Efficient and directive generation of two distinct endoderm lineages from human ESCs and iPSCs by differentiation stage-specific SOX17 transduction

The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively.     

Identification of an enhancer that controls up-regulation of fibronectin during differentiation of embryonic stem cells into extraembryonic endoderm

The extraembryonic endoderm is derived from inner cell mass cells of the blastocyst during early mouse embryogenesis. Formation of the extraembryonic endoderm, which later contributes to the yolk sac, appears to be a prerequisite for subsequent differentiation of the inner cell mass. While embryonic stem cells can be induced to differentiate into extraembryonic endoderm cells in vitro, the molecular mechanisms underlying this process are poorly understood. We used a promoter trap approach to search for genes that are expressed in embryonic stem cells and are highly up-regulated during differentiation to the extraembryonic endoderm fate. We showed that fibronectin fits this expression profile. Moreover we identified an enhancer in the 12th intron of the fibronectin locus that recapitulated the endogenous pattern of fibronectin expression. This enhancer carries Sox protein-binding sequences, and our analysis demonstrated that Sox7 and Sox17, which are highly expressed in the extraembryonic endoderm, were involved in enhancer activity.     

Regionalization of cell fates and cell movement in the endoderm of the mouse gastrula and the impact of loss of Lhx1(Lim1) function

Investigation of the developmental fates of cells in the endodermal layer of the early bud stage mouse embryo revealed a regionalized pattern of distribution of the progenitor cells of the yolk sac endoderm and the embryonic gut. By tracing the site of origin of cells that are allocated to specific regions of the embryonic gut, it was found that by late gastrulation, the respective endodermal progenitors are already spatially organized in anticipation of the prospective mediolateral and anterior-posterior destinations. The fate-mapping data further showed that the endoderm in the embryonic compartment of the early bud stage gastrula still contains cells that will colonize the anterior and lateral parts of the extraembryonic yolk sac. In the Lhx1(Lim1)-null mutant embryo, the progenitors of the embryonic gut are confined to the posterior part of the endoderm. In particular, the prospective anterior endoderm was sequestered to a much smaller distal domain, suggesting that there may be fewer progenitor cells for the anterior gut that is poorly formed in the mutant embryo. The deficiency of gut endoderm is not caused by any restriction in endodermal potency of the mutant epiblast cells but more likely the inadequate allocation of the definitive endoderm. The inefficient movement of the anterior endoderm, and the abnormal differentiation highlighted by the lack of Sox17 and Foxa2 expression, may underpin the malformation of the head of Lhx1 mutant embryos.     

Involvement of Ras in extraembryonic endoderm differentiation of embryonic stem cells

Embryonic stem (ES) cells, derived from the inner cell mass of blastocyst can differentiate into multiple cell lineages. In this study, we examined the possible involvement of Ras in ES cell differentiation. We found that Ras was activated upon formation of embryoid bodies (EBs), an initial step in ES cell differentiation. When expressed during EB differentiation, a dominant-negative mutant of Ras suppressed induction of marker genes for extraembryonic endoderm differentiation, including GATA-4, GATA-6, alpha-fetoprotein, and hepatocyte nuclear factor 3beta, while an activated mutant promoted their induction. Expression of a Ras mutant that selectively activates the Raf/MEK/Erk pathway also enhanced induction of extraembryonic endoderm markers, and treatment with a MEK inhibitor resulted in their decreased expression. In addition, Ras stimulated downregulation of Nanog, a suppressor of endoderm differentiation in ES cells. These data suggest that Ras activation during EB differentiation plays a crucial role in initiation of extraembryonic endoderm differentiation.     

Rat blastocyst-derived stem cells are precursors of embryonic and extraembryonic lineages

Despite recent advances in the derivation of rat embryonic stem cells, clear comprehension of the timing and mechanisms underlying rat early embryo lineage selection is lacking. We have previously shown the in vivo contribution of rat embryonic stem-like cells exclusively to developing extraembryonic tissues. To elucidate possible mechanisms governing the in vitro and in vivo behaviors of these rat blastocyst-derived stem cells, we evaluated their developmental capacity by using several approaches. Molecular marker analysis demonstrated the expression profile of genes characterizing not only pluripotency but also extraembryonic endoderm and trophoblast. In vitro differentiation through embryoid body formation showed in vitro pluripotent capacity through differentiation into derivatives of all three embryonic germ layers. Following either blastocyst injection, diploid or tetraploid aggregation, and embryo transfer, these rat blastocyst-derived stem cells also demonstrated in vivo multipotency through contribution to multiple developmentally distinct extraembryonic lineages. Features of phenotypic heterogeneity were revealed following examination of cell line morphology and culture behavior, as well as quantitative analysis of marker expression in discrete undifferentiated and differentiated populations of cells by flow cytometry. We demonstrate for the first time that stem cells derived from the rat blastocyst have the ability to contribute to the embryonic and extraembryonic lineages. Together, these results provide a valuable new model for rat stem cell biology and for the elucidation of early lineage selection in the embryo.     

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.     

Galpha13 activation rescues moesin-depletion induced apoptosis in F9 teratocarcinoma cells

Mouse F9 cells differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm in response to RA and dibutyryl (db-) cAMP. G protein signaling either blocks or mimics RA-induced differentiation, the latter signaling through the Wnt-beta-catenin pathway. In our study, we found that a constitutively active Galpha13 mutant induces F9 cells to differentiate into parietal endoderm in the absence of exogenous agents. Galpha13 expression and subsequent differentiation are accompanied by beta-catenin translocation to the nucleus. Differentiation and changes in cell morphology are supported by rearrangements to the F-actin cytoskeleton. ERM (ezrin-radixin-moesin) proteins, known to link F-actin to transmembrane receptors, are also redistributed during differentiation. Furthermore, morpholino antisense and shRNA approaches show that moesin expression is essential since its knockdown leads to altered F-actin distribution and subsequent apoptosis. Moesin-depleted cells, however, remain attached to the substrate when Galpha13 is constitutively expressed, but they do not differentiate into extraembryonic endoderm. Our study demonstrates a link between Galpha13 signaling that regulates differentiation of F9 cells through primitive to parietal endoderm and a moesin requirement for cell survival.     

Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies

We previously demonstrated that a member of the Hedgehog gene family, Indian hedgehog (Ihh), is expressed in the visceral endoderm of EC and ES cell embryoid bodies and mouse embryos. Overexpression studies suggested that Ihh was involved in visceral endoderm differentiation. We now provide evidence for a Hh response in the embryoid body core and in the mesothelial layer of the visceral yolk sac. We also demonstrate that treatment of ES embryoid bodies with the Hh antagonists cAMP and forskolin results in downregulation of the Hh response and altered embryoid body differentiation. The outer endoderm layer undergoes a transition to parietal endoderm while formation of an embryonic ectoderm layer surrounding a cavity is inhibited. These treatments also result in a decrease in the expression of markers for the mesoderm derivatives, blood and endothelial cells. We present a model to explain how Ihh and BMP signaling may regulate extraembryonic endoderm and embryonic ectoderm differentiation.     

BMP signaling induces visceral endoderm differentiation of XEN cells and parietal endoderm

The extraembryonic endoderm of mammals is essential for nutritive support of the fetus and patterning of the early embryo. Visceral and parietal endoderm are major subtypes of this lineage with the former exhibiting most, if not all, of the embryonic patterning properties. Extraembryonic endoderm (XEN) cell lines derived from the primitive endoderm of mouse blastocysts represent a cell culture model of this lineage, but are biased towards parietal endoderm in culture and in chimeras. In an effort to promote XEN cells to adopt visceral endoderm character we have mimicked different aspects of the in vivo environment. We found that BMP signaling promoted a mesenchymal-to-epithelial transition of XEN cells with up-regulation of E-cadherin and down-regulation of vimentin. Gene expression analysis showed the differentiated XEN cells most resembled extraembryonic visceral endoderm (exVE), a subtype of VE covering the extraembryonic ectoderm in the early embryo, and during gastrulation it combines with extraembryonic mesoderm to form the definitive yolk sac. We found that laminin, a major component of the extracellular matrix in the early embryo, synergised with BMP to promote highly efficient conversion of XEN cells to exVE. Inhibition of BMP signaling with the chemical inhibitor, Dorsomorphin, prevented this conversion suggesting that Smad1/5/8 activity is critical for exVE induction of XEN cells. Finally, we show that applying our new culture conditions to freshly isolated parietal endoderm (PE) from Reichert's membrane promoted VE differentiation showing that the PE is developmentally plastic and can be reprogrammed to a VE state in response to BMP. Generation of visceral endoderm from XEN cells uncovers the true potential of these blastocyst-derived cells and is a significant step towards modelling early developmental events ex vivo.     

Extraembryonic Endoderm cells as a model of endoderm development

In recent years the multipotent extraembryonic endoderm (XEN) stem cells have been the center of much attention. In vivo, XEN cells contribute to the formation of the extraembryonic endoderm, visceral and parietal endoderm and later on, the yolk sac. Recent data have shown that the distinction between embryonic and extraembryonic endoderm is not as strict as previously thought due to the integration, and not the displacement, of the visceral endoderm into the definitive embryonic endoderm. Therefore, cells from the extraembryonic endoderm also contribute to definitive endoderm. Many research groups focused on unraveling the potential and ability of XEN cells to both support differentiation and/or differentiate into endoderm‐like tissues as an alternative to embryonic stem (ES) cells. Moreover, the conversion of ES to XEN cells, shown recently without genetic manipulations, uncovers significant and novel molecular mechanisms involved in extraembryonic endoderm and definitive endoderm development. XEN cell lines provide a unique model for an early mammalian lineage that complements the established ES and trophoblast stem cell lines. Through the study of essential genes and signaling requirements for XEN cells in vitro, insights will be gained about the developmental program of the extraembryonic and embryonic endodermal lineage in vivo. This review will provide an overview on the current literature focusing on XEN cells as a model for primitive endoderm and possibly definitive endoderm as well as the potential of using these cells for therapeutic applications.     

Effects of Different Conditions of Retinoic Acid Treatment on Expression of MK Gene,which is Transiently Activated during Differentiation of Embryonal Carcinoma Cells1

MK gene was intensely expressed, when aggregates of HM-1 embryonal carcinoma (EC) cells were treated with retinoic acid for 2 days to induce the differntiation to nerve cells, myoblasts and extraembryonic endoderm cells. The conditions inhibiting nerve cell diffrentiation or extraembryonic endoderm cell differentiation affected MK gene expression only slightly. The maximum level of MK RNA was detected 2 days after initiation of retionic acid treatment, when cells were morphologically indistinguishable from undifferentiated EC cells. Thus, MK gene appears to be expressed in differentiating EC cells irrespective of the direction of differentiation. The degree of MK gene expression in sparsely cultured HM-1 cells correlated with the concentration of retinoic acid, especially between 10^-8 and 10^-7 M. When retinoic acid treatment was terminated after 1 day, the amount of MK RNA started to decrease. These two results are consistent with the view that retionic acid complexed with the receptor is directly involved in expression of MK gene.     

A strategy for immortalizing lines committed to endoderm, neuroectoderm or mesoderm from mouse teratocarcinoma

With the aim of immortalizing embryonic cells fixed at early embryonic stages, various plasmids carrying the SV40 early region were introduced into the mouse embryonal carcinomas (EC) F9 and 1003. Only the construction PK4, in which the SV40 oncogenes are placed under the control of the adenovirus E1A promoter, led to the immortalization of the cells at the onset of differentiation. Clones corresponding to committed precursors of each embryonic lineage (neuroectoderm, mesoderm and endoderm) were then selected with high efficiency according to the following strategy: selection of immature cells which: have lost EC cell markers, keep a stable phenotype, are immortalized by the expression of the SV40 oncogenes and are still able to differentiate along a restricted lineage in vitro or in vivo. Examples of an endodermal precursor (H7) which differentiates into extraembryonic and embryonic endoderm, of a neuroectodermic clone (ICII) committed to a serotoninergic differentiation, and of a mesodermal osteogenic clone (CI) which gives rise to bone in vivo and in vitro, are given.