Niels Geijsen

Derivation of embryonic germ cells and male gametes from embryonic stem cells.

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.

Sox2+ Adult Stem and Progenitor Cells Are Important for Tissue Regeneration and Survival of Mice

The transcription factor Sox2 maintains the pluripotency of early embryonic cells and regulates the formation of several epithelia during fetal development. Whether Sox2 continues to play a role in adult tissues remains largely unknown. We show here that Sox2 marks adult cells in several epithelial tissues where its expression has not previously been characterized, including the stomach, cervix, anus, testes, lens, and multiple glands. Genetic lineage tracing and transplantation experiments demonstrate that Sox2-expressing cells continuously give rise to mature cell types within these tissues, documenting their self-renewal and differentiation potentials. Consistent with these findings, ablation of Sox2(+) cells in mice results in a disruption of epithelial tissue homeostasis and lethality. Developmental fate mapping reveals that Sox2(+) adult stem cells originate from fetal Sox2(+) tissue progenitors. Thus, our results identify Sox2 expression in numerous adult endodermal and ectodermal stem cell compartments, which are critical for normal tissue regeneration and survival.

The Growth Factor Environment Defines Distinct Pluripotent Ground States in Novel Blastocyst-Derived Stem Cells

Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the postimplantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings indicate that novel stem cell lines, with unique functional and molecular properties, can be generated from murine blastocyst embryos. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.

The pluripotent genome in three dimensions is shaped around pluripotency factors

It is becoming increasingly clear that the shape of the genome importantly influences transcription regulation. Pluripotent stem cells such as embryonic stem cells were recently shown to organize their chromosomes into topological domains that are largely invariant between cell types. Here we combine chromatin conformation capture technologies with chromatin factor binding data to demonstrate that inactive chromatin is unusually disorganized in pluripotent stem-cell nuclei. We show that gene promoters engage in contacts between topological domains in a largely tissue-independent manner, whereas enhancers have a more tissue-restricted interaction profile. Notably, genomic clusters of pluripotency factor binding sites find each other very efficiently, in a manner that is strictly pluripotent-stem-cell-specific, dependent on the presence of Oct4 and Nanog protein and inducible after artificial recruitment of Nanog to a selected chromosomal site. We conclude that pluripotent stem cells have a unique higher-order genome structure shaped by pluripotency factors. We speculate that this interactome enhances the robustness of the pluripotent state.

The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

Efficient Intracellular Delivery of Native Proteins

Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.

Specificity in cytokine signal transduction: lessons learned from the IL-3/IL-5/GM-CSF receptor family

Cytokines mediate the transduction of proliferative, differentiation and survival signals in the hematopoietic system. Although the cytokine family is large and diverse, many different cytokines display broadly overlapping functions. This can be explained by the fact that cytokine receptors often share multiple subunits. Specificity in signal transduction can however be achieved through several mechanisms. This review focuses on how signal specificity can be achieved within the IL-3, IL-5 and GM-CSF receptor family. This is discussed in terms of receptor expression, recent advances in our understanding of intracellular signalling components, and analysis of null mutant knock-out mice.

Association of RACK1 and PKCβ with the common β-chain of the IL-5/IL-3/GM-CSF receptor

Granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5 belong to a family of cytokines that regulate proliferation, differentiation and function of haematopoietic cells. Their receptor consists of a ligand specific α-chain and a signal transducing β-chain (βc). While, the role of phosphotyrosine residues in the βc as mediators of downstream signalling cascades has been established, little is known about non-phosphotyrosine mediated events. To identify proteins interacting with βc, we screened a yeast two-hybrid library with the intracellular domain of βc. We found that RACK1, a molecule associating with activated PKC, PLCγ and Src kinases, associated with the membrane proximal region of βc in both yeast two-hybrid, immunoprecipitation and GST-pull-down assays. The association of RACK1 was constitutive, demonstrating no alteration upon cellular stimulation. Furthermore, upon stimulation of cells with IL-5 or PMA, a complex of βc and PKCβ was found. Together, these findings suggest a novel role for RACK1 as a possible adapter molecule associating with the intracellular domain of cytokine receptors.

In vitro generation of germ cells from murine embryonic stem cells

The demonstration of germ cell and haploid gamete development from embryonic stem cells (ESCs) in vitro has engendered a unique set of possibilities for the study of germ cell development and the associated epigenetic phenomenon. The process of embryoid body (EB) differentiation, like teratoma formation, signifies a spontaneous differentiation of ESCs into cells of all three germ layers, and it is from these differentiating aggregates of cells that putative primordial germ cells (PGCs) and more mature gametes can be identified and isolated. The differentiation system presented here requires the differentiation of murine ESCs into EBs and the subsequent isolation of PGCs as well as haploid male gametes from EBs at various stages of differentiation. It serves as a platform for studying the poorly understood process of germ cell allocation, imprint erasure and gamete formation, with 4–6 weeks being required to isolate PGCs as well as haploid cells.

Cytokine‐mediated cPLA2 phosphorylation is regulated by multiple MAPK family members

Cytosolic phospholipase A2 (cPLA2) plays a critical role in various neutrophil functions including the generation of leukotrienes and platelet‐activating factor release. Enzyme activity is regulated both by translocation to the membrane in a Ca2+‐dependent manner and serine phosphorylation by members of the mitogen‐activated protein kinase (MAPK) family. In this report, we have investigated the role of granulocyte/macrophage colony‐stimulating factor (GM‐CSF)‐mediated signalling pathways in the regulation of cPLA2. GM‐CSF‐induced cPLA2 phosphorylation was not affected by pharmacological inhibition of p38 MAPK, phosphatidylinositol 3‐kinase or Src. However, inhibition of extracellular signal‐regulated kinase (ERK) MAPK activation resulted in a partial inhibition of cPLA2 phosphorylation, revealed in a slower onset of phosphorylation. A cell line stably transfected with the GM‐CSF receptor was used to further analyze GM‐CSF‐mediated cPLA2 phosphorylation. Mutation of tyrosine residues 577 and 612 resulted in a delayed cPLA2 phosphorylation similar to the pharmacological ERK inhibition. Furthermore, inhibition of p38 MAPK in cells bearing the double mutant βc577/612 completely abrogated GM‐CSF‐induced cPLA2 phosphorylation. We conclude that GM‐CSF can mediate cPLA2 phosphorylation through the redundant activation of both p38 and ERK MAP kinases.