Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency.
ABSTRACT: Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.
Project description:Epiblast stem cells (EpiSCs) in mice and rats are primed pluripotent stem cells (PSCs). They barely contribute to chimeric embryos when injected into blastocysts. Reprogramming of EpiSCs to embryonic stem cell (ESC)-like cells (rESCs) may occur in response to LIF-STAT3 signaling; however, low reprogramming efficiency hampers potential use of rESCs in generating chimeras. Here, we describe dramatic improvement of conversion efficiency from primed to naive-like PSCs through upregulation of E-cadherin in the presence of the cytokine LIF. Analysis revealed that blocking nuclear localization of ?-CATENIN with small-molecule inhibitors significantly enhances reprogramming efficiency of mouse EpiSCs. Although activation of Wnt/?-catenin signals has been thought desirable for maintenance of naive PSCs, this study provides the evidence that inhibition of nuclear translocation of ?-CATENIN enhances conversion of mouse EpiSCs to naive-like PSCs (rESCs). This affords better understanding of gene regulatory circuits underlying pluripotency and reprogramming of PSCs.
Project description:Induced pluripotency depends on cooperativity between expression of defined factors and the culture environment. The latter also determines the pluripotent cell state, that is, naïve or primed. LIF-JAK/STAT3 signalling was recently shown to be a limiting factor for reprogramming to naïve pluripotency. Here we show that sufficient activation of JAK/STAT3 overcomes the reprogramming block of cell intermediates and enables somatic cell reprogramming in absence of otherwise essential pluripotency medium requisites. Activation of FGF-ERK signalling, which promotes exit of naïve pluripotent cells from self-renewal, does not prevent JAK/STAT3 induced post-implantation epiblast-derived stem cell conversion into naïve pluripotency. Moreover, even in the presence of FGF plus Activin, which instructs and maintains the primed state, JAK/STAT3 enforces naïve pluripotency in epiblast stem cells. We conclude that JAK/STAT3 signalling can be sufficient and dominant over antagonistic cues to enable the induction of a naïve pluripotent state.
Project description: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.
Project description:Pluripotent stem cells (PSCs) lie at the heart of modern regenerative medicine due to their properties of unlimited self-renewal in vitro and their ability to differentiate into cell types representative of the three embryonic germ layers-mesoderm, ectoderm and endoderm. The derivation of induced PSCs bypasses ethical concerns associated with the use of human embryonic stem cells and also enables personalized cell-based therapies. To exploit their regenerative potential, it is essential to have a firm understanding of the molecular processes associated with their induction from somatic cells. This understanding serves two purposes: first, to enable efficient, reliable and cost-effective production of excellent quality induced PSCs and, second, to enable the derivation of safe, good manufacturing practice-grade transplantable donor cells. Here, we review the reprogramming process of somatic cells into induced PSCs and associated mechanisms with emphasis on self-renewal, epigenetic control, mitochondrial bioenergetics, sub-states of pluripotency, naive ground state, naive and primed. A meta-analysis identified genes expressed exclusively in the inner cell mass and in the naive but not in the primed pluripotent state. We propose these as additional biomarkers defining naive PSCs.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.
Project description:Leukemia Inhibitory Factor (LIF)/Signal transducer and activator of transcription 3 (STAT3) signaling pathway maintains the stemness and pluripotency of mouse embryonic stem cells (mESCs). Detailed knowledge on key intermediates in this pathway as well as any parallel pathways is largely missing. We initiated our study by investigating the effect of small molecule Curcumin on various signalling pathways essential for self-renewal. Curcumin sustained the LIF independent self-renewal of mESCs and induced pluripotent stem cells (miPSCs) in a STAT3 activity dependent manner. Gene expression analysis showed LIF/STAT3 and redox signaling components to be majorly modulated. Amongst ROS genes, expression of Manganese Superoxide Dismutase (MnSOD) specifically relied on STAT3 signaling as evidenced by STAT3 inhibition and reporter assay. The silencing of MnSOD, but not Cu-ZnSOD expression, resulted in the loss of mESC pluripotency in presence of LIF, and the overexpression of MnSOD is sufficient for maintaining the expression of pluripotent genes in the absence of STAT3 signaling. Finally, we demonstrate MnSOD to stabilize the turnover of pluripotent proteins at the post-translational level by modulating proteasomal activity. In conclusion, our findings unravel a novel role of STAT3 mediated MnSOD in the self-renewal of mESCs.
Project description:Bone morphogenetic protein (BMP) signaling exerts paradoxical roles in pluripotent stem cells (PSCs); it sustains self-renewal of mouse embryonic stem cells (ESCs), while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs) in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors.
Project description:Activation of signal transducer and activator of transcription 3 (Stat3) by leukemia inhibitory factor (LIF) is required for maintaining self-renewal and pluripotency of mouse embryonic stem cells (mESCs). Here, we have confirmed transcription factor Forkhead Box m1 (Foxm1) as a LIF/Stat3 downstream target that mediates LIF/Stat3-dependent mESC self-renewal. The expression of Foxm1 relies on LIF signaling and is stimulated by Stat3 directly in mESCs. The knockdown of Foxm1 results in the loss of mESC pluripotency in the presence of LIF, and the overexpression of Foxm1 alone maintains mESC pluripotency in the absence of LIF and feeder layers, indicating that Foxm1 is a mediator of LIF/Stat3-dependent maintenance of pluripotency in mESCs. Furthermore, the inhibition of Foxm1 expression prevents the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells (iPSCs), suggesting that Foxm1 is essential for the reprogramming of somatic cells into iPSCs. Our results reveal an essential function of Foxm1 in the LIF/Stat3-mediated mESC self-renewal and the generation of iPSCs.
Project description:BACKGROUND:Human naïve pluripotency state cells can be derived from direct isolation of inner cell mass or primed-to-naïve resetting of human embryonic stem cells (hESCs) through different combinations of transcription factors, small molecular inhibitors, and growth factors. Long noncoding RNAs (lncRNAs) have been identified to be crucial in diverse biological processes, including pluripotency regulatory circuit of mouse pluripotent stem cells (PSCs), but few are involved in human PSCs' regulation of pluripotency and naïve pluripotency derivation. This study initially planned to discover more lncRNAs possibly playing significant roles in the regulation of human PSCs' pluripotency, but accidently identified a lncRNA whose knockdown in human PSCs induced naïve-like pluripotency conversion. METHODS:Candidate lncRNAs tightly correlated with human pluripotency were screened from 55 RNA-seq data containing human ESC, human induced pluripotent stem cell (iPSC), and somatic tissue samples. Then loss-of-function experiments in human PSCs were performed to investigate the function of these candidate lncRNAs. The naïve-like pluripotency conversion caused by CCDC144NL-AS1 knockdown (KD) was characterized by quantitative real-time PCR, immunofluorescence staining, western blotting, differentiation of hESCs in vitro and in vivo, RNA-seq, and chromatin immunoprecipitation. Finally, the signaling pathways in CCDC144NL-AS1-KD human PSCs were examined through western blotting and analysis of RNA-seq data. RESULTS:The results indicated that knockdown of CCDC144NL-AS1 induces naïve-like state conversion of human PSCs in the absence of additional transcription factors or small molecular inhibitors. CCDC144NL-AS1-KD human PSCs reveal naïve-like pluripotency features, such as elevated expression of naïve pluripotency-associated genes, increased developmental capacity, analogous transcriptional profiles to human naïve PSCs, and global reduction of repressive chromatin modification marks. Furthermore, CCDC144NL-AS1-KD human PSCs display inhibition of MAPK (ERK), accumulation of active β-catenin, and upregulation of some LIF/STAT3 target genes, and all of these are concordant with previously reported traits of human naïve PSCs. CONCLUSIONS:Our study unveils an unexpected role of a lncRNA, CCDC144NL-AS1, in the naïve-like state conversion of human PSCs, providing a new perspective to further understand the regulation process of human early pluripotency states conversion. It is suggested that CCDC144NL-AS1 can be potentially valuable for future research on deriving higher quality naïve state human PSCs and promoting their therapeutic applications.
Project description:Pluripotent stem cells (PSCs) can self-renew or differentiate from naive or more differentiated, primed, pluripotent states established by specific culture conditions. Increased intracellular ?-ketoglutarate (?KG) was shown to favor self-renewal in naive mouse embryonic stem cells (mESCs). The effect of ?KG or ?KG/succinate levels on differentiation from primed human PSCs (hPSCs) or mouse epiblast stem cells (EpiSCs) remains unknown. We examined primed hPSCs and EpiSCs and show that increased ?KG or ?KG-to-succinate ratios accelerate, and elevated succinate levels delay, primed PSC differentiation. ?KG has been shown to inhibit the mitochondrial ATP synthase and to regulate epigenome-modifying dioxygenase enzymes. Mitochondrial uncoupling did not impede ?KG-accelerated primed PSC differentiation. Instead, ?KG induced, and succinate impaired, global histone and DNA demethylation in primed PSCs. The data support ?KG promotion of self-renewal or differentiation depending on the pluripotent state.
Project description:Embryonic stem (ES) cell pluripotency is governed by OCT4-centric transcriptional networks. Conventional ES cells can be derived and maintained in vitro with media containing the cytokine leukemia inhibitory factor (LIF), which propagates the pluripotent state by activating STAT3 signaling, and simultaneous inhibition of glycogen synthase kinase-3 (GSK3) and MAP kinase/ERK kinase signaling. However, it is unclear whether overexpression of OCT4 is sufficient to overcome LIF-dependence. Here, we show that inducible expression of OCT4 (iOCT4) supports long-term LIF-independent self-renewal of ES cells cultured in media containing fetal bovine serum (FBS) and a glycogen synthase kinase-3 (GSK3) inhibitor, and in serum-free media. Global expression analysis revealed that LIF-independent iOCT4 ES cells and control ES cells exhibit similar transcriptional programs relative to epiblast stem cells (EpiSCs) and differentiated cells. Epigenomic profiling also demonstrated similar patterns of histone modifications between LIF-independent iOCT4 and control ES cells. Moreover, LIF-independent iOCT4 ES cells retain the capacity to differentiate in vitro and in vivo upon downregulation of OCT4 expression. These findings indicate that OCT4 expression is sufficient to sustain intrinsic signaling in a LIF-independent manner to promote ES cell pluripotency and self-renewal.