Dissecting the impact of differentiation stage, replicative history, and cell type composition on epigenetic clocks.

Epigenetic clocks, built on DNA methylation patterns of bulk tissues, are powerful age predictors, but their biological basis remains incompletely understood. Here, we conducted a comparative analysis of epigenetic age in murine muscle, epithelial, and blood cell types across lifespan. Strikingly, our results show that cellular subpopulations within these tissues, including adult stem and progenitor cells as well as their differentiated progeny, exhibit different epigenetic ages.

H3K36 methylation maintains cell identity by regulating opposing lineage programmes.

The epigenetic mechanisms that maintain differentiated cell states remain incompletely understood. Here we employed histone mutants to uncover a crucial role for H3K36 methylation in the maintenance of cell identities across diverse developmental contexts. Focusing on the experimental induction of pluripotency, we show that H3K36M-mediated depletion of H3K36 methylation endows fibroblasts with a plastic state poised to acquire pluripotency in nearly all cells.

Dissection of gastric homeostasis in vivo facilitates permanent capture of isthmus-like stem cells in vitro.

The glandular stomach is composed of two regenerative compartments termed corpus and antrum, and our understanding of the transcriptional networks that maintain these tissues is incomplete. Here we show that cell types with equivalent functional roles in the corpus and antrum share similar transcriptional states including the poorly characterized stem cells of the isthmus region. To further study the isthmus, we developed a monolayer two-dimensional (2D) culture system that is continually maintained by Wnt-responsive isthmus-like cells capable of differentiating into several gastric cell types.

Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells.

The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model.

Inducible histone K-to-M mutations are dynamic tools to probe the physiological role of site-specific histone methylation in vitro and in vivo.

Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality.

The RNA Helicase DDX6 Controls Cellular Plasticity by Modulating P-Body Homeostasis.

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner.

Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells.

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes.

An Intermediate Pluripotent State Controlled by MicroRNAs Is Required for the Naive-to-Primed Stem Cell Transition.

The embryonic stem cell (ESC) transition from naive to primed pluripotency is marked by major changes in cellular properties and developmental potential. ISY1 regulates microRNA (miRNA) biogenesis, yet its role and relevance to ESC biology remain unknown. Here, we find that highly dynamic ISY1 expression during the naive-to-primed ESC transition defines a specific phase of "poised" pluripotency characterized by distinct miRNA and mRNA transcriptomes and widespread poised cell contribution to mouse chimeras.

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs).

Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells.

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential.

DUSP9 Modulates DNA Hypomethylation in Female Mouse Pluripotent Stem Cells.

Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs.

Sox2 Suppresses Gastric Tumorigenesis in Mice.

Sox2 expression marks gastric stem and progenitor cells, raising important questions regarding the genes regulated by Sox2 and the role of Sox2 itself during stomach homeostasis and disease. By using ChIP-seq analysis, we have found that the majority of Sox2 targets in gastric epithelial cells are tissue specific and related to functions such as endoderm development, Wnt signaling, and gastric cancer. Unexpectedly, we found that Sox2 itself is dispensable for gastric stem cell and epithelial self-renewal, yet Sox2(+) cells are highly susceptible to tumorigenesis in an Apc/Wnt-driven mouse model.

Lce1 Family Members Are Nrf2-Target Genes that Are Induced to Compensate for the Loss of Loricrin.

Loricrin is a major component of the cornified cell envelope, a highly insoluble structure composed of covalently cross-linked proteins. Although loricrin knockout mice only exhibit a mild transient phenotype at birth, they show a marked delay in the formation of an epidermal barrier in utero. We recently discovered that induction of a compensatory response to repair the defective barrier is initiated by amniotic fluid via activation of NF-E2-related factor 2 and identified Sprr2d and Sprr2h as direct transcriptional targets.

Lineage conversion induced by pluripotency factors involves transient passage through an iPSC stage.

Brief expression of pluripotency-associated factors such as Oct4, Klf4, Sox2 and c-Myc (OKSM), in combination with differentiation-inducing signals, has been reported to trigger transdifferentiation of fibroblasts into other cell types. Here we show that OKSM expression in mouse fibroblasts gives rise to both induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) under conditions previously shown to induce only iNSCs. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells.

Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice.

The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula.

Amniotic fluid activates the nrf2/keap1 pathway to repair an epidermal barrier defect in utero.

The loss of loricrin, a major component of the cornified envelope, results in a delay of epidermal barrier formation. Therefore, the living layers of the epidermis are aberrantly exposed to late-stage amniotic fluid, which may serve as the signal to upregulate genes that functionally compensate for the loss of loricrin. Consistent with this hypothesis, metabolomic studies revealed marked changes in amniotic fluid between E14.

Aurora kinase-A deficiency during skin development impairs cell division and stratification.

Aurora kinase-A (Aurora-A) promotes timely entry into mitosis, centrosome maturation, and formation of bipolar spindles. To address the role of Aurora-A in skin development and homeostasis, we interbred a floxed Aurora-A (Aurora-A(fl)) mouse with the Cre-deleter strain, K14.Cre.

Nrf2 links epidermal barrier function with antioxidant defense.

The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes.