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.

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.

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.

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.