The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF)

LIF (leukaemia inhibitory factor) is commonly used to maintain mouse embryonic stem cells in an undifferentiated state. These cells spontaneously differentiate when allowed to aggregate in the absence of LIF, forming embryoid bodies in which early embryonic cell lineages develop. Using embryoid bodies cultured in the presence and absence of LIF, we show that although LIF inhibited the development of visceral and parietal endodermal cells, it did not affect the differentiation of the primitive endodermal cell precursors of these extraembryonic cell lineages. Furthermore, deposition of the basement membrane produced by the primitive endodermal cells, which separates them from the remaining cells of the embryoid body, still occurred. The differentiation of primitive ectodermal cells and their progeny was inhibited by LIF, as evidenced by the lack of expression of FGF-5, muscle, and neuronal markers. However, cavitation of the embryoid body and maintenance of the cells in contact with the primitive endodermal basement membrane as an epiblast epithelium still occurred normally in the presence of LIF. These results indicate that cavitation and formation of the epiblast epithelium are regulated by mechanisms distinct from those controlling the differentiation of epiblast cell lineages. Furthermore, although epithelium formation and cavitation do not require the differentiation of visceral endodermal cells, the results are consistent with the hypothesis that the primitive endodermal basement membrane is sufficient to induce the epithelialization of undifferentiated embryonic stem cells necessary for cavitation.     

Rac1 Is Essential for Basement Membrane-Dependent Epiblast Survival

During murine peri-implantation development, the egg cylinder forms from a solid cell mass by the apoptotic removal of inner cells that do not contact the basement membrane (BM) and the selective survival of the epiblast epithelium, which does. The signaling pathways that mediate this fundamental biological process are largely unknown. Here we demonstrate that Rac1 ablation in embryonic stem cell-derived embryoid bodies (EBs) leads to massive apoptosis of epiblast cells in contact with the BM. Expression of wild-type Rac1 in the mutant EBs rescues the BM-contacting epiblast, while expression of a constitutively active Rac1 additionally blocks the apoptosis of inner cells and cavitation, indicating that the spatially regulated activation of Rac1 is required for epithelial cyst formation. We further show that Rac1 is activated through integrin-mediated recruitment of the Crk-DOCK180 complex and mediates BM-dependent epiblast survival through activating the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Our results reveal a signaling cascade triggered by cell-BM interactions essential for epithelial morphogenesis.All epithelial sheets and tubes rest upon a basement membrane (BM), a thin mat of specialized extracellular matrix (ECM) consisting of laminins, type IV collagens, perlecan, and nidogens. The BM provides essential survival signals to protect epithelial cells from apoptosis, in addition to its role in cell adhesion, migration, proliferation, and polarity orientation. In the developing chick retina, removal of the retinal BM by collagenase digestion resulted in severe apoptosis of retinal neuroepithelial cells (17). In mice, targeted deletion of the genes for the BM component laminins or perlecan caused BM defects and various degrees of apoptosis of cells that attach to the BM (34, 41, 42). Also, mammary epithelial cells can survive for a long period of time when grown on a reconstituted basement membrane derived from Engelbreth-Holmof Swarm (EHS) tumor (Matrigel), but they die by apoptosis when grown on plastic, fibronectin, or type I collagen despite their firm attachment on these substrates (2, 11, 36). A similar response of keratinocytes to BM type IV collagen versus non-BM matrix proteins was observed in bioengineered human skin equivalents (40). These results suggest that the BM provides a unique microenvironment for the survival of associated epithelial cells.Embryoid body (EB) differentiation has been used to study epithelial morphogenesis and early embryogenesis. When cultured in suspension as small aggregates, mouse embryonic stem (ES) cells adhere strongly together and form spherical EBs. The outer cells of the EB differentiate to become endoderm cells, which secrete laminins, type IV collagen, perlecan, and other BM components that assemble into an underlying BM equivalent to the embryonic BM separating extraembryonic endoderm from the epiblast. Integrin α6β1 in the epiblast cells and integrin α5β1 in the endoderm cells redistribute from a pericellular location to a predominantly sub-basement membrane location (28). Following BM formation, the epiblast cells adjacent to the BM polarize to become a pseudostratified columnar epithelium (the epiblast epithelium), whereas the inner cells not in contact with the BM undergo apoptosis and are selectively removed by phagocytosis/autophagy, creating a proamniotic-like cavity. That the BM is essential for these sequential processes is evidenced by the observation that targeted deletion of the laminin γ1 gene in EBs blocks BM assembly, subsequent epiblast epithelialization, and then apoptosis-dependent cavitation (32, 42). These differentiation processes recapitulate peri-implantation development and provide a tractable in vitro model for the study of apoptosis and BM-dependent cell survival during epithelial morphogenesis.While BM-dependent cell survival is often coupled with apoptotic removal of centrally located cells not in contact with the BM during morphogenesis of epithelial cysts such as mammary glandular acini and embryonic mouse egg cylinders (7, 29), the molecular mechanisms underlying this fundamental process are poorly understood. Elegant studies on teratocarcinoma cell-derived EBs have suggested that formation of an epithelial cyst as they develop is the result of the interplay of two signals (7). One is a death signal from the endoderm that induces apoptosis of the centrally located cells to create a cavity; the other is a rescue signal mediated by contact with the BM and is required for the survival of the newly formed epiblast epithelium. Subsequent studies have revealed that bone morphogenetic protein 2 (BMP-2) is highly expressed in the endoderm and that expression of a dominant-negative (DN) BMP receptor in EBs blocked cavitation, suggesting BMP-2 to be a death factor (6). The survival signals from the interaction of the epiblast cells with the BM were studied by treating the EBs with polyclonal antiserum against membrane glycoproteins consisting of ECM adhesion receptors. The antiserum treatment induced programmed cell death in the BM-contacting epiblast layer. However, the identities of the receptors and the downstream signaling molecules involved have not been explored.In this study, we utilized EBs differentiated from genetically modified ES cells to investigate the mechanisms of BM-dependent cell survival. We show that targeted deletion of the Rac1 gene in EBs leads to massive apoptosis of epiblast cells in contact with the BM. Rac1 is activated in a BM- and integrin-dependent fashion. Stable expression of wild-type Rac1 in the mutant EBs rescues the BM-contacting epiblast, while expression of a constitutively active Rac1 also blocks the apoptosis of inner cells and cavitation. These results suggest that the spatial activation of Rac1 is essential not only for BM-dependent epiblast survival but also for apoptosis-mediated cavitation. We further show that Crk mediates Rac1 activation by recruiting the Rac1-specific activator DOCK180 to the cell-BM adhesions and that the phosphatidylinositol 3-kinase (PI3K)-Akt pathway acts downstream of Rac1 to promote BM-dependent survival. Collectively, our results have established a key role for Rac1 in embryonic epithelial morphogenesis and have uncovered a signaling pathway that mediates BM-dependent epithelial survival.     

Talin1 regulates integrin turnover to promote embryonic epithelial morphogenesis

Talin is a cytoskeletal protein that binds to integrin β cytoplasmic tails and regulates integrin activation. Talin1 ablation in mice disrupts gastrulation and causes embryonic lethality. However, the role of talin in mammalian epithelial morphogenesis is poorly understood. Here we demonstrate that embryoid bodies (EBs) differentiated from talin1-null embryonic stem cells are defective in integrin adhesion complex assembly, epiblast elongation, and lineage differentiation. These defects are accompanied by a significant reduction in integrin β1 protein levels due to accelerated degradation through an MG-132-sensitive proteasomal pathway. Overexpression of integrin β1 or MG-132 treatment in mutant EBs largely rescues the phenotype. In addition, epiblast cells isolated from talin1-null EBs exhibit impaired cell spreading and focal adhesion formation. Transfection of the mutant cells with green fluorescent protein (GFP)-tagged wild-type but not mutant talin1 that is defective in integrin binding normalizes integrin β1 protein levels and restores focal adhesion formation. Significantly, cell adhesion and spreading are also improved by overexpression of integrin β1. All together, these results suggest that talin1 binding to integrin promotes epiblast adhesion and morphogenesis in part by preventing integrin β1 degradation.     

Cell autonomous sorting and surface positioning in the formation of primitive endoderm in embryoid bodies

The differentiation and formation of the primitive endoderm in early embryos can be mimicked in vitro by the aggregation of embryonic stem cells to form embryoid bodies. We present morphological evidence that primitive endoderm cells often first locate in the interior of embryoid bodies and subsequently migrate to the surface. Cell mixing experiments indicate that surface positioning is an intrinsic property of endoderm epithelial cells. Moreover, Disabled-2 (Dab2) is required for surface sorting and positioning of the endoderm cells: when Dab2 expression was eliminated, the differentiated endoderm epithelial cells distributed throughout the interior of the embryoid bodies. Surprisingly, E-cadherin is dispensable for primitive endoderm differentiation and surface sorting in embryoid bodies. These results support the model that primitive endoderm cells first emerge in the interior of the inner cell mass and are subsequently sorted to the surface to form the primitive endoderm.     

Cell death activation during cavitation of embryoid bodies is mediated by hydrogen peroxide

The formation of the proamniotic cavity is the first indication of programmed cell death associated to a morphogenetic process in mammals. Although some growth factors have been implicated in proamniotic cavitation, very little is known about the intracellular mechanisms that control the cell death process itself. Reactive oxygen species (ROS) are potent activators of cell death, thus, in the present work we evaluated the role of ROS during the cavitation of embryoid bodies (EBs), a common model to study proamniotic cavitation. During cavitation, ROS concentration increases in the inner cells of EBs, and this ROS accumulation appears to be associated with the mitochondrial respiratory activity. In agreement with a role of ROS in cavitation, EBs derived from ES cells that overproduce catalase, an enzyme that specifically degrades hydrogen peroxide, do not cavitate, and caspase activation and cell death is markedly decreased. Notably, cell death, but not the rise in ROS, during EB cavitation is caspase-dependent. The apoptosis-inducing factor (Aif) is released from the mitochondria during cavitation, but EBs derived from Aif^−/y ES cells cavitate and ROS levels in the inner cells remain high. We conclude that hydrogen peroxide is a cell death activating signal essential for EB cavitation, suggesting that cell death during proamniotic cavitation is mediated by ROS.     

小鼠胚胎干细胞分化形成拟胚体过程中的细胞程序性死亡

为了检测小鼠胚胎干细胞 (embryonicstemcell ,ES细胞 )体外分化的拟胚体 (embryoidbodies ,EBs)形成过程中细胞程序性死亡 (programmedcelldeath ,PCD)的发生 ,通过悬滴、悬浮培养技术定向诱导未分化的ES细胞分化为拟胚体 ,并用RT PCR检测原始内胚层、原始外胚层、中胚层、内脏内胚层 4种分子标记物在EBs中的表达 .通过TUNEL染色、电镜、激光共聚焦显微镜及Western印迹以确定凋亡发生 .结果表明 :ES细胞体外分化为拟胚体并且表达各胚层相应的分子标记物 ;在拟胚体的发育过程中出现明显的空腔化过程 ,TUNEL染色及电镜观察到凋亡生成 ,同时线粒体膜电位 (ΔΨm)在拟胚体发育过程中降低 ,通过Western印迹检测到caspase3、caspase8的激活 .表明小鼠ES细胞所分化的拟胚体可以作为研究早期胚胎发育的实验模型 ,线粒体在拟胚体的细胞程序性死亡过程中发挥重要的作用 .为进一步利用拟胚体研究细胞程序性死亡及相关信号分子在小鼠胚胎发育早期的作用奠定了基础     

Formation of a Polarised Primitive Endoderm Layer in Embryoid Bodies Requires Fgfr/Erk Signalling

The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.     

Amine oxidases, programmed cell death, and tissue renewal

Embryonal carcinoma cells, with embryonic (ECaE) or trophectodermal (ECaT) potential, have been used in a colony assay to determine regulatory mechanisms in the blastocyst. The mechanism that regulates ECaE and results in chimera formation is dependent upon a soluble factor in blastocoele fluid and contact with trophectoderm. Two mechanisms contribute to the regulation of ECaT: one involves a factor in blastocoele fluid and the other contact with either trophectoderm or inner cell mass which results in differentiation of the cells into trophectoderm, and the other involves the killing of at least 40% of the cells by blastocoele fluid alone. This cytotoxic activity probably causes the programmed cell death that occurs in the inner cell mass during blastulation as it loses the potential to differentiate into trophectoderm. A toxic activity similar to that of normal blastocysts has been obtained from embryoid bodies. This activity is caused by amine oxidase-dependent catabolism of polyamines, and it is postulated that programmed cell death in the embryo and chalone activity in the adult may have similar mechanisms.     

Expression and biological role of laminin-1.

Of the approximately 15 laminin trimers described in mammals, laminin-1 expression seems to be largely limited to epithelial basement membranes. It appears early during epithelial morphogenesis in most tissues of the embryo, and remains present as a major epithelial laminin in some adult tissues. Previous organ culture studies with embryonic tissues have suggested that laminin-1 is important for epithelial development. Recent data using genetically manipulated embryonic stem (ES) cells grown as embryoid bodies provide strong support for the view of a specific role of laminin-1 in epithelial morphogenesis. One common consequence of genetic ablation of FGF signaling, beta1-integrin or laminin gamma1 chain expression in ES cells is the absence of laminin-1, which correlates with failure of BM assembly and epiblast differentiation. Partial but distinct rescue of epiblast differentiation has been achieved in all three mutants by exogenously added laminin-1. Laminin-1 contains several biologically active modules, but several are found in beta1 or gamma1 chains shared by at least 11 laminins. However, the carboxytermini of the alpha chains contain five laminin globular (LG) modules, distinct for each alpha chain. There is increasing evidence for a particular role of alpha1LG4 binding to its receptors for epithelial tubulogenesis. The biological roles of this and other domains of laminin-1 are currently being explored by genetic means. The pathways controlling laminin-1 synthesis have remained largely unknown, but recent advances raise the possibility that laminin-1 and collagen IV synthesis can be regulated by pro-survival kinases of the protein kinase B/Akt family.     

Matrix assembly,regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation

Laminin-1 is essential for early embryonic basement membrane assembly and differentiation. Several steps can be distinguished, i.e., the expression of laminin and companion matrix components, their accumulation on the cell surface and assembly into basement membrane between endoderm and inner cell mass, and the ensuing differentiation of epiblast. In this study, we used differentiating embryoid bodies derived from mouse embryonic stem cells null for gamma1-laminin, beta1-integrin and alpha/beta-dystroglycan to dissect the contributions of laminin domains and interacting receptors to this process. We found that (a) laminin enables beta1-integrin-null embryoid bodies to assemble basement membrane and achieve epiblast with beta1-integrin enabling expression of the laminin alpha1 subunit; (b) basement membrane assembly and differentiation require laminin polymerization in conjunction with cell anchorage, the latter critically dependent upon a heparin-binding locus within LG module-4; (c) dystroglycan is not uniquely required for basement membrane assembly or initial differentiation; (d) dystroglycan and integrin cooperate to sustain survival of the epiblast and regulate laminin expression; and (e) laminin, acting via beta1-integrin through LG1-3 and requiring polymerization, can regulate dystroglycan expression.     

Cell adhesive affinity does not dictate primitive endoderm segregation and positioning during murine embryoid body formation

The classical cell sorting experiments undertaken by Townes and Holtfreter described the intrinsic propensity of dissociated embryonic cells to self‐organize and reconcile into their original embryonic germ layers with characteristic histotypic positioning. Steinberg presented the differential adhesion hypothesis to explain these patterning phenomena. Here, we have reappraised these issues by implementing embryoid bodies to model the patterning of epiblast and primitive endoderm layers. We have used combinations of embryonic stem (ES) cells and their derivatives differentiated by retinoic acid treatment to model epiblast and endoderm cells, and wild‐type or E‐cadherin null cells to represent strongly or weakly adherent cells, respectively. One cell type was fluorescently labeled and reconstituted with another heterotypically to generate chimeric embryoid bodies, and cell sorting was tracked by time‐lapse video microscopy and confirmed by immunostaining. When undifferentiated wild‐type and E‐cadherin null ES cells were mixed, the resulting cell aggregates consisted of a core of wild‐type cells surrounded by loosely associated E‐cadherin null cells, consistent with the differential adhesion hypothesis. However, when mixed with undifferentiated ES cells, the differentiated primitive endoderm‐like cells sorted to the surface to form a primitive endoderm layer irrespective of cell‐adhesive strength, contradicting the differential adhesion hypothesis. We propose that the primitive endoderm cells reach the surface by random movement, and subsequently the cells generate an apical/basal polarity that prevents reentry. Thus, the ability to generate epithelial polarity, rather than adhesive affinity, determines the surface positioning of the primitive endoderm cells. genesis 47:579–589, 2009. © 2009 Wiley‐Liss, Inc.     

Differentiation of Pluripotent Embryonic Stem Cells in the Peritoneal Cavity of Irradiated Mice

We studied the behavior and differentiation of pluripotent embryonic stem cells of R1 mice in vivo. Undifferentiated embryonic stem cells and differentiating embryoid bodies implanted in the abdominal cavity of irradiated mice were shown to form tumors containing the derivatives of all germ layers. Cells of the embryoid bodies form tumors two weeks after implantation, while undifferentiated embryonic stem cells form tumors only by week three.     

Neoplastic embryoid bodies of embryonal carcinoma C44 as a source of blastocele-like fluid

Abstract. There is a cytotoxic activity in blastocele fluid that kills embryonal carcinoma cells with trophectodermal potential but spares those with embryonic potential [26]. This activity is present when programmed cell death occurs in the inner cell mass (ICM), and the ICM loses its trophectodermal potential [5, 8–10]. Because of the paucity of blastocele fluid, cystic embryoid bodies of embryonal carcinoma C44 were examined ultrastructurally and in tissue culture to determine if they corresponded to late blastocysts and if their fluid corresponded to blastocele fluid. No troph-ectoderm was demonstrated in the embryoid bodies, but embryonal carcinoma and endoderm were present, leading to the conclusion that the embryonal carcinoma corresponded to late ICM that had expressed endodermal potential. As a result the cyst fluid might have contained the toxic activity of blastocele fluid. The cyst fluid of C44 embryoid bodies did contain a soluble, low-molecular-weight, cytotoxic activity that preferentially killed embryonal carcinoma cells with trophectodermal potential while sparing those with embryonic potential. Enough of this fluid was available to determine the chemical nature of this toxic activity.     

An unexpected role for the clock protein timeless in developmental apoptosis

Background

Programmed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells. Recent work from our group demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, we identified Timeless (Tim), the mammalian ortholog of a Drosophila circadian rhythm protein, as a binding partner and substrate for c-Src and probed its role in the differentiation of mES cells.

Methodology/Principal Findings

To determine whether Tim is involved in ES cell differentiation, Tim protein levels were stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and programmed cell death during cavitation.

Conclusions/Significance

Our data demonstrate a novel function for the clock protein Tim during a key stage of early development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that circadian rhythms and early development may be intimately linked.     

Cytotoxic effects of etoposide at different stages of differentiation of embryoid bodies formed by mouse embryonic stem cells

The initial stages of in vitro differentiation of embryonic stem cells are considered as unique three-dimensional models of early development of mammals for basic, pharmacological, and toxicological studies. It has been previously shown (Gordeeva, 2012) that the assessment of embryotoxicity in the model of undifferentiated embryonic stem cells can be insufficiently accurate in predicting toxic effects on mammalian embryos. In view of this, we performed a comparative study of the damaging effects of the cytostatic etoposide in undifferentiated embryonic stem cells and embryoid bodies of different stages of differentiation that have similar three-dimensional structures with early embryos. The analysis of growth, cell death, and dynamics of differentiation of embryonic stem cells and embryoid bodies exposed to etoposide showed that the cytostatic and cytotoxic effects of etoposide are stage-specific. The damaging effects of etoposide were maximum in the undifferentiated embryonic stem cells and decreased with growth and differentiation of embryoid bodies. We suggest that the increase of embryoid body volume and overgrowth of extraembryonic endoderm layer lead to a decrease in the diffusion, transport and metabolism of chemical and bioactive substances and prevent the damaging effects.     

Migration of F9 parietal endoderm cells is regulated by the ERK pathway

Cell migration is regulated by the action of many signaling pathways that are activated in specific regions of migrating cells. Extracellular regulated kinase 1/2 (ERK) signaling can modulate the migration of cells by controlling the turnover of focal adhesions and the dynamics of actin polymerization. Focal adhesion turnover is necessary for cell migration, and the formation of strong actin stress fibers and mature focal adhesions puts the brakes on cell migration. We used F9 wild-type and vinculin null (vin-/-) parietal endoderm (PE) outgrowth to study the role of the ERK signaling pathway in cell migration. Upon plating of F9 embryoid bodies (EBs) onto laminin-coated dishes, PE cells migrate away from the EBs, providing an in vitro model for studying directed migration of this embryonic cell type. Our results suggest that the ERK pathway regulates PE cell migration by affecting the formation of focal adhesions and lamellipodia through the action of myosin light chain kinase (MLCK).