2 gene, which is required for the production of retinoic acid from vitamin A. Comparison of our mutant (aldh1a2um22) to neckless (aldh1a2i26), a previously identified aldh1a2 mutant, revealed similarities in residual endodermal gene expression. In contrast, treatment with DEAB (diethylaminobenzaldehyde), a competitive reversible inhibitor of Aldh enzymes, produces a more severe phenotype with complete loss of endodermal gene expression, indicating that a source of Aldh activity persists in both mutants. We find that mRNA from the aldh1a2um22 mutant allele is inactive, indicating that it represents a null allele. Instead, the residual Aldh activity is likely due to maternal aldh1a2, since we find that translation-blocking, but not splice-blocking, aldh1a2 morpholinos produce a phenotype similar to DEAB treatment. We conclude that Aldh1a2 is the primary Aldh acting during pancreas development and that maternal Aldh1a2 activity persists in aldh1a2um22 and aldh1a2i26 mutant embryos.

BMPs and Chordin regulate patterning of the directive axis in a sea anemone

The TGF-beta molecules Dpp/BMP2/4/7 and their antagonist Sog/Chd play a conserved role in establishing the dorso-ventral (DV) axis in bilaterians. Homologues of BMPs and the antagonist, Chordin, have been isolated from Cnidaria and show a striking asymmetric expression pattern with respect to the primary oral-aboral (OA) axis. We used Morpholino knockdowns of Nematostella dpp (bmp2/4), bmp5-8, chordin, and tolloid to investigate their function during early development of the sea anemone Nematostella vectensis. Molecular analysis of the BMP Morpholino phenotypes revealed an upregulated and radialized expression of bmps and chordin in ectoderm and endoderm indicating a negative feedback loop. Our data further suggest that BMP signaling is required for symmetry breaking of bmp and chordin expression during gastrulation. While bmps and chordin marker genes of the ectodermal OA axis extended aborally, other ectodermal markers of the OA axis were not significantly affected. By contrast, expression of other endodermal marker genes marking both the OA and the directive axis were abolished. Our data suggest that the logic of BMP2/4 signaling and the BMP antagonist, Chordin, differs significantly between Cnidaria and Bilateria, yet the double negative feedback loop detected in Nematostella bears systemic similarities with part of the regulatory network of the DV axis patterning system in amphibians.

VEGF and IHH rescue definitive hematopoiesis in Gata-4 and Gata-6–deficient murine embryoid bodies

Murine embryonic stem cells can be differentiated into embryoid bodies (EBs), which serve as an in vitro model recapitulating many aspects of embryonic yolk sac hematopoiesis. Differentiation of embryonic stem cells deficient in either Gata-4 or Gata-6 results in EBs with disrupted visceral endoderm (VE). While lack of VE has detrimental effects on hematopoiesis in vivo, it is unclear whether lack of VE affects hematopoiesis in EBs. Therefore, we compared Gata-4 null (G4N) and Gata-6 null (G6N) EBs with wild-type EBs to assess their ability to commit to hematopoietic cells. EB VE formation was examined using cell-sorting techniques and analysis visceral endoderm gene expression. Hematopoietic progenitor potential of EBs cultured under various conditions was assessed using colony-forming assays. Definitive erythroid, granulocyte-macrophage, and mixed colonies were significantly reduced in G4N and G6N EBs compared to wild-type EBs. Vascular endothelial growth factor (VEGF) expression and secretion were also reduced in both G4N and G6N EBs, consistent with VE serving as a site of VEGF production. Addition of exogenous VEGF(165), to EB cultures completely rescued definitive colony-forming cells in G4N and G6N EBs. This rescue response could be blocked by addition of soluble Flk-1 Fc to EB cultures. Similarly, addition of exogenous Indian hedgehog to EB cultures also recovers the diminishment in definitive hematopoiesis in a reversible manner. These results suggest that the absence of VE in G4N and G6N EBs does not prevent emergence of definitive progenitors from EBs. However, the decreased level of VEGF and Indian hedgehog production in VE devoid G4N and G6N EBs attenuates definitive hematopoietic progenitor cell expansion.

In vitro Differentiation of Human Cord Blood-Derived Unrestricted Somatic Stem Cells into Hepatocyte-Like Cells on Poly(ε-Caprolactone) Nanofiber Scaffolds

Tissue engineering of implantable cellular constructs is an emerging cellular therapy for hepatic disease. In this study, we tested the ability of poly(ε-caprolactone) (PCL) nanofiber scaffold to support and maintain hepatic differentiation of human cord blood-derived unrestricted somatic stem cells (USSCs) in vitro. USSCs, self-renewing pluripotent cells, were isolated from human cord blood. The electrospun PCL nanofiber porous scaffold was constructed of uniform, randomly oriented nanofibers. USSCs were seeded onto PCL nanofiber scaffolds, and were induced to differentiate into hepatogenic lineages by culturing with differentiation factors for 6 weeks. RT-PCR analysis of endoderm and hepatic-specific gene expression, immunohistochemical detection of cytokeratin 18 (CK-18), α-fetoprotein, albumin, glycogen storage and indocyanine green uptake confirmed the differentiation of USSCs into endoderm and hepatocyte-like cells. In the present study, we show that hepatocyte-like cells differentiated from USSCs on the PCL nanofiber scaffold can be candidate for tissue engineering and cell therapy of hepatic tissues.

MicroRNA expression patterns and function in endodermal differentiation of human embryonic stem cells.

Background/AimsmicroRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. Human embryonic stem cells (hESC), which exhibit the characteristics of pluripotency and self-renewal, may serve as a model to study the role of miRNAs in early human development. We aimed to determine whether endodermally-differentiated hESC demonstrate a unique miRNA expression pattern, and whether overexpression of endoderm-specific miRNA may affect hESC differentiation.MethodsmiRNA expression was profiled in undifferentiated and NaButyrate-induced differentiated hESC of two lines, using microarray and quantitative RT-PCR. Then, the effect of lentiviral-based overexpression of liver-specific miR-122 on hESC differentiation was analyzed, using genomewide gene microarrays.ResultsThe miRNA profiling revealed expression of three novel miRNAs in undifferentiated and differentiated hESC. Upon NaButyrate induction, two of the most upregulated miRNAs common to both cell lines were miR-24 and miR-10a, whose target genes have been shown to inhibit endodermal differentiation. Furthermore, induction of several liver-enriched miRNAs, including miR-122 and miR-192, was observed in parallel to induction of endodermal gene expression. Stable overexpression of miR-122 in hESC was unable to direct spontaneous differentiation towards a clear endodermal fate, but rather, delayed general differentiation of these cells.ConclusionsOur results demonstrate that expression of specific miRNAs correlates with that of specific genes upon differentiation, and highlight the potential role of miRNAs in endodermal differentiation of hESC.

The RNA-binding protein XSeb4R: a positive regulator of VegT mRNA stability and translation that is required for germ layer formation in Xenopus

VegT represents a localized maternal determinant essentially required for endoderm formation in Xenopus. Here, we report on the identification of the RNA-binding protein XSeb4R as a positive regulator of VegT. XSeb4R interacts directly with the 3'-untranslated region of VegT mRNA, stabilizes it, and stimulates translation. Ablation of XSeb4R activity results in impairment of endoderm and mesoderm formation, while ectopic expression of XSeb4R in ectodermal cells induces endodermal and mesodermal gene expression. These observations unravel a novel mode of VegT regulation at the post-transcriptional level that is essential for germ layer formation in Xenopus.