Davood Sabour

Conserved and Divergent Roles of FGF Signaling in Mouse Epiblast Stem Cells and Human Embryonic Stem Cells

Mouse epiblast stem cells (EpiSCs) are cultured with FGF2 and Activin A, like human embryonic stem cells (hESCs), but the action of the associated pathways in EpiSCs has not been well characterized. Here, we show that activation of the Activin pathway promotes self-renewal of EpiSCs via direct activation of Nanog, whereas inhibition of this pathway induces neuroectodermal differentiation, like in hESCs. In contrast, the different roles of FGF signaling appear to be only partially conserved in the mouse. Our data suggest that FGF2 fails to cooperate with SMAD2/3 signaling in actively promoting EpiSC self-renewal through Nanog, in contrast to its role in hESCs. Rather, FGF appears to stabilize the epiblast state by dual inhibition of differentiation to neuroectoderm and of media-induced reversion to a mouse embryonic stem cell-like state. Our data extend the current model of cell fate decisions concerning EpiSCs by clarifying the distinct roles played by FGF signaling.

Inhibition of TGFβ Signaling Promotes Ground State Pluripotency

Embryonic stem (ES) cells are considered to exist in a ground state if shielded from differentiation triggers. Here we show that FGF4 and TGFβ signaling pathway inhibitors, designated R2i, not only provide the ground state pluripotency in production and maintenance of naïve ES cells from blastocysts of different mouse strains, but also maintain ES cells with higher genomic integrity following long-term cultivation compared with the chemical inhibition of the FGF4 and GSK3 pathways, known as 2i. Global transcriptome analysis of the ES cells highlights augmented BMP4 signaling pathway. The crucial role of the BMP4 pathway in maintaining the R2i ground state pluripotency is demonstrated by BMP4 receptor suppression, resulting in differentiation and cell death. In conclusion, by inhibiting TGFβ and FGF signaling pathways, we introduce a novel defined approach to efficiently establish the ground state pluripotency.

Identification of genes specific to mouse primordial germ cells through dynamic global gene expression

Molecular mechanisms underlying the commitment of cells to the germ cell lineage during mammalian embryogenesis remain poorly understood due to the limited availability of cellular materials to conduct in vitro analyses. Although primordial germ cells (PGCs)--precursors to germ cells--have been generated from embryonic stem cells (ESCs)--pluripotent stem cells derived from the inner cell mass of the blastocyst of the early embryo in vitro-the simultaneous expression of cell surface receptors and transcription factors complicates the detection of PGCs. To date, only a few genes that mark the onset of germ cell commitment in the epiblast--the outer layer of cells of the embryo--including tissue non-specific alkaline phosphatase (TNAP), Blimp1, Stella and Fragilis--have been used with some success to detect PGC formation in in vitro model systems. Here, we identified 11 genes (three of which are novel) that are specifically expressed in male and female fetal germ cells, both in vivo and in vitro, but are not expressed in ESCs. Expression of these genes allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro.

Reprogramming and the mammalian germline: the Weismann barrier revisited

The germline represents a unique cell type that can transmit genetic material to the next generation. During early embryonic development, somatic cells give rise to a small population of cells known as germ cells, which eventually differentiate into mature gametes. Germ cells undergo a process of removing and resetting relevant epigenetic information, mainly by DNA demethylation. This extensive epigenetic reprogramming leads to the conversion of germ cells into immortal cells that can pass on the genome to the next generation. In the absence of germline-specific reprogramming, germ cells would preserve the old, parental epigenetic memory, which would prevent the transfer of heritable information to the offspring. On the contrary, somatic cells cannot reset epigenetic information by preserving the full methylation pattern on imprinting genes. In this review, we focus on the capacity of germ cells and somatic cells (soma) to transfer genetic information to the next generation, and thus revisit the Weismann theory of heredity.

Autologous Pluripotent Stem Cells Generated from Adult Mouse Testicular Biopsy

IntroductionPrimordial germ cells (PGCs) are the precursors of the maleand female gametes. At 6.25 days post coitum (dpc), PGCsare specified in the proximal epiblast [21] and then migratethrough the hindgut into the developing gonads [27]. At12.5 dpc, most PGCs have reached the gonads; aftercolonizing the gonads, female germ cells enter meiosiswhereas male germ cells undergo mitotic arrest [25].However, under certain conditions, male germ cells continueproliferating and give rise to embryonal carcinoma cells(ECs). ECs were the first pluripotent stem cells to be isolatedand could give rise to teratomas containing tissues from allthree germ layers [37]. In addition, after being cultured inspecific culture conditions, isolated PGCs can also bededifferentiated into pluripotent cells in vitro, termedembryonic germ (EG) cells [33]. Thus, highly specializedcells, such as the germ cells, can spontaneously dedifferen-tiate to a pluripotent state both in vitro and in vivo.Spermatogonial stem cells (SSCs) are found in postnataltestis and are unipotent, as they can only give rise to sperm[2, 9]. SSCs can be established as in vitro stem cell lines,namely germline stem cells (GSCs), which can restorespermatogenesis after transplantation into sterile testis [1].Previous studies have demonstrated that neonatal GSC linescould be dedifferentiated into multipotent embryonic stemcells (ESC)-like cells [10, 34]. We have recently reportedthat adult GSC lines could be reprogrammed not only tomultipotency, but also to pluripotency. The generatedgermline-derived pluripotent stem (gPS) cells were similarto embryonic stem cells (ESCs) with respect to develop-mental potential both in vitro and in vivo [18]. Themouse model shows that the conversion process is robustand reproducible and induced in defined culture con-

Distinct Signaling Requirements for the Establishment of ESC Pluripotency in Late-Stage EpiSCs

Summary It has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling.

Ultrastructural characterization of mouse embryonic stem cell-derived oocytes and granulosa cells.

Germ cells are a unique population of cells responsible for transmitting genetic information from one generation to the next. Our understanding of the key mechanisms underlying germ cell development in vivo remains scarce because of insufficient amounts of cell materials available for conducting biological studies. The establishment of in vitro differentiation models that support the generation of germ cells from mouse pluripotent stem cells provides an alternative means for studying reproductive development. The detection and analysis of stem cell-derived germ cells, however, present technical challenges. Methods for determining the developmental stage of germ cells ex vivo, such as gene expression and/or immunochemical analyses are inadequate, frequently necessitating the use of alternative, elaborate methods to prove germ cell identity. We have generated putative oocytes and granulosa cells in vitro from mouse embryonic stem cells and utilized electron microscopy to characterize these cells. Here, we report on the striking ultrastructural similarity of in vitro-generated oocytes and granulosa cells to in vivo oocytes developing within follicles.

Germ Cell Nuclear Factor Regulates Gametogenesis in Developing Gonads

Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.

Overlapping genes may control reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) and breast cancer stem cells

Recent findings suggest the possibility that tumors originate from cancer cells with stem cell properties. The cancer stem cell (CSC) hypothesis provides an explanation for why existing cancer therapies often fail in eradicating highly malignant tumors and end with tumor recurrence. Although normal stem cells and CSCs both share the capacity for self-renewal and multi-lineage differentiation, suggesting that CSC may be derived from normal SCs, the cellular origin of transformation of CSCs is debatable. Research suggests that the tightly controlled balance of self-renewal and differentiation that characterizes normal stem cell function is dis-regulated in cancer. Additionally, recent evidence has linked an embryonic stem cell (ESC)-like gene signature with poorly differentiated high-grade tumors, suggesting that regulatory pathways controlling pluripotency may in part contribute to the somatic CSC phenotype. Here, we introduce expression profile bioinformatic analyses of mouse breast cells with CSC properties, mouse embryonic stem (mES) and induced pluripotent stem (iPS) cells with an emphasis on how study of pluripotent stem cells may contribute to the identification of genes and pathways that facilitate events associated with oncogenesis. Global gene expression analysis from CSCs and induced pluripotent stem cell lines represent an ideal model to study cancer initiation and progression and provide insight into the origin cancer stem cells. Additionally, insight into the genetic and epigenomic mechanisms regulating the balance between self-renewal and differentiation of somatic stem cells and cancer may help to determine whether different strategies used to generate iPSCs are potentially safe for therapeutic use.

Blockage of the Epithelial-to-Mesenchymal Transition Is Required for Embryonic Stem Cell Derivation

Summary Pluripotent cells emanate from the inner cell mass (ICM) of the blastocyst and when cultivated under optimal conditions immortalize as embryonic stem cells (ESCs). The fundamental mechanism underlying ESC derivation has, however, remained elusive. Recently, we have devised a highly efficient approach for establishing ESCs, through inhibition of the MEK and TGF-β pathways. This regimen provides a platform for dissecting the molecular mechanism of ESC derivation. Via temporal gene expression analysis, we reveal key genes involved in the ICM to ESC transition. We found that DNA methyltransferases play a pivotal role in efficient ESC generation. We further observed a tight correlation between ESCs and preimplantation epiblast cell-related genes and noticed that fundamental events such as epithelial-to-mesenchymal transition blockage play a key role in launching the ESC self-renewal program. Our study provides a time course transcriptional resource highlighting the dynamics of the gene regulatory network during the ICM to ESC transition.