Stem Cell Reports最新文献

In mammals, while X chromosome inactivation (XCI) balances the dosage of X-linked gene expression between sexes, upregulation of active-X balances the dosage of monoallelic X-linked genes with biallelic autosomal genes (AA). Here, we have investigated the role of m6A RNA methylation in the maintenance of XCI and X-to-autosome (X-to-A) dosage compensation in early embryonic lineages: epiblast stem cells (EpiSCs), trophoblast stem cells (TSCs), and extraembryonic endoderm stem cells (XENs). We find that the depletion of m6A RNA methylation in these cells does not affect the maintenance of inactive-X silencing. Moreover, we show that m6A marks are less enriched on X-linked transcripts than the autosomal transcripts in early embryonic lineages. Notably, we demonstrate that the extent of X-to-A dosage compensation varies with m6A methylation level. Finally, we show that the depletion of m6A partly disrupts X-to-A dosage compensation in a cell-type-specific manner. Together, our study provides significant insight into the role of m6A RNA methylation in dosage compensation.

As members of the CCT family, CCT5 and CCT7 play pivotal roles in telomerase trafficking, with their depletion resulting in TCAB1 protein loss and impaired telomere maintenance. However, their functional significance in embryonic stem cells (ESCs) state transitions remains incompletely understood. Here, we demonstrate that CCT5 or CCT7 deficiency disrupts telomere length homeostasis, triggering DNA damage response pathways and inducing epigenetic reprogramming. This cascade enhances cellular plasticity, activates repeat elements and 2-cell transcriptional programs, and facilitates the generation of 2-cell-like cells, suggesting that CCT5 and CCT7 may serve as epigenetic barriers restricting the transition from pluripotency to totipotency. Additionally, CCT5/7 stabilizes pluripotency through Wnt/β-catenin signaling: CCT7 directly binds β-catenin to facilitate nuclear translocation, while CCT5 dissociates E-cadherin/β-catenin complexes. These findings underscore the dual role of CCT5 and CCT7 in maintaining telomere integrity and regulating pluripotent state dynamics.

Cardiomyocytes differentiated in vitro from human induced pluripotent stem cells (iPSC-CMs) are increasingly used in studies of disease mechanisms, drug development, toxicity testing, and regenerative medicine. Alternative splicing (AS) plays a pivotal role in cardiac development. However, the extent to which iPSC-CMs recapitulate native cardiac splicing patterns remains poorly understood. Here, we provide a comprehensive temporal map of AS regulation during human cardiac development. iPSC-derived cardiomyocytes globally recapitulate the transcriptome of prenatal cardiomyocytes, yet their splicing profiles remain heterogeneous, with certain events reflecting early embryonic patterns and others resembling those of later-stage fetal hearts. Moreover, we uncover altered splicing events in iPSC-CMs, including mis-splicing of splicing factors. In conclusion, we present a resource of AS dynamics throughout human cardiac development and a catalog of splicing markers to assess cardiomyocyte maturation in vitro. Our findings provide critical insights into the limitations of iPSC-CM models and their utility in cardiovascular research.

Asthma is a chronic inflammatory airway disease characterized by defective epithelial repair, resulting from metabolic dysregulation in facultative progenitor cells. Here, we investigate how pyruvate metabolism in airway club cells controls epithelial differentiation and allergic airway inflammation. Single-cell transcriptomics revealed elevated glycolytic activity in club and goblet cells from patients with asthma. In an ovalbumin (OVA)-induced asthma model, conditional deletion of Mpc2-but not Ldha-in club cells impaired club-to-goblet cell differentiation, reduced CLCA3 and Foxa3 expression, and attenuated eosinophilic inflammation and Il-13 expression. Mpc2 loss increased Cxcl17 expression in club cells, promoting Cxcl17-Cxcr4 signaling with alveolar macrophages that suppressed CCL17-mediated type 2 inflammation. Neutralizing CCL17 phenocopied the Mpc2 knockout by reducing airway inflammation and goblet cell differentiation. These findings reveal a metabolic-immune crosstalk underlying asthma pathogenesis and identify mitochondrial pyruvate metabolism as a therapeutic target to limit epithelial remodeling and type 2 inflammation.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes. In rodents, PGCs readily form self-renewing embryonic germ cell (EGC) lines in vitro. Although human PGCs undergo a similar conversion during germ cell tumorigenesis, no comparable in vitro system has yet been established in humans. Here we report that hPGC-like cells (hPGCLCs) undergo conversion to human EGC-like cells (hEGCLCs) using the inductive signals previously identified in mice. This feeder-free culture system allows efficient derivation of hEGCLCs that are transcriptionally similar to human induced pluripotent stem cells and can give rise to hPGCLCs once more demonstrating the interconvertibility of pluripotent states. This is also evident at the chromatin level, as the initial DNA demethylation that occurs in hPGCLCs is reversed in hEGCLCs. This new in vitro model provides a highly tractable system to study human pluripotent and early developmental transitions, including those driving germ cell tumorigenesis and epigenetic inheritance.

Lacrimal glands (LGs) serve as pivotal exocrine glands crucial for protecting the ocular surface. Dysfunction in LG cell composition or secretion is implicated in dry eye disease (DED). While autophagy plays a vital role in tissue homeostasis in many organs, how it affects LG development and secretory function is not known. Here, we have undertaken a genetic study by utilizing autophagy-deficient human embryonic stem cells (hESCs) and differentiating them into LG-like organoids. Autophagy-deficient LG-like organoids exhibited improper development and secretion, along with increased protein aggregation, proliferation, and cell death. These phenotypes were associated with an accumulation of PAX6, a transcription factor crucial for brain and eye development, which we identified as an autophagy substrate. Pharmacological interventions with nicotinamide mononucleotide (NMN) and melatonin were able to rescue the cellular dysfunction in autophagy-deficient LG-like organoids. Together, our study highlights the role of autophagy in LG along with potential therapeutic interventions for DED.

Xenotransplantation enables the interrogation of human neuron-specific vulnerabilities to Alzheimer's pathology within a physiologically relevant in vivo context. While amyloid-beta (Aβ) is known to disrupt synaptic integrity, it remains uncertain whether the synaptotoxicity observed in vitro accurately models the disease. Here, we establish a xenotransplantation paradigm in which human neurons integrate into the brains of amyloid precursor protein (APP) transgenic mice that develop amyloid plaques. Using a genetically encoded pre-synaptic reporter, we label human pre-synapses post engraftment to assess early-stage pathology. We demonstrate that extracellular Aβ plaques induce localized synaptic damage in human neurons, characterized by local pre-synaptic loss and the formation of dystrophic neurites. Notably, this pathology is restricted to the plaque microenvironment and does not result in widespread pre-synaptic degeneration. Our findings establish this human-mouse chimera model as a platform for dissecting Aβ-induced synaptic pathology and reveal that extracellular Aβ exerts compartmentalized yet impactful toxicity on human pre-synapses.

The human genome encodes thousands of long non-coding RNAs (lncRNAs), transcripts of over 200 nucleotides that lack protein-coding potential. lncRNAs are emerging as key players in diverse cellular processes, particularly in tissue-specific contexts, yet their functionality remained poorly understood. Here, we performed a CRISPR interference (CRISPRi) screen in human embryonic stem cells (hESCs), identifying over 100 essential and about 150 growth-restricting lncRNAs. We show that growth-modifying lncRNAs display distinctive properties, including unique expression signatures, genomic structure, evolutionary conservation, chromosomal distribution, and potential involvement in teratoma formation. Notably, we uncovered two primate-conserved, uncharacterized, essential lncRNAs that regulate neighboring pluripotency transcription factors: lncOCT4, which positively regulates OCT4 and induces p53-mediated apoptosis upon knockdown, and lncVRTN, which acts as a putative negative regulator of VRTN, affecting cell fate determination. These findings shed light on the contribution of lncRNAs to the human-specific pluripotency network and provide insights into lncRNA-mediated regulation of hESC growth and differentiation.

Primordial germ cells (PGCs) can give rise to pluripotent embryonic germ cells (EGCs) in rodents. Leitch and colleagues in this issue successfully derived human EGC-like cells from PGC-like cells and applied comprehensive multi-omic profiling to resolve their pluripotent and epigenetic dynamics, providing new insights into potential mechanisms underlying human germ cell tumorigenesis.

The common marmoset (Callithrix jacchus) is a genetically modifiable non-human primate increasingly used in biomedical research. Here, we established a method for deriving embryonic stem cells (ESCs) from blastocysts generated by somatic cell nuclear transfer (SCNT) in the marmoset. Injection of histone demethylase Kdm4d mRNA enabled efficient reprogramming of somatic nuclei, allowing blastocyst formation in 14.5% from fibroblasts. Combining this method with a G9a/EHMT2 histone methyltransferase inhibitor improved blastocyst quality and allowed derivation of nuclear transfer ESCs (ntESCs), including wild-type and GFP-transgenic lines. These ntESCs exhibited normal karyotypes and pluripotency. Nuclear and mitochondrial DNA analyses confirmed their nuclear donor origin and cytoplasmic inheritance from recipient oocytes. Transcriptome analysis identified abnormally expressed genes in ntESCs present in a line-dependent and independent manner, suggesting partial reprogramming resistance. Our study establishes a marmoset SCNT method enabling derivation of ntESCs and provides a new platform for preserving and engineering marmoset genetic resources.