Daisuke Shimosato

Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells

The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct–Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct–Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct–Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.

Interaction between Oct3/4 and Cdx2 Determines Trophectoderm Differentiation

Trophectoderm (TE), the first differentiated cell lineage of mammalian embryogenesis, forms the placenta, a structure unique to mammalian development. The differentiation of TE is a hallmark event in early mammalian development, but molecular mechanisms underlying this first differentiation event remain obscure. Embryonic stem (ES) cells can be induced to differentiate into the TE lineage by forced repression of the POU-family transcription factor, Oct3/4. We show here that this event can be mimicked by overexpression of Caudal-related homeobox 2 (Cdx2), which is sufficient to generate proper trophoblast stem (TS) cells. Cdx2 is dispensable for trophectoderm differentiation induced by Oct3/4 repression but essential for TS cell self-renewal. In preimplantation embryos, Cdx2 is initially coexpressed with Oct3/4 and they form a complex for the reciprocal repression of their target genes in ES cells. This suggests that reciprocal inhibition between lineage-specific transcription factors might be involved in the first differentiation event of mammalian development.

A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells

The cytokine leukaemia inhibitory factor (LIF) integrates signals into mouse embryonic stem (ES) cells to maintain pluripotency. Although the Jak–Stat3 pathway is essential and sufficient to mediate LIF signals, it is still unclear how these signals are linked to the core circuitry of pluripotency-associated transcription factors, consisting of Oct3/4 (also called Pou5f1), Sox2 and Nanog. Here we show that two LIF signalling pathways are each connected to the core circuitry via different transcription factors. In mouse ES cells, Klf4 is mainly activated by the Jak–Stat3 pathway and preferentially activates Sox2, whereas Tbx3 is preferentially regulated by the phosphatidylinositol-3-OH kinase–Akt and mitogen-activated protein kinase pathways and predominantly stimulates Nanog. In the absence of LIF, artificial expression of Klf4 or Tbx3 is sufficient to maintain pluripotency while maintaining the expression of Oct3/4. Notably, overexpression of Nanog supports LIF-independent self-renewal of mouse ES cells in the absence of Klf4 and Tbx3 activity. Therefore, Klf4 and Tbx3 are involved in mediating LIF signalling to the core circuitry but are not directly associated with the maintenance of pluripotency, because ES cells keep pluripotency without their expression in the particular context.

Identification and characterization of subpopulations in undifferentiated ES cell culture.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) and the epiblast, and have been suggested to be a homogeneous population with characteristics intermediate between them. These cells express Oct3/4 and Rex1 genes, which have been used as markers to indicate the undifferentiated state of ES cells. Whereas Oct3/4 is expressed in totipotent and pluripotent cells in the mouse life cycle, Rex1 expression is restricted to the ICM, and is downregulated in pluripotent cell populations in the later stages, i.e. the epiblast and primitive ectoderm (PrE). To address whether ES cells comprise a homogeneous population equivalent to a certain developmental stage of pluripotent cells or a heterogeneous population composed of cells corresponding to various stages of differentiation, we established knock-in ES cell lines in which genes for fluorescent proteins were inserted into the Rex1 and Oct3/4 gene loci to visualize the expression of these genes. We found that undifferentiated ES cells included at least two different populations, Rex1+/Oct3/4+ cells and Rex1-/Oct3/4+ cells. The Rex1-/Oct3/4+ and Rex1+/Oct3/4+ populations could convert into each other in the presence of LIF. In accordance with our assumption that Rex1+/Oct3/4+ cells and Rex1-/Oct3/4+ cells have characteristics similar to those of ICM and early-PrE cells, Rex1+/Oct3/4+ cells predominantly differentiated into primitive ectoderm and contributed to chimera formation, whereas Rex1-/Oct3/4+ cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.

Activin-Nodal signaling is involved in propagation of mouse embryonic stem cells

Embryonic stem (ES) cells are self-renewing cells that maintain pluripotency to differentiate into all types of cells. Because of their potential to provide a variety of tissues for use in regenerative medicine, there is great interest in the identification of growth factors that govern these unique properties of ES cells. However, the signaling pathways controlling ES cell proliferation remain largely unknown. Since transforming growth factor β (TGFβ) superfamily members have been implicated in the processes of early embryogenesis, we investigated their roles in ES cell self-renewal. Inhibition of activin-Nodal-TGFβ signaling by Smad7 or SB-431542 dramatically decreased ES cell proliferation without decreasing ES pluripotency. By contrast, inhibition of bone morphogenetic protein (BMP) signaling by Smad6 did not exhibit such effects, suggesting that activin-Nodal-TGFβ signaling, but not BMP signaling, is indispensable for ES cell propagation. In serum-free culture, supplementation of recombinant activin or Nodal, but not TGFβ or BMP, significantly enhanced ES cell propagation without affecting pluripotency. We also found that activin-Nodal signaling was constitutively activated in an autocrine fashion in serum-free cultured ES cells, and that inhibition of such endogenous signaling by SB-431542 decreased ES cell propagation in serum-free conditions. These findings suggest that endogenously activated autocrine loops of activin-Nodal signaling promote ES cell self-renewal.

Krüppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.

The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.

An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit.

The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.

Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors acquire the character of XEN cells

BackgroundThree types of cell lines have been established from mouse blastocysts: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extra-embryonic endoderm (XEN) cells, which have the potential to differentiate into their respective cognate lineages. ES cells can differentiate in vitro not only into somatic cell lineages but into extra-embryonic lineages, including trophectoderm and extra-embryonic endoderm (ExEn) as well. TS cells can be established from ES cells by the artificial repression of Oct3/4 or the upregulation of Cdx2 in the presence of FGF4 on feeder cells. The relationship between these embryo-derived XEN cells and ES cell-derived ExEn cell lines remains unclear, although we have previously reported that overexpression of Gata4 or Gata6 induces differentiation of mouse ES cells into extra-embryonic endoderm in vitro.ResultsA system in which GATA factors were conditionally activated revealed that the cells continue to proliferate while expressing a set of extra-embryonic endoderm markers, and, following injection into blastocysts, contribute only to the extra-embryonic endoderm lineage in vivo. Although the in vivo contribution is limited to cells of parietal endoderm lineage, Gata-induced extra-embryonic endoderm cells (gExEn) can be induced to differentiate into visceral endoderm-like cells in vitro by repression of Gata6. During early passage, the propagation of gExEn cells is dependent on the expression of the Gata6 transgene. These cells, however, lose this dependency following establishment of endogenous Gata6 expression.ConclusionWe show here that Gata-induced extra-embryonic endoderm cells derived from ES cells mimic the character of XEN cells. These findings indicate that Gata transcription factors are sufficient for the derivation and propagation of XEN-like extra-embryonic endoderm cells from ES cells.

Sox7 is dispensable for primitive endoderm differentiation from mouse ES cells

BackgroundPrimitive endoderm is a cell lineage segregated from the epiblast in the blastocyst and gives rise to parietal and visceral endoderm. Sox7 is a member of the SoxF gene family that is specifically expressed in primitive endoderm in the late blastocyst, although its function in this cell lineage remains unclear.ResultsHere we characterize the function of Sox7 in primitive endoderm differentiation using mouse embryonic stem (ES) cells as a model system. We show that ectopic expression of Sox7 in ES cells has a marginal effect on triggering differentiation into primitive endoderm-like cells. We also show that targeted disruption of Sox7 in ES cells does not affect differentiation into primitive endoderm cells in embryoid body formation as well as by forced expression of Gata6.ConclusionsThese data indicate that Sox7 function is supplementary and not essential for this differentiation from ES cells.