Osmolar Modulation Drives Reversible Cell Cycle Exit and Human Pluripotent Cell Differentiation via NF-κВ and WNT Signaling.

Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53-independent quiescent state in immature hepatoma cells and in pluripotent stem cell-derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity-regulated growth arrest followed by cell maturation, mediated by activation of NF-κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long-term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely.

Automated Generation of hiPSC-Derived Hepatic Progeny by Cost-Efficient Compounds.

Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) hold great promise for liver disease modeling, drug discovery, and drug toxicity screens. Yet, several hurdles still need to be overcome, including among others decrease in the cost of goods to generate HLCs and automation of the differentiation process. We here describe that the use of an automated liquid handling system results in highly reproducible HLC differentiation from hPSCs. This enabled us to screen 92 chemicals to replace expensive growth factors at each step of the differentiation protocol to reduce the cost of goods of the differentiation protocol by approximately 79%. In addition, we also evaluated several recombinant extracellular matrices to replace Matrigel. We demonstrated that differentiation of hPSCs on Laminin-521 using an optimized small molecule combination resulted in HLCs that were transcriptionally identical to HLCs generated using the growth factor combinations. In addition, the HLCs created using the optimized small molecule combination secreted similar amounts of albumin and urea, and relatively low concentrations of alfa-fetoprotein, displayed similar CYP3A4 functionality, and a similar drug toxicity susceptibility as HLCs generated with growth factor cocktails. The broad applicability of the new differentiation protocol was demonstrated for 4 different hPSC lines. This allowed the creation of a scalable, xeno-free, and cost-efficient hPSC-derived HLC culture, suitable for high throughput disease modeling and drug screenings, or even for the creation of HLCs for regenerative therapies.

Dual functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation.

Gastrulation begins when the epiblast forms the primitive streak or becomes definitive ectoderm. During this lineage bifurcation, the DNA dioxygenase TET1 has bipartite functions in transcriptional activation and repression, but the mechanisms remain unclear. By converting mouse embryonic stem cells (ESCs) into neuroprogenitors, we defined how Tet1-/- cells switch from neuroectoderm fate to form mesoderm and endoderm. We identified the Wnt repressor Tcf7l1 as a TET1 target that suppresses Wnt/ß-catenin and Nodal signalling. ESCs expressing catalytic dead TET1 retain neural potential but activate Nodal and subsequently Wnt/ß-catenin pathways to generate also mesoderm and endoderm. At CpG-poor distal enhancers, TET1 maintains accessible chromatin at neuroectodermal loci independently of DNA demethylation. At CpG-rich promoters, DNA demethylation by TET1 affects the expression of bivalent genes. In ESCs, a non-catalytic TET1 cooperation with Polycomb represses primitive streak genes; post-lineage priming, the interaction becomes antagonistic at neuronal genes, when TET1's catalytic activity is further involved by repressing Wnt signalling. The convergence of repressive DNA and histone methylation does not inhibit neural induction in Tet1-deficient cells, but some DNA hypermethylated loci persist at genes with brain-specific functions. Our results reveal versatile switching of non-catalytic and catalytic TET1 activities based on genomic context, lineage and developmental stage.

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency.

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells.

A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here, we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. EXMCs are mesenchymal, can arise from an epiblast intermediate, and are capable of self-renewal. Thus, EXMCs arising via primate-specific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects.

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

Wnt Signaling in the Breast: From Development to Disease.

The Wnt cascade is a primordial developmental signaling pathway that plays a myriad of essential functions throughout development and adult homeostasis in virtually all animal species. Aberrant Wnt activity is implicated in embryonic and tissue morphogenesis defects, and several diseases, most notably cancer. The role of Wnt signaling in mammary gland development and breast cancer initiation, maintenance, and progression is far from being completely understood and is rather shrouded in controversy. In this review, we dissect the fundamental role of Wnt signaling in mammary gland development and adult homeostasis and explore how defects in its tightly regulated and intricated molecular network are interlinked with cancer, with a focus on the breast.

Wnt/ß-Catenin Inhibition Disrupts Carboplatin Resistance in Isogenic Models of Triple-Negative Breast Cancer.

Triple-Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype, characterized by limited treatment options and higher relapse rates than hormone-receptor-positive breast cancers. Chemotherapy remains the mainstay treatment for TNBC, and platinum salts have been explored as a therapeutic alternative in neo-adjuvant and metastatic settings. However, primary and acquired resistance to chemotherapy in general and platinum-based regimens specifically strongly hampers TNBC management. In this study, we used carboplatin-resistant in vivo patient-derived xenograft and isogenic TNBC cell-line models and detected enhanced Wnt/ß-catenin activity correlating with an induced expression of stem cell markers in both resistant models. In accordance, the activation of canonical Wnt signaling in parental TNBC cell lines increases stem cell markers' expression, formation of tumorspheres and promotes carboplatin resistance. Finally, we prove that Wnt signaling inhibition resensitizes resistant models to carboplatin both in vitro and in vivo, suggesting the synergistic use of Wnt inhibitors and carboplatin as a therapeutic option in TNBC. Here we provide evidence for a prominent role of Wnt signaling in mediating resistance to carboplatin, and we establish that combinatorial targeting of Wnt signaling overcomes carboplatin resistance enhancing chemotherapeutic drug efficacy.

Keep the fate: how chromatin regulators safeguard embryonic stem cell identity.

Segregation of cells that form the embryo from those that produce the surrounding extra-embryonic tissues is critical for early mammalian development, but the regulatory layers governing these first cell fate decisions remain poorly understood. Recent work in The EMBO Journal identifies two chromatin regulators, Hdac3 and Dax1, that synergistically restrict the developmental potential of mouse embryonic stem cells and act as a lineage barrier to primitive endoderm formation.

Canonical Wnt Pathway Controls mESC Self-Renewal Through Inhibition of Spontaneous Differentiation via ß-Catenin/TCF/LEF Functions.

The Wnt/ß-catenin signaling pathway is a key regulator of embryonic stem cell (ESC) self-renewal and differentiation. Constitutive activation of this pathway has been shown to increase mouse ESC (mESC) self-renewal and pluripotency gene expression. In this study, we generated a novel ß-catenin knockout model in mESCs to delete putatively functional N-terminally truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as ß-catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a ß-catenin-dependent manner. Finally, ß-catenin (ΔC) isoforms can rescue ß-catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner.

Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration.

Defective cell migration causes delayed wound healing (WH) and chronic skin lesions. Autologous micrograft (AMG) therapies have recently emerged as a new effective and affordable treatment able to improve wound healing capacity. However, the precise molecular mechanism through which AMG exhibits its beneficial effects remains unrevealed. Herein we show that AMG improves skin re-epithelialization by accelerating the migration of fibroblasts and keratinocytes. More specifically, AMG-treated wounds showed improvement of indispensable events associated with successful wound healing such as granulation tissue formation, organized collagen content, and newly formed blood vessels. We demonstrate that AMG is enriched with a pool of WH-associated growth factors that may provide the starting signal for a faster endogenous wound healing response. This work links the increased cell migration rate to the activation of the extracellular signal-regulated kinase (ERK) signaling pathway, which is followed by an increase in matrix metalloproteinase expression and their extracellular enzymatic activity. Overall we reveal the AMG-mediated wound healing transcriptional signature and shed light on the AMG molecular mechanism supporting its potential to trigger a highly improved wound healing process. In this way, we present a framework for future improvements in AMG therapy for skin tissue regeneration applications.

Controlled ploidy reduction of pluripotent 4n cells generates 2n cells during mouse embryo development.

Cells with high ploidy content are common in mammalian extraembryonic and adult tissues. Cell-to-cell fusion generates polyploid cells during mammalian development and tissue regeneration. However, whether increased ploidy can be occasionally tolerated in embryonic lineages still remains largely unknown. Here, we show that pluripotent, fusion-derived tetraploid cells, when injected in a recipient mouse blastocyst, can generate diploid cells upon ploidy reduction. The generated diploid cells form part of the adult tissues in mouse chimeras. Parental chromosomes in pluripotent tetraploid cells are segregated through tripolar mitosis both randomly and nonrandomly and without aneuploidy. Tetraploid-derived diploid cells show a differentiated phenotype. Overall, we discovered an unexpected process of controlled genome reduction in pluripotent tetraploid cells. This mechanism can ultimately generate diploid cells during mouse embryo development and should also be considered for cell fusion-mediated tissue regeneration approaches.

Activator Protein-1 Transcriptional Activity Drives Soluble Micrograft-Mediated Cell Migration and Promotes the Matrix Remodeling Machinery.

Impaired wound healing and tissue regeneration have severe consequences on the patient's quality of life. Micrograft therapies are emerging as promising and affordable alternatives to improve skin regeneration by enhancing the endogenous wound repair processes. However, the molecular mechanisms underpinning the beneficial effects of the micrograft treatments remain largely unknown. In this study, we identified the active protein-1 (AP-1) member Fos-related antigen-1 (Fra-1) to play a central role in the extracellular signal-regulated kinase- (ERK-) mediated enhanced cell migratory capacity of soluble micrograft-treated mouse adult fibroblasts and in the human keratinocyte cell model. Accordingly, we show that increased micrograft-dependent in vitro cell migration and matrix metalloprotease activity is abolished upon inhibition of AP-1. Furthermore, soluble micrograft treatment leads to increased expression and posttranslational phosphorylation of Fra-1 and c-Jun, resulting in the upregulation of wound healing-associated genes mainly involved in the regulation of cell migration. Collectively, our work provides insights into the molecular mechanisms behind the cell-free micrograft treatment, which might contribute to future advances in wound repair therapies.

Generation of a human induced pluripotent stem cell-based model for tauopathies combining three microtubule-associated protein TAU mutations which displays several phenotypes linked to neurodegeneration.

INTRODUCTION: Tauopathies are neurodegenerative diseases characterized by TAU protein-related pathology, including frontotemporal dementia and Alzheimer's disease among others. Mutant TAU animal models are available, but none of them faithfully recapitulates human pathology and are not suitable for drug screening. METHODS: To create a new in vitro tauopathy model, we generated a footprint-free triple MAPT-mutant human induced pluripotent stem cell line (N279K, P301L, and E10+16 mutations) using clustered regularly interspaced short palindromic repeats-FokI and piggyBac transposase technology. RESULTS: Mutant neurons expressed pathogenic 4R and phosphorylated TAU, endogenously triggered TAU aggregation, and had increased electrophysiological activity. TAU-mutant cells presented deficiencies in neurite outgrowth, aberrant sequence of differentiation to cortical neurons, and a significant activation of stress response pathways. RNA sequencing confirmed stress activation, demonstrated a shift toward GABAergic identity, and an upregulation of neurodegenerative pathways. DISCUSSION: In summary, we generated a novel in vitro human induced pluripotent stem cell TAU-mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening.

The Pleiotropic Effects of the Canonical Wnt Pathway in Early Development and Pluripotency.

The technology to derive embryonic and induced pluripotent stem cells from early embryonic stages and adult somatic cells, respectively, emerged as a powerful resource to enable the establishment of new in vitro models, which recapitulate early developmental processes and disease. Additionally, pluripotent stem cells (PSCs) represent an invaluable source of relevant differentiated cell types with immense potential for regenerative medicine and cell replacement therapies. Pluripotent stem cells support self-renewal, potency and proliferation for extensive periods of culture in vitro. However, the core pathways that rule each of these cellular features specific to PSCs only recently began to be clarified. The Wnt signaling pathway is pivotal during early embryogenesis and is central for the induction and maintenance of the pluripotency of PSCs. Signaling by the Wnt family of ligands is conveyed intracellularly by the stabilization of ß-catenin in the cytoplasm and in the nucleus, where it elicits the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors. Interestingly, in PSCs, the Wnt/ß-catenin-TCF/LEF axis has several unrelated and sometimes opposite cellular functions such as self-renewal, stemness, lineage commitment and cell cycle regulation. In addition, tight control of the Wnt signaling pathway enhances reprogramming of somatic cells to induced pluripotency. Several recent research efforts emphasize the pleiotropic functions of the Wnt signaling pathway in the pluripotent state. Nonetheless, conflicting results and unanswered questions still linger. In this review, we will focus on the diverse functions of the canonical Wnt signaling pathway on the developmental processes preceding embryo implantation, as well as on its roles in pluripotent stem cell biology such as self-renewal and cell cycle regulation and somatic cell reprogramming.

Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus.

Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/ß-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/ß-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15Ink4b, p16Ink4a and p19Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of ß-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors.

PDGFRα+ Cells in Embryonic Stem Cell Cultures Represent the In Vitro Equivalent of the Pre-implantation Primitive Endoderm Precursors.

In early mouse pre-implantation development, primitive endoderm (PrE) precursors are platelet-derived growth factor receptor alpha (PDGFRα) positive. Here, we demonstrated that cultured mouse embryonic stem cells (mESCs) express PDGFRα heterogeneously, fluctuating between a PDGFRα+ (PrE-primed) and a platelet endothelial cell adhesion molecule 1 (PECAM1)-positive state (epiblast-primed). The two surface markers can be co-detected on a third subpopulation, expressing epiblast and PrE determinants (double-positive). In vitro, these subpopulations differ in their self-renewal and differentiation capability, transcriptional and epigenetic states. In vivo, double-positive cells contributed to epiblast and PrE, while PrE-primed cells exclusively contributed to PrE derivatives. The transcriptome of PDGFRα+ subpopulations differs from previously described subpopulations and shows similarities with early/mid blastocyst cells. The heterogeneity did not depend on PDGFRα but on leukemia inhibitory factor and fibroblast growth factor signaling and DNA methylation. Thus, PDGFRα+ cells represent the in vitro counterpart of in vivo PrE precursors, and their selection from cultured mESCs yields pure PrE precursors.

Temporal perturbation of the Wnt signaling pathway in the control of cell reprogramming is modulated by TCF1.

Cyclic activation of the Wnt/ß-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with ß-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/ß-catenin signaling needs to be off during the early reprogramming phases of mouse embryonic fibroblasts (MEFs) into iPSCs. In MEFs undergoing reprogramming, senescence genes are repressed and mesenchymal-to-epithelial transition is favored. This is correlated with a repressive activity of TCF1, which contributes to the silencing of Wnt/ß-catenin signaling at the onset of reprogramming. In contrast, the Wnt pathway needs to be active in the late reprogramming phases to achieve successful reprogramming. In conclusion, continued activation or inhibition of the Wnt/ß-catenin signaling pathway is detrimental to the reprogramming of MEFs; instead, temporal perturbation of the pathway is essential for efficient reprogramming, and the "Wnt-off" state can be considered an early reprogramming marker.

Resetting epigenetic signatures to induce somatic cell reprogramming.

Somatic cell reprogramming consists of the induction of a complex sequence of events that results in the modification of the developmental state of the cell. It is now routinely possible to reprogram fully differentiated cells back to pluripotent cells, and to transdifferentiate cells of a given type in cells of a totally different lineage origin. However, whether there are key initiating factors that are distinct from those that control stem-cell renewal and that can initiate the reprogramming process remains unknown. In contrast, what is clear is that, by modifying the epigenetic status of a cell, its reprogramming can be initiated. Here, we review the current literature that shows how the plasticity of a cell can be modulated by modifying its epigenetic status, and we discuss how epigenetic barriers can be removed, to induce an efficient reprogramming process.

Regulation of self-renewal and reprogramming by TCF factors.

Wnt/ß-catenin canonical pathway has very important roles both in the regulation of self-renewal and lineage differentiation of pluripotent stem cells, as well as in the reprogramming of somatic cells. In the absence of Wnt stimulation, the target genes of the pathway are repressed or down-regulated. Conversely, when ß-catenin is stabilized there is an accumulation of cytoplasmic and nuclear ß-catenin that allows up-regulation of its target genes. However, the molecular mechanisms downstream of ß-catenin that regulate self-renewal of embryonic stem cells remain unclear. It has been proposed that ß-catenin acts through members of the T-cell factor (TCF) family. Recent studies have confirmed the role of Tcf3 (Tcf7l1) as a transcriptional repressor in embryonic stem cells, and they have also proposed a new mechanistic view as to how activation of the Wnt/ß-catenin pathway can overcome this repression.