Nerve regeneration and circuit reconstruction remain a challenge following spinal cord injury (SCI). Corticospinal pyramidal neurons possess strong axon projection ability. In this study, human induced pluripotent stem cells (iPSCs) were differentiated into pyramidal neuronal precursors (PNPs) by addition of small molecule dorsomorphin into the culture. iPSC-derived PNPs were transplanted acutely into a rat contusion SCI model on the same day of injury. Following engraftment, the SCI rats showed significantly improved motor functions compared with vehicle control group as revealed by behavioral tests. Eight weeks following engraftment, the PNPs matured into corticospinal pyramidal neurons and extended axons into distant host spinal cord tissues, mostly in a caudal direction. Host neurons rostral to the lesion site also grew axons into the graft. Possible synaptic connections as a bridging relay may have been formed between host and graft-derived neurons, as indicated by pre- and post-synaptic marker staining and the regulation of chemogenetic regulatory systems. PNP graft showed an anti-inflammatory effect at the injury site and could bias microglia/macrophages towards a M2 phenotype. In addition, PNP graft was safe and no tumor formation was detected after transplantation into immunodeficient mice and SCI rats. The potential to reconstruct a neuronal relay circuitry across the lesion site and to modulate the microenvironment in SCI makes PNPs a promising cellular candidate for treatment of SCI.

Many long noncoding RNAs (lncRNAs) have been identified through siRNA-based screening as essential regulators of embryonic stem cell (ESC) pluripotency. However, the biological and molecular functions of most lncRNAs remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 lncRNAs previously reported to promote pluripotency in mouse ESCs. Unexpectedly, all of these lncRNAs were dispensable for pluripotency maintenance and proliferation in mouse ESCs when disrupted individually or in combination. Single-cell transcriptomic analysis also showed that the knockout of these lncRNAs has a minimal impact on pluripotency gene expression and cell identity. We further showed that several small hairpin RNAs (shRNAs) previously used to knock down lncRNAs caused the downregulation of pluripotency genes in the corresponding lncRNA-knockout ESCs, indicating that off-target effects likely responsible for the pluripotency defects caused by these shRNAs. Interestingly, linc1343-knockout and linc1343-knockdown ESCs failed to form cystic structures and exhibited high expression of pluripotency genes during embryoid body (EB) differentiation. By reintroducing RNA products generated from the linc1343 locus, we found that two snoRNAs, Snora73a and Snora73b, but not lncRNAs, could rescue pluripotency silencing defects during EB differentiation of linc1343 knockout ESCs. Our results suggest that the 8 previously annotated pluripotency-regulating lncRNAs have no overt functions in conventional ESC culture; however, we identified snoRNA products derived from an annotated lncRNA locus as essential regulators for silencing pluripotency genes.

The widespread application of nanoparticles (NPs) in various fields has raised health concerns, especially in reproductive health. Our research has shown zinc oxide nanoparticles (ZnONPs) exhibit the most significant toxicity to pre-implantation embryos in mice compared to other common NPs. In patients undergoing assisted reproduction technology (ART), a significant negative correlation was observed between Zn concentration and clinical outcomes. Therefore, this study explores the impact of ZnONPs exposure on pre-implantation embryonic development and its underlying mechanisms. We revealed that both in vivo and in vitro exposure to ZnONPs impairs pre-implantation embryonic development. Moreover, ZnONPs were found to reduce the pluripotency of mouse embryonic stem cells (mESCs), as evidenced by teratoma and diploid chimera assays. Employing multi-omics approaches, including RNA-Seq, CUT&Tag, and ATAC-seq, the embryotoxicity mechanisms of ZnONPs were elucidated. The findings indicate that ZnONPs elevate H3K9me3 levels, leading to increased heterochromatin and consequent inhibition of gene expression related to development and pluripotency. Notably, Chaetocin, a H3K9me3 inhibitor, sucessfully reversed the embryotoxicity effects induced by ZnONPs. Additionally, the direct interaction between ZnONPs and H3K9me3 was verified through pull-down and immunoprecipitation assays. Collectively, these findings offer new insights into the epigenetic mechanisms of ZnONPs toxicity, enhancing our understanding of their impact on human reproductive health.

Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.

Exosome-mediated horizontal and vertical transmission of subgroup J avian leukosis virus (ALV-J) in poultry flocks can lead to growth inhibition and severe immunosuppression. However, there are few reports on the early infection of chicken embryonic stem cells (cESCs) with ALV-J. In this study, we confirmed that early infection with ALV-J can accelerate the differentiation of cESCs and promote the secretion of exosomes. To investigate the modulation strategy of ALV-J in cESCs, circRNA sequencing was performed for further analysis. A total of 305 differentially expressed circRNAs (DECs) were obtained, including 71 upregulated DECs. Circ-CCDC7 was found to be the most upregulated DEC and was assessed by qRT-PCR, with the result consistent with the result of circRNA-seq. Based on qRT-PCR, gga-miR-6568-3p was found to be the target of the top 3 DECs, including circ-CCDC7, and the stem cell marker gene Pax7 was identified as the target gene of gga-miR-6568-3p. This study demonstrated that exosomal circ-CCDC7/gga-miR-6568-3p/Pax7 accelerates the differentiation of cESCs after early infection with ALV-J.

Mouse embryonic stem cells (mESCs) sporadically transition to a transient totipotent state that resembles blastomeres of the two-cell (2C) embryo stage, which has been proposed to contribute to exceptional genomic stability, one of the key features of mESCs. However, the biological significance of the rare population of 2C-like cells (2CLCs) in ESC cultures remains to be tested. Here we generated an inducible reporter cell system for specific elimination of 2CLCs from the ESC cultures to disrupt the equilibrium between ESCs and 2CLCs. We show that removing 2CLCs from the ESC cultures leads to dramatic accumulation of DNA damage, genomic mutations, and rearrangements, indicating impaired genomic instability. Furthermore, 2CLCs removal results in increased apoptosis and reduced proliferation of mESCs in both serum/LIF and 2i/LIF culture conditions. Unexpectedly, p53 deficiency results in defective response to DNA damage, leading to early accumulation of DNA damage, micronuclei, indicative of genomic instability, cell apoptosis, and reduced self-renewal capacity of ESCs when devoid of 2CLCs in cultures. Together, our data reveal that transition to the privileged 2C-like state is a major component of the intrinsic mechanisms that maintain the exceptional genomic stability of mESCs for long-term self-renewal.

UNASSIGNED: The committed differentiation fate regulation has been a difficult problem in the fields of stem cell research, evidence showed that nanomaterials could promote the differentiation of stem cells into specific cell types. Layered double hydroxide (LDH) nanoparticles possess the regulation function of stem cell fate, while the underlying mechanism needs to be investigated. In this study, the process of embryonic stem cells (ESCs) differentiate to neural progenitor cells (NPCs) by magnesium aluminum LDH (MgAl-LDH) was investigated.
UNASSIGNED: MgAl-LDH with diameters of 30, 50, and 100 nm were synthesized and characterized, and their effects on the cytotoxicity and differentiation of NPCs were detected in vitro. Dot blot and MeRIP-qPCR were performed to detect the level of m6A RNA methylation in nanoparticles-treated cells.
UNASSIGNED: Our work displayed that LDH nanoparticles of three different sizes were biocompatible with NPCs, and the addition of MgAl-LDH could significantly promote the process of ESCs differentiate to NPCs. 100 nm LDH has a stronger effect on promoting NPCs differentiation compared to 30 nm and 50 nm LDH. In addition, dot blot results indicated that the enhanced NPCs differentiation by MgAl-LDH was closely related to m6A RNA methylation process, and the major modification enzyme in LDH controlled NPCs differentiation may be the m6A RNA methyltransferase METTL3. The upregulated METTL3 by LDH increased the m6A level of Sox1 mRNA, enhancing its stability.
UNASSIGNED: This work reveals that MgAl-LDH nanoparticles can regulate the differentiation of ESCs into NPCs by increasing m6A RNA methylation modification of Sox1.

Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive.
By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes.
Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.

As the most well-studied modification in mRNA, m6A has been shown to regulate multiple biological processes, including RNA degradation, processing, and translation. Recent studies showed that m6A modification is enriched in chromatin-associated RNAs and nascent RNAs, suggesting m6A might play regulatory roles in chromatin contexts. Indeed, in the past several years, a number of studies have clarified how m6A and its modulators regulate different types of chromatin states. Specifically, in the past 2-3 years, several studies discovered the roles of m6A and/or its modulators in regulating constitutive and facultative heterochromatin, shedding interesting lights on RNA-dependent heterochromatin formation in mammalian cells. This review will summarize and discuss the mechanisms underlying m6A\'s regulation in different types of heterochromatin, with a specific emphasis on the regulation in mammalian embryonic stem cells, which exhibit distinct features of multiple heterochromatin marks.

Haploid cells are a kind of cells with only one set of chromosomes. Compared with traditional diploid cells, haploid cells have unique advantages in gene screening and drug-targeted therapy, due to their phenotype being equal to the genotype. Embryonic stem cells are a kind of cells with strong differentiation potential that can differentiate into various types of cells under specific conditions in vitro. Therefore, haploid embryonic stem cells have the characteristics of both haploid cells and embryonic stem cells, which makes them have significant advantages in many aspects, such as reproductive developmental mechanism research, genetic screening, and drug-targeted therapy. Consequently, establishing haploid embryonic stem cell lines is of great significance. This paper reviews the progress of haploid embryonic stem cell research and briefly discusses the applications of haploid embryonic stem cells.