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G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and regulate various physiological and pathological processes. Despite extensive studies, the roles of GPCRs in mouse embryonic stem cells (mESCs) remain poorly understood. Here, we show that GPR160, a class A member of GPCRs, is dramatically downregulated concurrent with mESC differentiation into embryoid bodies in vitro. Knockdown of Gpr160 leads to downregulation of the expression of pluripotency-associated transcription factors and upregulation of the expression of lineage markers, accompanying with the arrest of the mESC cell-cycle in the G0/G1 phase. RNA-seq analysis shows that GPR160 participates in the JAK/STAT signaling pathway crucial for maintaining ESC stemness, and the knockdown of GPRGpr160 results in the downregulation of STAT3 phosphorylation level, which in turn is partially rescued by colivelin, a STAT3 activator. Consistent with these observations, GPR160 physically interacts with JAK1, and cooperates with leukemia inhibitory factor receptor (LIFR) and gp130 to activate the STAT3 pathway. In summary, our results suggest that GPR160 regulates mESC self-renewal and pluripotency by interacting with the JAK1-LIFR-gp130 complex to mediate the JAK1/STAT3 signaling pathway., Competing Interests: Conflict of interest The authors declare no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hematopoietic stem cells (HSCs) are stem cells that can differentiate into various blood cells and have long-term self-renewal capacity. At present, HSC transplantation is an effective therapeutic means for many malignant hematological diseases, such as aplastic hematological diseases and autoimmune diseases. The hematopoietic microenvironment affects the proliferation, differentiation, and homeostasis of HSCs. The regulatory effect of the hematopoietic microenvironment on HSCs is complex and has not been thoroughly studied yet. In this study, we focused on mononuclear cells (MNCs), which provided an important microenvironment for HSCs and established a methodological system for identifying cellular composition by means of multiple technologies and methods. First, single-cell RNA sequencing (scRNA-seq) technology was used to investigate the cellular composition of cells originating from different microenvironments during different stages of hematopoiesis, including mouse fetal liver mononuclear cells (FL-MNCs), bone marrow mononuclear cells (BM-MNCs), and in vitro -cultured fetal liver stromal cells. Second, bioinformatics analysis showed a higher proportion and stronger proliferation of the HSCs in FL-MNCs than those in BM-MNCs. On the other hand, macrophages in in vitro -cultured fetal liver stromal cells were enriched to about 76%. Differential gene expression analysis and Gene Ontology (GO) functional enrichment analysis demonstrated that fetal liver macrophages have strong cell migration and actin skeleton formation capabilities, allowing them to participate in the hematopoietic homeostasis through endocytosis and exocytosis. Last, various validation experiments such as quantitative real-time PCR (qRT-PCR), ELISA, and confocal image assays were performed on randomly selected target genes or proteins secreted by fetal liver macrophages to further demonstrate the potential relationship between HSCs and the cells inhabiting their microenvironment. This system, which integrates multiple methods, could be used to better understand the fate of these specific cells by determining regulation mechanism of both HSCs and macrophages and could also be extended to studies in other cellular models.

Objectives: Mesoderm, derived from a new layer between epiblast and hypoblast during gastrulation, can differentiate into various tissues, including muscles, bones, kidneys, blood, and the urogenital system. However, systematic elucidation of mesoderm characteristics and specific markers remains a challenge. This study aims to screen and identify candidate genes important for mesoderm development., Materials and Methods: Cells originating from the three germ layers were obtained by laser capture microdissection, followed by microcellular RNA sequencing. Mesoderm-specific differentially expressed genes (DEGs) were identified by using a combination of three bioinformatics pipelines. Candidate mesoderm-specific genes expression were verified by real-time quantitative polymerase chain reaction analysis and immunohistochemistry. Functional analyses were verified by ESCs-EBs differentiation and colony-forming units (CFUs) assay., Results: A total of 1962 differentially expressed mesoderm genes were found, out of which 50 were candidate mesoderm-specific DEGs which mainly participate in somite development, formation of the primary germ layer, segmentation, mesoderm development, and pattern specification process by GO analysis. Representative genes Cdh2, Cdh11, Jag1, T, Fn-1, and Pcdh7 were specifically expressed in mesoderm among the three germ layers. Pcdh7 as membrane-associated gene has hematopoietic-relevant functions identified by ESCs-EBs differentiation and CFUs assay., Conclusions: Spatial transcriptomic profiling with multi-method analysis and confirmation revealed candidate mesoderm progenitors. This approach appears to be efficient and reliable and can be extended to screen and validate candidate genes in various cellular systems., (© 2022 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Objectives: Early embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal-to-zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high- or low- competence mouse strains., Materials and Methods: In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real-time quantitative PCR (RT-qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment., Results: The fertilization and 2-cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty-nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT-qPCR. Prkce knockout mice showed a reduced 2-cell rate (Prkce +/+ 80.1% vs. Prkce -/- 32.4%) that could be rescued by Prkce cRNA injection (2-cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation., Conclusions: The transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2-cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high-quality mature oocytes., (© 2022 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Down's syndrome (DS) is one of the most commonly known disorders with multiple congenital disabilities. Besides severe cognitive impairment and intellectual disability, individuals with DS also exhibit additional phenotypes of variable penetrance and severity, with one or more comorbid conditions, including Alzheimer's disease, congenital heart disease, or leukemia. Various vital genes and regulatory networks had been studied to reveal the pathogenesis of the disease. Nevertheless, very few studies have examined alternative splicing. Alternative splicing (AS) is a regulatory mechanism of gene expression when making one multi-exon protein-coding gene produce more than one unique mature mRNA. We employed the GeneChip Human Transcriptome Array 2.0 (HTA 2.0) for the global gene analysis with hiPSCs from DS and healthy individuals. Examining differentially expressed genes (DEGs) in these groups and focusing on specific transcripts with AS, 466 up-regulated and 722 down-regulated genes with AS events were identified. These genes were significantly enriched in biological processes, such as cell adhesion, cardiac muscle contraction, and immune response, through gene ontology (GO) analysis of DEGs. Candidate genes, such as FN1 were further explored for potentially playing a key role in DS. This study provides important insights into the potential role that AS plays in DS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Li, Yang, Cai, Yang, Zhang, Zeng, Ma and Zeng.)

Objectives: This study explored whether TALENs-mediated non-homologous end joining (NHEJ) targeting the mutation site can correct the aberrant β-globin RNA splicing, and ameliorate the β-thalassaemia phenotype in β 654 mice., Material and Methods: TALENs vectors targeted to the human β-globin gene (HBB) IVS2-654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALENs + /β 654 mice. The gene editing and off-target effects were analysed by sequencing analysis. β-globin expression was identified by RT-PCR and Western blot analysis. Various clinical indices including haematologic parameters and tissue pathology were examined to determine the therapeutic effect in these TALENs + /β 654 mice., Results: Sequencing analysis revealed that the HBB IVS2-654C >T point mutation was deleted in over 50% of the TALENs + /β 654 mice tested, and off-target effects were not detected. RT-PCR and Western blot analysis confirmed the expression of normal β-globin in TALENs + /β 654 mice. The haematologic parameters were significantly improved as compared with their affected littermates. The proportion of nucleated cells in bone marrow was considerably decreased, splenomegaly with extramedullary haematopoiesis was reduced, and significant decreases in iron deposition were seen in spleen and liver of the TALENs + /β 654 mice., Conclusion: These results suggest effective treatment of the anaemia phenotype in TALENs + /β 654 mice following deletion of the mutation site by TALENs, demonstrating a simple and straightforward strategy for gene therapy of β 654 -thalassaemia in the future., (© 2018 John Wiley & Sons Ltd.)

Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs., (© 2015 International Federation for Cell Biology.)

Since Yamanaka successfully reprogrammed murine fibroblasts into iPSCs in 2006, iPSCs technology has drawn much attention worldwide. Although iPSCs provides tremendous possibilities for both basic research and regenerative medicine, it has meanwhile potential risks, e.g. tumorigenicity. Scientists, therefore, have made efforts in clarifying the mechanism of the cause for iPSCs tumorigenicity and the way how to reduce the risk. The results of some researches reveal some of tumorigenic factors, e.g. the partial similarity of gene expression profiles between cancer cells and iPSCs, the accumulation of the genetic damages in the course of reprogramming process, and mutation in the cellular culture. As a consequence, numerous methods for reducing iPSCs tumorigenicity have been explored, such as minimized use of the reprogramming factors at the controlled manner, and the selection of the expression vector or parental cells. In this paper, the cause of iPSCs tumorigenicity and the current achievements on preventing iPSCs tumorigenesis are reviewed.

Although β-thalassemia is one of the most common human genetic diseases, there is still no effective treatment other than bone marrow transplantation. Induced pluripotent stem cells have been considered good candidates for the future repair or replacement of malfunctioning organs. As a basis for developing transgenic induced pluripotent stem cell therapies for thalassemia, β(654) induced pluripotent stem cells from a β(654) -thalassemia mouse transduced with the normal human β-globin gene, and the induced pluripotent stem cells with an erythroid-expressing reporter GFP were used to produce chimeric mice. Using these chimera models, we investigated changes in various pathological indices including hematologic parameters and tissue pathology. Our data showed that when the chimerism of β(654) induced pluripotent stem cells with the normal human β-globin gene in β(654) mice is over 30%, the pathology of anemia appeared to be reversed, while chimerism ranging from 8% to 16% provided little improvement in the typical β-thalassemia phenotype. Effective alleviation of thalassemia-related phenotypes was observed when chimerism with the induced pluripotent stem cells owning the erythroid-expressing reporter GFP in β(654) mouse was greater than 10%. Thus, 10% or more expression of the exogenous normal β-globin gene reduces the degree of anemia in our β-thalassemia mouse model, whereas treatment with β(654) induced pluripotent stem cells which had the normal human β-globin gene had stable therapeutic effects but in a more dose-dependent manner., (Copyright© Ferrata Storti Foundation.)