The regulative capability of single cells to give rise to all primary embryonic lineages is termed pluripotency. Observations of fluctuating gene expression and phenotypic heterogeneity in vitro have ...fostered a conception of pluripotency as an intrinsically metastable and precarious state. However, in the embryo and in defined culture environments the properties of pluripotent cells change in an orderly sequence. Two phases of pluripotency, called naïve and primed, have previously been described. In this Hypothesis article, a third phase, called formative pluripotency, is proposed to exist as part of a developmental continuum between the naïve and primed phases. The formative phase is hypothesised to be enabling for the execution of pluripotency, entailing remodelling of transcriptional, epigenetic, signalling and metabolic networks to constitute multi-lineage competence and responsiveness to specification cues.
The nature of embryonic stem cells Martello, Graziano; Smith, Austin
Annual review of cell and developmental biology,
01/2014, Letnik:
30
Journal Article
Recenzirano
Odprti dostop
Mouse embryonic stem (ES) cells perpetuate in vitro the broad developmental potential of naïve founder cells in the preimplantation embryo. ES cells self-renew relentlessly in culture but can reenter ...embryonic development seamlessly, differentiating on schedule to form all elements of the fetus. Here we review the properties of these remarkable cells. Arising from the stability, homogeneity, and equipotency of ES cells, we consider the concept of a pluripotent ground state. We evaluate the authenticity of ES cells in relation to cells in the embryo and examine their utility for dissecting mechanisms that confer pluripotency and that execute fate choice. We summarize current knowledge of the transcription factor circuitry that governs the ES cell state and discuss the opportunity to expose molecular logic further through iterative computational modeling and experimentation. Finally, we present a perspective on unresolved questions, including the challenge of deriving ground state pluripotent stem cells from non-rodent species.
In the mouse blastocyst, epiblast cells are newly formed shortly before implantation. They possess a unique developmental plasticity, termed naive pluripotency. For development to proceed, this naive ...state must be subsumed by multi-lineage differentiation within 72 h following implantation. In vitro differentiation of naive embryonic stem cells (ESCs) cultured in controlled conditions provides a tractable system to dissect and understand the process of exit from naive pluripotency and entry into lineage specification. Exploitation of this system in recent large-scale RNAi and mutagenesis screens has uncovered multiple new factors and modules that drive or facilitate progression out of the naive state. Notably, these studies show that the transcription factor network that governs the naive state is rapidly dismantled prior to upregulation of lineage specification markers, creating an intermediate state that we term formative pluripotency. Here, we summarize these findings and propose a road map for state transitions in ESC differentiation that reflects the orderly dynamics of epiblast progression in the embryo.
The ground state of pluripotency Wray, Jason; Kalkan, Tuzer; Smith, Austin G
Biochemical Society transactions,
08/2010, Letnik:
38, Številka:
4
Journal Article
Recenzirano
Pluripotency is defined as the capacity of individual cells to initiate all lineages of the mature organism in response to signals from the embryo or cell culture environment. A pluripotent cell has ...no predetermined programme; it is a blank slate. This is the foundation of mammalian development and of ES (embryonic stem) cell biology. What are the design principles of this naïve cell state? How is pluripotency acquired and maintained? Suppressing activation of ERKs (extracellular-signal-regulated kinases) is critical to establishing and sustaining ES cells. Inhibition of GSK3 (glycogen synthase kinase 3) reinforces this effect. We review the effect of selective kinase inhibitors on pluripotent cells and consider how these effects are mediated. We propose that ES cells represent a ground state, meaning a basal proliferative state that is free of epigenetic restriction and has minimal requirements for extrinsic stimuli. The stability of this state is reflected in the homogeneity of ES cell populations cultured in the presence of small-molecule inhibitors of MEK (mitogen-activated protein kinase/ERK kinase) and GSK3.
Daylight Saving Time (DST) impacts over 1.5 billion people, yet many of its impacts on practicing populations remain uncertain. Exploiting the discrete nature of DST transitions and a 2007 policy ...change, I estimate the impact of DST on fatal automobile crashes. My results imply that from 2002–2011 the transition into DST caused over 30 deaths at a social cost of $275 million annually. Employing four tests to decompose the aggregate effect into an ambient light or sleep mechanism, I find that shifting ambient light only reallocates fatalities within a day, while sleep deprivation caused by the spring transition increases risk.
Pluripotency denotes the flexible capacity of single cells to give rise to all somatic lineages and typically also the germline. Mouse ES cells and post‐implantation epiblast‐derived stem cells ...(EpiSC) are widely used pluripotent cell culture systems. These two in vitro stem cell types have divergent characteristics. They are considered as representative of distinct developmental stages, distinguished by using the terms “naïve” and “primed”. A binary description is an over‐simplification, however. Here, we discuss an intermediate stage of pluripotency that we term “formative”. Formative pluripotency features a gene regulatory network switch from the naïve state and comprises capacitation of enhancers, signaling pathways and epigenetic machinery in order to install competence for lineage specification.
Currently, pluripotent states are categorized by two terms, “naïve” and “primed”. Formative pluripotency is an intermediate transitional phase which is important for the capacitation of naive cells to respond to differentiation signals.
Impact of reconstituted cytosol on protein stability Sarkar, Mohona; Smith, Austin E.; Pielak, Gary J.
Proceedings of the National Academy of Sciences - PNAS,
11/2013, Letnik:
110, Številka:
48
Journal Article
Recenzirano
Odprti dostop
Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. ...Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMR-detected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions.
Embryonic stem (ES) cells are used extensively in biomedical research and as a model with which to study early mammalian development, but their exact origin has been subject to much debate. They are ...routinely derived from pre-implantation embryos, but it has been suggested that the cells that give rise to ES cells might arise from epiblast cells that are already predisposed to a primordial germ cell (PGC) fate, which then progress to ES cell status via the PGC lineage. Based on recent findings, we propose here that ES cells can be derived directly from early epiblast cells and that ES cells might arise via two different routes that are dictated by their culture conditions.
Transcription factor Stat3 directs self‐renewal of pluripotent mouse embryonic stem (ES) cells downstream of the cytokine leukemia inhibitory factor (LIF). Stat3 upregulates pivotal transcription ...factors in the ES cell gene regulatory network to sustain naïve identity. Stat3 also contributes to the rapid proliferation of ES cells. Here, we show that Stat3 increases the expression of mitochondrial‐encoded transcripts and enhances oxidative metabolism. Chromatin immunoprecipitation reveals that Stat3 binds to the mitochondrial genome, consistent with direct transcriptional regulation. An engineered form of Stat3 that localizes predominantly to mitochondria is sufficient to support enhanced proliferation of ES cells, but not to maintain their undifferentiated phenotype. Furthermore, during reprogramming from primed to naïve states of pluripotency, Stat3 similarly upregulates mitochondrial transcripts and facilitates metabolic resetting. These findings suggest that the potent stimulation of naïve pluripotency by LIF/Stat3 is attributable to parallel and synergistic induction of both mitochondrial respiration and nuclear transcription factors.
Synopsis
The LIF/STAT3 pathway promotes naïve pluripotency and proliferation in embryonic stem cells. To do this, in addition to its nuclear role, STAT3 translocates to the mitochondrion, regulates mitochondrial transcription and enhances mitochondrial respiration.
The LIF/Stat3 axis promotes mitochondrial gene expression and respiration in ES cells.
Mitochondrial respiration determines optimal ES cell proliferation.
LIF‐dependent up‐regulation of mitochondrial activity contributes to induction of naïve pluripotency.
The LIF/STAT3 pathway promotes naïve pluripotency and proliferation in embryonic stem cells. To do this, in addition to its nuclear role, STAT3 translocates to the mitochondrion, regulates mitochondrial transcription and enhances mitochondrial respiration.
Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and ...of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naïve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naïve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.
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