During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from ...areas including the primitive streak. However, it is unknown whether this restriction accompanies, at the individual cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of epiblast stem cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, mouse EpiSCs express gastrulation stage regional markers in self-renewing conditions. Here, we examined the differentiation potential of cells expressing such lineage markers. We show that undifferentiated EpiSC cultures contain a major subfraction of cells with reversible early primitive streak characteristics, which is mutually exclusive to a neural-like fraction. Using in vitro differentiation assays and embryo grafting we demonstrate that primitive streak-like EpiSCs are biased towards mesoderm and endoderm fates while retaining pluripotency. The acquisition of primitive streak characteristics by self-renewing EpiSCs is mediated by endogenous Wnt signalling. Elevation of Wnt activity promotes restriction towards primitive streak-associated lineages with mesendodermal and neuromesodermal characteristics. Collectively, our data suggest that EpiSC pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula stage epiblast.
The molecular mechanisms governing the transition from hematopoietic stem cells (HSCs) to lineage-committed progenitors remain poorly understood. Transcription factors (TFs) are powerful cell ...intrinsic regulators of differentiation and lineage commitment, while cytokine signaling has been shown to instruct the fate of progenitor cells. However, the direct regulation of differentiation-inducing hematopoietic TFs by cell extrinsic signals remains surprisingly difficult to establish. PU.1 is a master regulator of hematopoiesis and promotes myeloid differentiation. Here we report that tumor necrosis factor (TNF) can directly and rapidly upregulate PU.1 protein in HSCs in vitro and in vivo. We demonstrate that in vivo, niche-derived TNF is the principal PU.1 inducing signal in HSCs and is both sufficient and required to relay signals from inflammatory challenges to HSCs.
•TNF integrates inflammatory signals to directly regulate the lineage instructing transcription factor PU.1 in HSCs.
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Transcription factor (TF) networks are thought to regulate embryonic stem cell (ESC) pluripotency. However, TF expression dynamics and regulatory mechanisms are poorly understood. We use reporter ...mouse ESC lines allowing non-invasive quantification of Nanog or Oct4 protein levels and continuous long-term single-cell tracking and quantification over many generations to reveal diverse TF protein expression dynamics. For cells with low Nanog expression, we identified two distinct colony types: one re-expressed Nanog in a mosaic pattern, and the other did not re-express Nanog over many generations. Although both expressed pluripotency markers, they exhibited differences in their TF protein correlation networks and differentiation propensities. Sister cell analysis revealed that differences in Nanog levels are not necessarily accompanied by differences in the expression of other pluripotency factors. Thus, regulatory interactions of pluripotency TFs are less stringently implemented in individual self-renewing ESCs than assumed at present.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Continuous analysis of single cells, over several cell divisions and for up to weeks at a time, is crucial to deciphering rare, dynamic and heterogeneous cell responses, which would otherwise be ...missed by population or single-cell snapshot analysis. Although the field of long-term single-cell imaging, tracking and analysis is constantly advancing, several technical challenges continue to hinder wider implementation of this important approach. This is a particular problem for mammalian cells, where in vitro observation usually remains the only possible option for uninterrupted long-term, single-cell observation. Efforts must focus not only on identifying and maintaining culture conditions that support normal cellular behavior while allowing high-resolution imaging over time, but also on developing computational methods that enable semiautomatic analysis of the data. Solutions in microscopy hard- and software, computer vision and specialized theoretical methods for analysis of dynamic single-cell data will enable important discoveries in biology and beyond.
Embryonic stem cells (ESCs) display heterogeneous expression of pluripotency factors such as Nanog when cultured with serum and leukemia inhibitory factor (LIF). In contrast, dual inhibition of the ...signaling kinases GSK3 and MEK (2i) converts ESC cultures into a state with more uniform and high Nanog expression. However, it is so far unclear whether 2i acts through an inductive or selective mechanism. Here, we use continuous time-lapse imaging to quantify the dynamics of death, proliferation, and Nanog expression in mouse ESCs after 2i addition. We show that 2i has a dual effect: it both leads to increased cell death of Nanog low ESCs (selective effect) and induces and maintains high Nanog levels (inductive effect) in single ESCs. Genetic manipulation further showed that presence of NANOG protein is important for cell viability in 2i medium. This demonstrates complex Nanog-dependent effects of 2i treatment on ESC cultures.
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•Continuous long-term single-cell quantification of 2i effects on murine ESCs•2i enriches for a Nanog high population through a selective cell death effect•2i also upregulates Nanog expression and prevents its downregulation•The viability of Nanog−/− cells is compromised in 2i
Schroeder and colleagues show that GSK3 and MEK inhibition (2i) has several simultaneous effects on murine ESC cultures. They applied long-term time-lapse imaging to quantify cell death, proliferation, and Nanog expression dynamics in single ESCs. This demonstrated that 2i treatment of ESCs leads to more uniform and high Nanog expression due to both selective and inductive effects.
Since the first generation of induced pluripotent stem cells (iPSCs), several reprogramming systems have been used to study its molecular mechanisms. However, the system of choice largely affects the ...reprogramming efficiency, influencing our view on the mechanisms. Here, we demonstrate that reprogramming triggered by less efficient polycistronic reprogramming cassettes not only highlights mesenchymal-to-epithelial transition (MET) as a roadblock but also faces more severe difficulties to attain a pluripotent state even post-MET. In contrast, more efficient cassettes can reprogram both wild-type and Nanog−/− fibroblasts with comparable efficiencies, routes, and kinetics, unlike the less efficient reprogramming systems. Moreover, we attribute a previously reported variation in the N terminus of KLF4 as a dominant factor underlying these critical differences. Our data establish that some reprogramming roadblocks are system dependent, highlighting the need to pursue mechanistic studies with close attention to the systems to better understand reprogramming.
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•Distinct reprogramming cassettes yield different reprogramming intermediates•MET is not a major rate-limiting step in reprogramming with high KLF4 expression•A lack of endogenous Nanog becomes trivial in efficient reprogramming systems•Roadblocks toward iPSCs depend on the reprogramming systems
In this article, Kaji and colleagues demonstrate that different reprogramming systems with distinct reprogramming efficiencies yield clearly distinguishable intermediate populations and, thus, potentially biased mechanistic views. For example, mesenchymal-to-epithelial transition and lack of endogenous Nanog are obstacles for iPSC generation only in inefficient reprogramming systems. This work highlights the importance of re-assessing molecular mechanisms of reprogramming in the literature heeding the reprogramming systems.
The generation of induced pluripotent stem (iPS) cells presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the ...precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. Although several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPS cells. The rapid expansion of minor reprogrammed cells in the heterogeneous population can also obscure investigation of relevant transition processes. Understanding the biological mechanisms essential for successful iPS cell generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that in mouse embryonic fibroblasts, reprogramming follows an orderly sequence of stage transitions, marked by changes in the cell-surface markers CD44 and ICAM1, and a Nanog-enhanced green fluorescent protein (Nanog-eGFP) reporter. RNA-sequencing analysis of these populations demonstrates two waves of pluripotency gene upregulation, and unexpectedly, transient upregulation of several epidermis-related genes, demonstrating that reprogramming is not simply the reversal of the normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and the improved understanding of the reprogramming process will lead to new reprogramming strategies.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The elongating mouse anteroposterior axis is supplied by progenitors with distinct tissue fates. It is not known whether these progenitors confer anteroposterior pattern to the embryo. We have ...analysed the progenitor population transcriptomes in the mouse primitive streak and tail bud throughout axial elongation. Transcriptomic signatures distinguish three known progenitor types (neuromesodermal, lateral/paraxial mesoderm and notochord progenitors; NMPs, LPMPs and NotoPs). Both NMP and LPMP transcriptomes change extensively over time. In particular, NMPs upregulate Wnt, Fgf and Notch signalling components, and many Hox genes as progenitors transit from production of the trunk to the tail and expand in number. In contrast, the transcriptome of NotoPs is stable throughout axial elongation and they are required for normal axis elongation. These results suggest that NotoPs act as a progenitor niche whereas anteroposterior patterning originates within NMPs and LPMPs.
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