scholarly journals Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
L Tombor ◽  
D John ◽  
S.F Glaser ◽  
G Luxan ◽  
E Forte ◽  
...  
Keyword(s):  
Single Cell ◽  
Time Course ◽  
Metabolic Adaptation ◽  
Marker Genes ◽  
Funding Source ◽  
Paracrine Factors ◽  
Mesenchymal Transition ◽  
Mesenchymal Marker ◽  
Baseline Levels

Abstract   After myocardial infarct (MI), followed by ischemia and scar formation, interstitial cells play key roles in the adaptation to injury. Endothelial cells (ECs), for instance, can clonally expand, migrate into the infarct area and facilitate crucial functions promoting revascularization, reestablishment of oxygen supply and secretion of paracrine factors. Moreover, ECs can transiently undergo changes towards a mesenchymal phenotype (Endothelial-to-mesenchymal transition; EndMT). Whether this process contributes to long-term cardiac fibrosis or helps to facilitate post-ischemic vessel growth remains controversial. Here, we aim to delineate kinetics and characteristics of phenotypic changes in ECs with single cell RNA-sequencing (scRNA-seq). We performed a time course (homeostasis or 0 day (d), 1d, 3d, 5d, 7d, 14d, 28d post-MI) in mice and isolated the non-cardiomyocyte fraction for scRNA-seq (n=35,312 cells). Pecam1/Cdh5 double positive ECs showed expression of apoptosis, hypoxia and inflammation markers at 3d. Bioinformatic cell cycle analysis predicted high association with proliferative capacities at 3d, indicative of EC turnover post-MI. Metabolism, recently linked to regulate EndMT, was altered. We found genes of the glycolysis and the TCA-cycle pathway upregulated at 1d to 3d, and a decrease of fatty acid signaling genes. At 3d, mesenchymal markers Fn1, Vim, S100a4, Serpine1 transiently increased compared to homeostasis (>1.6-fold, p<0.05) together with a reduction of EC genes such as Pecam1. Interestingly, mesenchymal transition was transient and returned to baseline levels at 28d after MI. Cell fate trajectory analysis confirmed these findings by identifying an EC state characterized by high proliferation and mesenchymal but low EC properties. At 3d to 7d the majority of the ECs were assigned to this state, based on their transcriptomic profile. We additionally used Cdh5-CreERT2; R26-mT/mG mice followed by scRNA-seq to trace the fate of ECs. Bioinformatic analysis of GFP-positive ECs confirmed the gain in mesenchymal marker but revealed no full transition to the mesenchymal state at later timepoints. This suggests a transient mesenchymal activation of ECs rather than a complete lineage transition. We further induced EndMT with TGF-β2 in ECs in vitro and observed reversibility of the phenotype after withdrawal of the stimulus. After treatment, ECs upregulated various mesenchymal marker genes. Withdrawal of TGF-β2 at 3d or 7d, reverted expression to baseline levels. We further determined DNA methylation of EndMT gene loci to assess if TGF-β2 leads to a true fate change but did not observe changes after TGF-β2 stimulation and withdrawal. Taken together, our data suggests that ECs undergo a transient mesenchymal activation concomitant with a metabolic adaptation early after MI but do not acquire a long-term mesenchymal fate. This activation may facilitate EC migration and clonal expansion to regenerate the vascular network. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Center of Cardiovascular Research (DZHK), Deutsche Forschungsgemeinschaft (DFG) CRC1366 Project B4

scholarly journals Single-cell RNA sequencing of human fetal epicardium reveals novel markers and regulators of EMT

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T.J Streef ◽  
T Van Herwaarden ◽  
A.M Smits ◽  
M.J Goumans
Keyword(s):  
Cell Culture ◽  
Single Cell ◽  
Rna Sequencing ◽  
Public Institution ◽  
Funding Source ◽  
Injury Response ◽  
Post Injury ◽  
Paracrine Factors ◽  
Adult Heart ◽  
Single Cell Rna Sequencing

Abstract Background The heart is covered by the epicardium, consisting of epithelial cells and a mesenchymal layer. The epicardium has been shown to be essential during cardiac development by contributing cells through epithelial-to-mesenchymal transition (EMT) and the secretion of paracrine factors. In the adult, the epicardium conveys a cardioprotective response after myocardial infarction, albeit suboptimal compared to the epicardial contribution to heart development. Although the developing epicardium has been characterised in mice and zebrafish, knowledge on the human fetal epicardium derives mostly from cell culture models. Therefore, direct analysis of the human fetal epicardium is vital as it provides new insights into the cellular and biochemical interactions within the developing heart, which can potentially contribute to enhancing the post-injury response. Aim To study the human fetal epicardium using single-cell RNA sequencing (scRNA seq) in order to determine its cellular compositionThe data are further explored to e.g.identify regulators of epicardial EMT. Methods Epicardial layers were isolated from four fetal human hearts (14–15 weeks gestation, obtained under informed consent and according to local ethical approval). Tissue was digested, and single live cells were sorted into 384-wells plates and sequenced. Data analysis was performed using R-packages RaceID3 and StemID2. Findings were validated using qPCR and immunohistochemistry. Results Analysis of 2024 cells reveals a clear clustering of the epicardial epithelium and the mesenchymal population. Importantly, we found that “classical” markers, such as Wilms' Tumor 1 and T-box transcription factor 18, are not specific enough to reliably identify the epicardium, but our analysis has provided markers that do allow for robust identification of the epicardium. Additionally, we were able to identify epicardial subpopulations based on their expression profile, and we are currently investigating these using immunohistochemistry in human fetal and adult heart tissue sections. To establish the regulation of epicardial activation we are focussing on the process of EMT within our dataset using RaceID2. From our analysis, several regulators of epicardial EMT are proposed that will be followed up on in vitro. Conclusions We identify various novel markers of the fetal epithelial epicardium, as well as characterizing markers of the mesenchymal layer. We also identified novel factors involved in epicardial EMT, and these are currently being validated in our cell-culture model. These data can provide new insights into the post-injury response in the adult heart. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Dutch Heart Foundation

scholarly journals Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lukas S. Tombor ◽  
David John ◽  
Simone F. Glaser ◽  
Guillermo Luxán ◽  
Elvira Forte ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Endothelial Cell Migration ◽  
Metabolic Adaptation ◽  
Endothelial Cell Proliferation ◽  
Mesenchymal Transition ◽  
Single Cell Rna Sequencing

AbstractEndothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

scholarly journals Mapping lung cancer epithelial-mesenchymal transition states and trajectories with single-cell resolution

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Loukia G. Karacosta ◽  
Benedict Anchang ◽  
Nikolaos Ignatiadis ◽  
Samuel C. Kimmey ◽  
Jalen A. Benson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Single Cell ◽  
Cancer Progression ◽  
Time Course ◽  
Epithelial Mesenchymal Transition ◽  
Clinical Samples ◽  
Neural Net ◽  
Mesenchymal Transition ◽  
Phenotypic Profile

AbstractElucidating the spectrum of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) states in clinical samples promises insights on cancer progression and drug resistance. Using mass cytometry time-course analysis, we resolve lung cancer EMT states through TGFβ-treatment and identify, through TGFβ-withdrawal, a distinct MET state. We demonstrate significant differences between EMT and MET trajectories using a computational tool (TRACER) for reconstructing trajectories between cell states. In addition, we construct a lung cancer reference map of EMT and MET states referred to as the EMT-MET PHENOtypic STAte MaP (PHENOSTAMP). Using a neural net algorithm, we project clinical samples onto the EMT-MET PHENOSTAMP to characterize their phenotypic profile with single-cell resolution in terms of our in vitro EMT-MET analysis. In summary, we provide a framework to phenotypically characterize clinical samples in the context of in vitro EMT-MET findings which could help assess clinical relevance of EMT in cancer in future studies.

scholarly journals Single-Cell Transcriptomes Reveal Characteristic Features of Mouse Hepatocytes with Liver Cholestatic Injury

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1069 ◽  
Author(s):  
Chang ◽  
Tian ◽  
Ji ◽  
Zhou ◽  
Hou ◽  
...  
Keyword(s):  
Single Cell ◽  
Disease Process ◽  
Marker Genes ◽  
Mesenchymal Transition ◽  
Injury Model ◽  
Duct Ligation ◽  
Study Gene Expression ◽  
Mouse Hepatocytes ◽  
Characteristic Features ◽  
Parenchymal Cells

Hepatocytes are the main parenchymal cells of the liver and play important roles in liver homeostasis and disease process. The heterogeneity of normal hepatocytes has been reported, but there is little knowledge about hepatocyte subtype and distinctive functions during liver cholestatic injury. Bile duct ligation (BDL)-induced mouse liver injury model was employed, and single-cell RNA sequencing was performed. Western blot and qPCR were used to study gene expression. Immunofluoresence was employed to detect the expressions of marker genes in hepatocytes. We detected a specific hepatocyte cluster (BDL-6) expressing extracellular matrix genes, indicating these hepatocytes might undergo epithelia-mesenchymal transition. Hepatocytes of BDL-6 also performed tissue repair functions (such as angiogenesis) during cholestatic injury. We also found that four clusters of cholestatic hepatocytes (BDL-2, BDL-3, BDL-4, and BDL-5) were involved in inflammatory process in different ways. To be specific, BDL-2/3/5 were inflammation-regulated hepatocytes, while BDL-4 played a role in cell chemotaxis. Among these four clusters, BDL-5 was special. because the hepatocytes of BDL-5 were proliferating hepatocytes. Our analysis provided more knowledge of hepatocyte distinctive functions in injured liver and gave rise to future treatment aiming at hepatocytes.

scholarly journals Epigenetic adaptation prolongs photoreceptor survival during retinal degeneration

2019 ◽  
Author(s):  
Rachayata Dharmat ◽  
Sangbae Kim ◽  
Hehe Liu ◽  
Shangyi Fu ◽  
Yumei Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Exponential Distribution ◽  
Time Course ◽  
Chromatin State ◽  
Retinal Cell ◽  
Marker Genes ◽  
Specific Marker ◽  
Stretched Exponential ◽  
Rod Cells ◽  
Stretched Exponential Distribution

AbstractNeural degenerative diseases often display a progressive loss of cells as a stretched exponential distribution. The mechanisms underlying the survival of a subset of genetically identical cells in a population beyond what is expected by chance alone remains unknown. To gain mechanistic insights underlying prolonged cellular survival, we used Spata7 mutant mice as a model and performed single-cell transcriptomic profiling of retinal tissue along the time course of photoreceptor degeneration. Intriguingly, rod cells that survive beyond the initial rapid cell apoptosis phase progressively acquire a distinct transcriptome profile. In these rod cells, expression of photoreceptor-specific phototransduction pathway genes is downregulated while expression of other retinal cell type-specific marker genes is upregulated. These transcriptomic changes are achieved by modulation of the epigenome and changes of the chromatin state at these loci, as indicated by immunofluorescence staining and single-cell ATAC-seq. Consistent with this model, when induction of the repressive epigenetic state is blocked by in vivo histone deacetylase inhibition, all photoreceptors in the mutant retina undergo rapid degeneration, strongly curtailing the stretched exponential distribution. Our study reveals an intrinsic mechanism by which neural cells progressively adapt to genetic stress to achieve prolonged survival through epigenomic regulation and chromatin state modulation.

scholarly journals Effects of catheter ablation on left atrial performance in different types of atrial fibrillation: a strain study

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Katbeh ◽  
T De Potter ◽  
P Geelen ◽  
E Stefanidis ◽  
K Iliodromitis ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Catheter Ablation ◽  
Time Course ◽  
Public Institution ◽  
P Value ◽  
Funding Source ◽  
Functional Changes ◽  
Af Recurrence

Abstract Background Atrial structural and functional changes may develop as a result of catheter ablation (CA) in patients with paroxysmal and persistent atrial fibrillation (AF). However, the relation between AF recurrence and atrial performance following CA is still under debate. Our aim is to describe the long-term effects of CA on LA remodeling and its correlates to the maintenance of sinus rhythm (SR). Methods We prospectively enrolled 178 consecutive patients (age: 63±9 years, 35% females) with paroxysmal AF undergoing first-CA (67%) or redo-CA (22%), and 20 individuals (11%) with long-standing persistent AF (PAF) undergoing first CA. All patients underwent comprehensive transthoracic echocardiography at baseline and at 12-month follow-up, including the assessment of reservoir and contractile strain (LAS) using two dimensional speckle tracking echocardiography in all three apical views. The study population was divided in two sub-groups according to AF recurrence during follow-up. Results During one-year follow-up, 144 (81%) patients maintained SR whereas 34 (19%) patients had AF recurrence [first-CA group 16 (13%), redo-CA group 8 (20%) and PAF group 10 (50%)]. Improvement of LAS was observed only in patients with paroxysmal and long-standing persistent AF who underwent the first CA and who remained in SR (Figure 1A, 1C). In contrast, recurrent AF was associated with absence of LAS improvement (Figure 1A, 1C). Different time course of LA performance was observed in the redo-CA group, i.e. LAS remained unchanged from baseline regardless of long-term maintenance of SR (Figure 1B). Moreover, at follow-up, no significant differences in LAS between redo-CA patients with SR versus AF were observed. Of note, in patients with long-standing persistent AF and SR, follow-up LAS increased to values observed in the redo-CA group. Conclusion LA performance following CA is strongly affected by complex interplay between extent of atrial electro-structural remodeling and CA procedure. Repeated wide CA might affects negatively LA compliance and contractility despite SR restoration. Figure 1. Reservoir and contractile LAS at Baseline and 12-month follow-up in the First-CA (1A), the Redo-CA (1B) and the long-standing persistent AF (1C) groups in patients who maintained SR versus patients who had AF recurrence. *p value <0.05 (baseline vs. follow-up). Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): International PhD programme in Cardiovascular Pathophysiology and Therapeutics (CardioPaTh).

scholarly journals Single-cell RNA sequencing of human fetal epicardium reveals novel markers and regulators of EMT

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
T J Streef ◽  
T Van Herwaarden ◽  
M J Goumans ◽  
A M Smits
Keyword(s):  
Cell Culture ◽  
Single Cell ◽  
Rna Sequencing ◽  
Heart Development ◽  
Injury Response ◽  
Post Injury ◽  
Paracrine Factors ◽  
Mesenchymal Transition ◽  
Adult Heart ◽  
Single Cell Rna Sequencing

Abstract Background The heart is covered by the epicardium, consisting of epithelial cells and a mesenchymal layer. The epicardium has been shown to be essential during cardiac development by contributing cells through epithelial-to-mesenchymal transition (EMT) and the secretion of paracrine factors. In the adult, the epicardium conveys a cardioprotective response after myocardial infarction, albeit suboptimal compared to the epicardial contribution to heart development. Although the developing epicardium has been characterised in mice and zebrafish, knowledge on the human fetal epicardium derives mostly from cell culture models. Therefore, direct analysis of the human fetal epicardium is vital as it provides new insights into the cellular and biochemical interactions within the developing heart, which can potentially contribute to enhancing the post-injury response. Aim To study the human fetal epicardium using single-cell RNA sequencing (scRNA seq) in order to determine its cellular composition. The data are further explored to e.g. identify regulators of epicardial EMT. Methods Epicardial layers were isolated from four fetal human hearts (14–15 weeks gestation, obtained under informed consent and according to local ethical approval). Tissue was digested, and single live cells were sorted into 384-wells plates and sequenced. Data analysis was performed using R-packages RaceID3 and StemID2. Findings were validated using qPCR and immunohistochemistry. Results Analysis of 2073 cells reveals a clear clustering of the epicardial epithelium and the mesenchymal population. Importantly, we found that “classical” markers, such as Wilms' Tumor 1 and T-box transcription factor 18, are not specific enough to reliably identify the epicardium, but our analysis has provided markers that do allow for robust identification of the epicardium. Additionally, we were able to identify epicardial subpopulations based on their expression profile and validated these using immunohistochemistry in human fetal and adult heart tissue sections. To establish the regulation of epicardial activation we are focussing on the process of EMT within our dataset using RaceID2. From our analysis, several regulators of epicardial EMT are proposed that will be followed up on in vitro. Conclusions We identify various novel markers of the fetal epithelial epicardium, as well as characterizing markers of the mesenchymal layer. We also identified novel factors involved in epicardial EMT, and these are currently being validated in our cell-culture model. These data can provide new insights into the post-injury response in the adult heart. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation

scholarly journals Mapping Lung Cancer Epithelial-Mesenchymal Transition States and Trajectories with Single-Cell Resolution

2019 ◽  
Author(s):  
Loukia G. Karacosta ◽  
Benedict Anchang ◽  
Nikolaos Ignatiadis ◽  
Samuel C. Kimmey ◽  
Jalen A. Benson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Single Cell ◽  
Cancer Progression ◽  
Time Course ◽  
Epithelial Mesenchymal Transition ◽  
Clinical Samples ◽  
Neural Net ◽  
Mesenchymal Transition ◽  
Phenotypic Profile

ABSTRACTElucidating a continuum of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) states in clinical samples promises new insights in cancer progression and drug response. Using mass cytometry time-course analysis, we resolve lung cancer EMT states through TGFβ-treatment and identify through TGFβ-withdrawal, an MET state previously unrealized. We demonstrate significant differences between EMT and MET trajectories using a novel computational tool (TRACER) for reconstructing trajectories between cell states. Additionally, we construct a lung cancer reference map of EMT and MET states referred to as the EMT-MET STAte MaP (STAMP). Using a neural net algorithm, we project clinical samples onto the EMT-MET STAMP to characterize their phenotypic profile with single-cell resolution in terms of ourin vitroEMT-MET analysis. In summary, we provide a framework that can be extended to phenotypically characterize clinical samples in the context ofin vitrostudies showing differential EMT-MET traits related to metastasis and drug sensitivity.

A small molecule screen identifies novel inducers of EMT that may increase epicardium-driven repair of the heart

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
E Dronkers ◽  
T Van Herwaarden ◽  
M.J Goumans ◽  
A.M Smits
Keyword(s):  
Small Molecule ◽  
Ex Vivo ◽  
Smooth Muscle Actin ◽  
Cardiac Injury ◽  
Funding Source ◽  
Paracrine Factors ◽  
Mesenchymal Transition ◽  
Adult Heart ◽  
Small Molecule Screen

Abstract Background Since the adult heart has minimal capacity to repair itself, myocardial infarction often leads to pathological remodelling and consequently the development of heart failure. Unfortunately, no therapy is available to restore damaged heart tissue. The epicardium, the outer layer of the heart, plays an important role during the development of the heart via the contribution of cells and paracrine factors to the myocardium. In this process, epicardial epithelial-to-mesenchymal transition (EMT) is an essential step. In the adult heart, developmental epicardial behaviour is recapitulated upon injury, characterized by upregulation of e.g. EMT-related genes. However, this process is less efficient than fetal activation. We hypothesize that the reparative capacity of the heart can be enhanced by optimizing the participation of epicardium to repair, particularly through increasing the occurrence of EMT. The aim of this study is to find epicardial EMT-inducing compounds using a small molecule screen which will ultimately improve epicardium-driven repair after cardiac injury. Methods Epicardial cells were derived from human heart auricles that are routinely removed during thoracic surgery. These epicardial derived cells (EPDCs) were cultured as epithelial-like cells with a cobblestone morphology in the presence of a TGFβ inhibitor. Upon stimulation with TGFβ ligand (1ng/mL), EPDCs undergo EMT, characterized by a switch from cobblestone to spindle-shaped cells and upregulation of α-Smooth Muscle Actin (αSMA). Using these cells, a phenotypic screen was performed in 384 wells format using the LOPAC1280 small molecule library. EMT was assessed by αSMA immunostaining. The screen was performed three times (2x 10 μM and 1x 5 μM) to include interpatient variability. From each screen the top 20 ranked compounds were validated in multiple primary epicardial cell isolations. Results From the 1280 tested compounds, at least 54 compounds displayed a clear upregulation of αSMA compared to the negative controls. Next, after validation in individual patient isolations, five compounds were selected for further analysis. These five compounds reproducibly induce EMT, as shown by a dose-dependent decrease of epithelial marker E-Cadherin, and an increase of mesenchymal markers (SMA, Periostin) and EMT transcription factors (Snail, Slug). The compounds were not toxic to cardiomyocytes and fibroblasts and 4 out of 5 compounds were not toxic to endothelial cells. Currently, the compounds are tested for their ability to induce invasion in ex vivo hearts. Furthermore, we will determine the effect on cardiac repair in vivo by incorporating the compounds into slowly releasing, biodegradable patches that are applied onto the damaged murine heart. Conclusion A small molecule screen revealed five novel epicardial EMT-inducing compounds that have the potential to increase the contribution of the epicardium to the repair of the heart. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Dutch Heart Foundation

scholarly journals Single-cell transcriptomics, scRNA-Seq and C1 CAGE discovered distinct phases of pluripotency during naïve-to-primed conversion in mice

2020 ◽  
Author(s):  
Michael Böttcher ◽  
Yuhki Tada ◽  
Jonathan Moody ◽  
Masayo Kondo ◽  
Hiroki Ura ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Single Cell ◽  
Epithelial Mesenchymal Transition ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Transition Process ◽  
Marker Genes ◽  
Sudden Increase ◽  
Mesenchymal Transition

AbstractBackgroundTwo types of mammalian pluripotent stem cells (PSC), i.e. naïve and primed possess distinct cellular characteristics. It is largely unknown how these differences are generated during naïve-to-primed transition process. We have established a robust in vitro transition system using a Wnt inhibitor for the first time and analyzed dynamic changes in cellular status via single-cell RNA-sequencing and C1 CAGE analyses.ResultsAnalysis of known marker genes suggested that the cell transition process progresses as expected. However, cluster analyses revealed a sudden increase in expression profile diversities three and four days after induction of the transition. These expression diversities can be reconciled by the presence of two subpopulations with distinct transcription profiles emerging at these time points. One of the subpopulations appears transiently, and surprisingly these cells showed a global downregulation of gene expression. Moreover, initiation of random X chromosome inactivation (XCI) coincides with the appearance of these transient cells. The other subpopulation can be maintained as a stem cell line and possesses expression profiles more similar to those of primed epiblast stem cells (EpiSC) than embryonic stem cells (ESC). However, there are important differences in gene expression related to epithelial-mesenchymal transition (EMT), suggesting that this subpopulation may represent a novel pluripotent state that has an intermediate cellular phenotype between ESC and EpiSC.ConclusionsThese findings should contribute to our understanding of the establishment and maintenance of distinct differentiation statuses of mammalian PSCs and provide new insights into the pluripotency spectrum in general.

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