Anestis Tsakiridis

In Vivo Differentiation Potential of Epiblast Stem Cells Revealed by Chimeric Embryo Formation

Chimera formation after blastocyst injection or morula aggregation is the principal functional assay of the developmental potential of mouse embryonic stem cells (ESCs). This property, which demonstrates functional equivalence between ESCs and the preimplantation epiblast, is not shared by epiblast stem cell (EpiSC) lines. Here, we show that EpiSCs derived either from postimplantation embryos or from ESCs in vitro readily generate chimeras when grafted to postimplantation embryos in whole embryo culture. EpiSC derivatives integrate and differentiate to derivatives of all three embryonic germ layers and primordial germ cells. In contrast, grafted ESCs seldom proliferate in postimplantation embryos, and fail to acquire the identity of their host-derived neighbors. EpiSCs do not incorporate efficiently into embryonic day 8.5 embryos, a stage by which pluripotency has been lost. Thus, chimera formation by EpiSCs requires a permissive environment, the postimplantation epiblast, and demonstrates functional equivalence between this cell type and EpiSCs.

In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity.

Timed pulses of WNT and FGF signaling convert human and mouse pluripotent stem cells into neuromesodermal progenitors that can be directed to differentiate into spinal cord and paraxial mesoderm cells

Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors

During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from areas including the primitive streak. However, it is unknown whether this restriction accompanies, at the individual cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of epiblast stem cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, mouse EpiSCs express gastrulation stage regional markers in self-renewing conditions. Here, we examined the differentiation potential of cells expressing such lineage markers. We show that undifferentiated EpiSC cultures contain a major subfraction of cells with reversible early primitive streak characteristics, which is mutually exclusive to a neural-like fraction. Using in vitro differentiation assays and embryo grafting we demonstrate that primitive streak-like EpiSCs are biased towards mesoderm and endoderm fates while retaining pluripotency. The acquisition of primitive streak characteristics by self-renewing EpiSCs is mediated by endogenous Wnt signalling. Elevation of Wnt activity promotes restriction towards primitive streak-associated lineages with mesendodermal and neuromesodermal characteristics. Collectively, our data suggest that EpiSC pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula stage epiblast.

Microsphere-Mediated Protein Delivery into Cells

Delivering the goods: By coupling proteins to varyingly sized polymeric microspheres, it is possible to deliver them to cells in an easy and effective way. For this study a fluorescent protein (EGFP) and a functional enzyme (β‐galactosidase) were coupled to these particles. Evaluation of the cellular uptake after “beadfection” shows that the functionality and activity of these proteins were not adversely affected through coupling to the carrier system; this shows that their functional structure is retained.

Microsphere-based tracing and molecular delivery in embryonic stem cells.

Embryonic stem (ES) cells are in vitro cell lines that can differentiate into all lineages of the fetus and the adult. Despite the versatility of genetic manipulation in murine ES cells, these approaches are time-consuming and rely on inefficient transient cellular delivery systems that can only be applied to undifferentiated ES cell cultures. Here we describe a polystyrene microsphere-based system designed to efficiently deliver biological materials into both undifferentiated and differentiating ES cells. Our results demonstrate that these microspheres can be successfully employed for simultaneous cellular labeling and controlled transfer of various cargos such as fluorophores, proteins and nucleic acids into ES cells without any significant toxicity or loss of pluripotency. This versatile delivery system is also effective in other stem cell lines derived from early embryos, trophoblast and neural stem cells.

Assessing the bipotency of in vitro-derived neuromesodermal progenitors

Retrospective clonal analysis in the mouse has demonstrated that the posterior spinal cord neurectoderm and paraxial mesoderm share a common bipotent progenitor. These neuromesodermal progenitors (NMPs) are the source of new axial structures during embryonic rostrocaudal axis elongation and are marked by the simultaneous co-expression of the transcription factors T(Brachyury) (T(Bra)) and Sox2. NMP-like cells have recently been derived from pluripotent stem cells in vitro following combined stimulation of Wnt and fibroblast growth factor (FGF) signaling. Under these conditions the majority of cultures consist of T(Bra)/Sox2 co-expressing cells after 48-72 hours of differentiation. Although the capacity of these cells to generate posterior neural and paraxial mesoderm derivatives has been demonstrated at the population level, it is unknown whether a single in vitro-derived NMP can give rise to both neural and mesodermal cells. Here we demonstrate that T(Bra) positive cells obtained from mouse epiblast stem cells (EpiSCs) after culture in NMP-inducing conditions can generate both neural and mesodermal clones. This finding suggests that, similar to their embryonic counterparts, in vitro-derived NMPs are truly bipotent and can thus be exploited as a model for studying the molecular basis of developmental cell fate decisions.

Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells

Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous genes polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this relative positional independence is linked to the human β-actin promoter and is most likely a result of its transcriptional activity in ES cells. Taken together our data indicate that these vectors are an effective tool for insertional mutagenesis that can be used for either gene trapping or gene targeting.

A wider context for gene trap mutagenesis.

Gene trapping is a technology originally developed for the simultaneous identification and mutation of genes by random integration in embryonic stem (ES) cells. While gene trapping was developed before efficient and high-throughput gene targeting, a significant proportion of the publically available mutant ES cell lines and mice were generated through a number of large-scale gene trapping initiatives. Moreover, elements of gene trap vectors continue to be incorporated into gene targeting vectors as a means to increase the efficiency of homologous recombination. Here, we review the current state of gene trapping technology and the applications of specific types of gene trap vector. As a component of this analysis, we consider the behavior of specific vector types both from the perspective of their application and how they can inform our annotation of the mammalian transcriptome. We consider the utility of gene trap vectors as tools for cell-based expression analysis, targeted screening in embryonic differentiation, and for use in cell lines derived from different lineages.

METHODS IN ENZYMOLOGY, VOL 477: GUIDE TO TECHNIQUES IN MOUSE DEVELOPMENT, PART B: MOUSE MOLECULAR GENETICS, SECOND EDITION

Gene trapping is a technology originally developed for the simultaneous identification and mutation of genes by random integration in embryonic stem (ES) cells. While gene trapping was developed before efficient and high-throughput gene targeting, a significant proportion of the publically available mutant ES cell lines and mice were generated through a number of large-scale gene trapping initiatives. Moreover, elements of gene trap vectors continue to be incorporated into gene targeting vectors as a means to increase the efficiency of homologous recombination. Here, we review the current state of gene trapping technology and the applications of specific types of gene trap vector. As a component of this analysis, we consider the behavior of specific vector types both from the perspective of their application and how they can inform our annotation of the mammalian transcriptome. We consider the utility of gene trap vectors as tools for cell-based expression analysis, targeted screening in embryonic differentiation, and for use in cell lines derived from different lineages.

Intrinsic factors and the embryonic environment influence the formation of extragonadal teratomas during gestation

BackgroundPluripotent cells are present in early embryos until the levels of the pluripotency regulator Oct4 drop at the beginning of somitogenesis. Elevating Oct4 levels in explanted post-pluripotent cells in vitro restores their pluripotency. Cultured pluripotent cells can participate in normal development when introduced into host embryos up to the end of gastrulation. In contrast, pluripotent cells efficiently seed malignant teratocarcinomas in adult animals. In humans, extragonadal teratomas and teratocarcinomas are most frequently found in the sacrococcygeal region of neonates, suggesting that these tumours originate from cells in the posterior of the embryo that either reactivate or fail to switch off their pluripotent status. However, experimental models for the persistence or reactivation of pluripotency during embryonic development are lacking. MethodsWe manually injected embryonic stem cells into conceptuses at E9.5 to test whether the presence of pluripotent cells at this stage correlates with teratocarcinoma formation. We then examined the effects of reactivating embryonic Oct4 expression ubiquitously or in combination with Nanog within the primitive streak (PS)/tail bud (TB) using a transgenic mouse line and embryo chimeras carrying a PS/TB-specific heterologous gene expression cassette respectively.ResultsHere, we show that pluripotent cells seed teratomas in post-gastrulation embryos. However, at these stages, induced ubiquitous expression of Oct4 does not lead to restoration of pluripotency (indicated by Nanog expression) and tumour formation in utero, but instead causes a severe phenotype in the extending anteroposterior axis. Use of a more restricted T(Bra) promoter transgenic system enabling inducible ectopic expression of Oct4 and Nanog specifically in the posteriorly-located primitive streak (PS) and tail bud (TB) led to similar axial malformations to those induced by Oct4 alone. These cells underwent induction of pluripotency marker expression in Epiblast Stem Cell (EpiSC) explants derived from somitogenesis-stage embryos, but no teratocarcinoma formation was observed in vivo.ConclusionsOur findings show that although pluripotent cells with teratocarcinogenic potential can be produced in vitro by the overexpression of pluripotency regulators in explanted somitogenesis-stage somatic cells, the in vivo induction of these genes does not yield tumours. This suggests a restrictive regulatory role of the embryonic microenvironment in the induction of pluripotency.