Wnt/?-catenin signaling promotes self-renewal and inhibits the primed state transition in naive human embryonic stem cells.
ABSTRACT: In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/?-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/?-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of ?-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/?-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/?-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.
Project description:<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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 ?-catenin, and upregulation of some LIF/STAT3 target genes, and all of these are concordant with previously reported traits of human naïve PSCs.<h4>Conclusions</h4>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:WNT signaling is involved in maintaining stem cells in an undifferentiated state; however, it is often unclear which WNTs and WNT receptors are mediating these activities. Here we examined the role of the WNT receptor FZD7 in maintaining human embryonic stem cells (hESCs) in an undifferentiated and pluripotent state. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated cell populations, and interfering with its expression or function, either by short hairpin RNA-mediated knockdown or with a fragment antigen binding (Fab) molecule directed against FZD7, disrupts the pluripotent state of hESCs. The FZD7-specific Fab blocks signaling by Wnt3a protein by down-regulating FZD7 protein levels, suggesting that FZD7 transduces Wnt signals to activate Wnt/?-catenin signaling. These results demonstrate that FZD7 encodes a regulator of the pluripotent state and that hESCs require endogenous WNT/?-catenin signaling through FZD7 to maintain an undifferentiated phenotype.
Project description:In mice, inhibition of both the fibroblast growth factor (FGF) mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/Erk) and the Wnt signaling inhibitor glycogen synthase-3? (GSK3?) enables the derivation of mouse embryonic stem cells (mESCs) from nonpermissive strains in the presence of leukemia inhibitory factor (LIF). Whereas mESCs are in an uncommitted naïve state, human embryonic stem cells (hESCs) represent a more advanced state, denoted as primed pluripotency. This burdens hESCs with a series of characteristics, which, in contrast to naïve ESCs, makes them not ideal for key applications such as cell-based clinical therapies and human disease modeling. In this study, different small molecule combinations were applied during human ESC derivation. Hereby, we aimed to sustain the naïve pluripotent state, by interfering with various key signaling pathways. First, we tested several combinations on existing, 2i (PD0325901 and CHIR99021)-derived mESCs. All combinations were shown to be equally adequate to sustain the expression of naïve pluripotency markers. Second, these conditions were tested during hESC derivation. Overall, the best results were observed in the presence of medium supplemented with 2i, LIF, and the noncanonical Wnt signaling agonist Wnt5A, alone and combined with epinephrine. In these conditions, outgrowths repeatedly showed an ESC progenitor-like morphology, starting from day 3. Culturing these "progenitor cells" did not result in stable, naïve hESC lines in the current conditions. Although Wnt5A could not promote naïve hESC derivation, we found that it was sustaining the conversion of established hESCs toward a more naïve state. Future work should aim to distinct the effects of the various culture formulations, including our Wnt5A-supplemented medium, reported to promote stable naïve pluripotency in hESCs.
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:Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.
Project description:Cardiac development requires coordinated biphasic regulation of the WNT/β-catenin signaling pathway. By intersecting gene expression and loss-of-function siRNA screens we identified Alpha Protein Kinase 2 (ALPK2) as a candidate negative regulator of WNT/β-catenin signaling in cardiogenesis. In differentiating human embryonic stem cells (hESCs), ALPK2 is highly induced as hESCs transition from mesoderm to cardiac progenitors. Using antisense knockdown and CRISPR/Cas9 mutagenesis in hESCs and zebrafish, we demonstrate that ALPK2 promotes cardiac function and cardiomyocyte differentiation. Quantitative phosphoproteomics, protein expression profiling, and β-catenin reporter assays demonstrate that loss of ALPK2 led to stabilization of β-catenin and increased WNT signaling. Furthermore, cardiac defects attributed to ALPK2 depletion can be rescued in a dose-dependent manner by direct inhibition of WNT signaling through the small molecule XAV939. Together, these results demonstrate that ALPK2 regulates β-catenin-dependent signaling during developmental commitment of cardiomyocytes.
Project description:Conventional human pluripotent stem cells (hPSCs), known for being in a primed state, are pivotal for both basic research and clinical applications since such cells produce various types of differentiated cells. Recent reports on PSCs shed light on the pluripotent hierarchy of stem cells and have promoted the exploration of new stem cell states along with their culture systems. Human naïve PSCs are expected to provide further knowledge of early developmental mechanisms and improvements for differentiation programmes in the regenerative therapy of conventionally primed PSCs. However, practical challenges exist in using naïve-state PSCs such as determining the conditions for hypoxic culture condition and showing limited stable cellular proliferation. Here, we have developed new leukemia inhibitory factor dependent PSCs by applying our previous work, the combination of dibenzazepine and a DOT1L inhibitor to achieve the stable culture of naïve-state PSCs. The potential of these cells to differentiate into all three germ layers was shown both in vitro and in vivo. Such new naïve-state PSCs formed dome-shaped colonies at a faster rate than conventional, primed-state human induced PSCs and could be maintained for an extended period in the absence of hypoxic culture conditions. We also identified relatively high expression levels of naïve cell markers. Thus, non-hypoxia treated, leukemia inhibitory factor-dependent PSCs are anticipated to have characteristics similar to those of naïve-like PSCs, and to enhance the utility value of PSCs. Such naïve PSCs may allow the molecular characterization of previously undefined naïve human PSCs, and to ultimately contribute to the use of human pluripotent stem cells in regenerative medicine and disease modelling.
Project description:In the liver, Wnt/?-catenin signaling is involved in regulating zonation and hepatocyte proliferation during homeostasis. We examined Wnt gene expression and signaling after injury, and we show by in situ hybridization that Wnts are activated by acute carbon tetrachloride (CCl4 ) toxicity. Following injury, peri-injury hepatocytes become Wnt-responsive, expressing the Wnt target gene axis inhibition protein 2 (Axin2). Lineage tracing of peri-injury Axin2+ hepatocytes shows that during recovery the injured parenchyma becomes repopulated and repaired by Axin2+ descendants. Using single-cell RNA sequencing, we show that endothelial cells are the major source of Wnts following acute CCl4 toxicity. Induced loss of ?-catenin in peri-injury hepatocytes results in delayed repair and ultimately injury-induced lethality, while loss of Wnt production from endothelial cells leads to a delay in the proliferative response after injury. Conclusion: Our findings highlight the importance of the Wnt/?-catenin signaling pathway in restoring tissue integrity following acute liver toxicity and establish a role of endothelial cells as an important Wnt-producing regulator of liver tissue repair following localized liver injury.
Project description:Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.
Project description: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. 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 previous reported traits of human naïve PSCs.