At E2.5, an extended focus image is shown. Representative images are shown for each stage. (A) HV transgenic embryos were collected at E2.5 and cultured in vitro for different time periods before live confocal microscopy imaging. Thus, the combination of 2i and LIF promoted the expansion of individual totipotent cells reminiscent of the morula or early blastocyst stage, before lineage restrictions have occurred.Ĭulture in 2i Does Not Eliminate Endoderm Marker Expression in the Early Embryo This population demonstrated an enhanced capacity to generate extraembryonic cell types, including trophoblast, in vitro, and single cells from this fraction were totipotent when assessed by morula aggregation in vivo. We show that embryos and ESCs cultured in 2i are heterogeneous and contain a fraction of cells coexpressing markers of both embryonic and extraembryonic lineages. Here, we utilize ESCs containing this reporter, and a transgenic reporter mouse derived from them, to explore the nature of the ground state and investigate the cell-intrinsic role of LIF in this defined context. We previously described a sensitive reporter for the endoderm marker Hex utilizing a reiterated IRES element to translationally amplify expression of the fluorescent protein Venus, encoded downstream of Hex in the endogenous locus ( Canham et al., 2010). Culture in 2i is often supplemented with LIF, which not only supports self-renewal of ESCs but also has a function in extraembryonic development, where it promotes trophoblast proliferation, differentiation, and invasion ( Poehlmann et al., 2005 Prakash et al., 2011 Takahashi et al., 2003). These inhibitors shield ESCs from differentiation-inducing signals and are thought to generate a homogeneous early epiblast-like ground state for embryonic but not extraembryonic development ( Nichols et al., 2009 Wray et al., 2010, 2011).
ESCs can also be cultured in minimal medium with MEK (mitogen-activated protein or extracellular signal-regulated kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) ( Ying et al., 2008). Under these conditions, numerous genes are expressed in a heterogeneous manner ( Canham et al., 2010 Chambers et al., 2007 Hayashi et al., 2008 Kobayashi et al., 2009 Singh et al., 2007 Toyooka et al., 2008), implying that ESC cultures may harbor cells with distinct functional potentials. This suggests that ESC cultures contain a mixture of cells resembling precursors of embryonic epiblast and extraembryonic tissues but that epiblast-like precursors have a competitive advantage when reintroduced into chimeras.ĮSCs can be maintained in the presence of LIF and either BMP4 or serum ( Ying et al., 2003a). Additionally, although ESCs have been shown to be pluripotent only in standard injections, they can generate extraembryonic primitive endoderm (PE) in vitro ( Hayashi et al., 2010 Xu et al., 2002) and, in rare events, contribute to both embryonic and extraembryonic lineages in vivo ( Beddington and Robertson, 1989 Canham et al., 2010 Lallemand and Brûlet, 1990 Macfarlan et al., 2012 Suemori et al., 1990). However, because this definition is based on retrospective function, the precise cellular phenotype of pluripotent cells is unknown, especially as there has been little characterization of the functional potential of single cells. In mouse, this is assessed by their ability to differentiate into all embryonic but not extraembryonic lineages when reintroduced into morulae or blastocysts. They are classically defined as pluripotent. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.Įmbryonic stem cells (ESCs) are karyotypically normal cells derived from the inner cell mass (ICM) or epiblast of peri-implantation embryos. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs.
Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages.
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