STEM CELLS最新文献

To enable robust expression of transgenes in stem cells, recombinase mediated cassette exchange at safe harbour loci is frequently adopted. The choice of recombinase enzyme is a critical parameter to ensure maximum efficiency and accuracy of the integration event. We have explored the serine recombinase family of site-specific integrases and have directly compared the efficiency of PhiC31, W-beta and Bxb1 integrase for targeted transgene integration at the Gt(ROSA)26Sor locus in mouse embryonic stem cells. All three integrases were found to be suitable for efficient engineering and long-term expression of each integrase was compatible with pluripotency, as evidenced by germline transmission. Bxb1 integrase was found to be 2-3 times more efficient than PhiC31 and W-beta. The Bxb1 system was adapted for cassette exchange at the AAVS1 locus in human induced pluripotent stem (iPS) cells, and the two commonly used ubiquitous promoters, CAG and Ef1α (EIF1A), were tested for their suitability in driving expression of the integrated transgenic cargo. AAVS1-integrated Ef1α promoter led to a very mosaic pattern of expression in targeted hiPS cells, whereas the AAVS1-integrated CAG promoter drove consistent and stable expression. To validate the system for the integration of functional machinery, the Bxb1 integrase system was used to integrate CAG-driven CRISPR-activation and CRISPR-inhibition machinery in human iPS cells and robust sgRNA-induced up- and down-regulation of target genes was demonstrated.

The function and mechanism of pri-miRNA N6-methyladenosine (m6A) modification in promoting miRNA maturation and regulating osteoblastic differentiation are not fully understood. The aim of this study was to investigate the role and regulatory mechanism of miRNA shear maturation regulated by methyltransferase like 3 (METTL3) in human adipose-derived stem cell (hASC) osteogenesis. Firstly, we found METTL3 promoted osteogenesis both in vivo and in vitro. Subsequently, three pri-miRNAs with the most significant methylated peaks were identified through methylated RNA immunoprecipitation sequencing (MeRIP-seq). Through quantitative real-time polymerase chain reaction (qRT-PCR), MeRIP-qPCR and co-immunoprecipitation (CO-IP), it was determined that METTL3 promoted the processing of hsa-miR-4526 by mediating pri-miR4526/5190 m6A modification. Subsequent in vivo and in vitro experiments demonstrated that hsa-miR-4526 promoted osteogenesis. Dual luciferase reporter assay was performed to verify that hsa-miR-4526 regulated osteogenic differentiation through TUBB3. It was found that TUBB3 can inhibit hASC osteogenesis. Further rescue experiments confirmed that METTL3 inhibited TUBB3 expression through hsa-miR-4526, thereby regulating osteogenic differentiation. RNA-seq revealed that TUBB3 may be involved in cell metabolism, calcium enrichment, osteoclast differentiation, and other pathways. Our study is the first to investigate the mechanism of pri-miRNA m6A modification in regulating hASC osteogenesis, presenting a novel idea and method for repairing bone defects.

Background: Amniotic mesenchymal stem cells (AMSCs) have been demonstrated as effective in tissue repair and regeneration. Trophoblast dysfunction is associated with several types of pregnancy complications. The aim of this study is to investigate the effects of AMSCs on the biological activities of human trophoblasts, as well as their molecular mechanisms.

Methods: Exosomes were isolated from AMSC supernatants, and characterized and quantified by transmission electron microscopy (TEM) , nanoparticle tracking analysis (NTA) and Western blotting assay. Immunofluorescence assay was performed to detect the uptake of AMSCs-derived exomes (AMSC-Exos) by human trophoblasts. Human trophoblasts were subjected to transcriptome analysis after being co-cultured with AMSC-Exos. Lentiviral transfection was performed to construct the human trophoblast cell lines with stable HRK knockdown or overexpression. Immunohistochemistry was used to detect the HRK expression in preeclampsia (PE) patients. CCK8 and Transwell assays were respectively used to detect the trophoblast proliferation and migration. TUNEL flow cytometry assay was used to detect the apoptosis in trophoblasts. qRT-PCR and Western blotting assays were used to detect the mRNA and protein levels of the genes. Dual luciferase reporter assays were used to detect the changes in gene-transcript levels.

Results: AMSC-Exos could be absorbed by human trophoblasts. Transcriptome analysis showed that HRK was significantly reduced in human trophoblasts co-cultured with AMSC-Exos. HRK inhibited cell proliferation and migration in human trophoblasts and promoted their apoptosis via p53 upregulation. miR-18a-5p, present at high levels in AMSC-Exos, improved trophoblast proliferation and migration, and inhibited their apoptosis by inhibiting the HRK expression.

Conclusion: miR-18a-5p present in AMSC-Exos could be absorbed by trophoblasts, and in turn, improved their proliferation and migration and inhibited their apoptosis by HRK down-regulation.

Pluripotent stem cells provide opportunities for treating injuries and previously incurable diseases. A major concern is the immunogenicity of stem cells and their progeny. Here, we have dissected the molecular mechanisms that allow natural killer (NK) cells to respond to human pluripotent stem cells, investigating a wide selection of activating and inhibitory NK cell receptors and their ligands. Reporter cells expressing the activating receptor NKG2D responded strongly to embryonic stem (ES) cell lines and induced pluripotent stem (iPS) cell lines, whereas reporter cells expressing the activating receptors NKp30, NKp46, KIR2DS1, KIR2DS2 and KIR2DS4 did not respond. Human ES and iPS cells invariably expressed several ligands for NKG2D. Expression of HLA-C and HLA-E was lacking or low, insufficient to trigger reporter cells expressing the inhibitory receptors KIR2DL1, -2DL2 or -2DL3. Similar results were obtained for the pluripotent embryonic carcinoma cell lines NTERA-2 and 2102Ep, and also iPS cell-derived neural progenitor cells. Importantly, neural progenitor cells and iPS cell-derived motoneurons also expressed B7H6, the ligand for the activating receptor NKp30. In line with these observations, IL-2 stimulated NK cells showed robust cytotoxic responses to ES and iPS cells as well as to iPS cell-derived motoneurons. No significant differences in cytotoxicity levels were observed between KIR/HLA matched and mismatched combinations of NK cells and pluripotent targets. Together, these data indicate that pluripotent stem cells and their neural progeny are targets for NK cell killing both by failing to sufficiently express ligands for inhibitory receptors and by expression of ligands for activating receptors.

Human induced pluripotent stem cells (iPSCs) provide powerful cellular models of Alzheimer's disease (AD) and offer many advantages over non-human models, including the potential to reflect variation in individual-specific pathophysiology and clinical symptoms. Previous studies have demonstrated that iPSC-neurons from individuals with Alzheimer's disease (AD) reflect clinical markers, including β-amyloid (Aβ) levels and synaptic vulnerability. However, despite neuronal loss being a key hallmark of AD pathology, many risk genes are predominantly expressed in glia, highlighting them as potential therapeutic targets. In this work iPSC-derived astrocytes were generated from a cohort of individuals with high versus low levels of the inflammatory marker YKL-40, in their cerebrospinal fluid (CSF). iPSC-derived astrocytes were treated with exogenous Aβ oligomers and high content imaging demonstrated a correlation between astrocytes that underwent the greatest morphology change from patients with low levels of CSF-YKL-40 and more protective APOE genotypes. This finding was subsequently verified using similarity learning as an unbiased approach. This study shows that iPSC-derived astrocytes from AD patients reflect key aspects of the pathophysiological phenotype of those same patients, thereby offering a novel means of modelling AD, stratifying AD patients and conducting therapeutic screens.

Premature osteoporosis due to parathyroid hormone-related peptide (PTHrP) dysfunction presents significant bone health challenges. This study explores the role of p16-mediated cellular senescence in this condition using a Pthrp knock-in (KI) mouse model lacking the nuclear localization sequence and C-terminus of PTHrP. We generated p16⁻⁄⁻KI mice and compared them with wild-type, p16⁻⁄⁻, and KI mice. The genetic ablation of p16 in KI mice extended their lifespan, increased body size, and weight. Micro-CT analysis revealed a significant increase in bone volume, while histological and immunohistochemical studies revealed enhanced chondrocyte proliferation and osteoblast function in p16⁻⁄⁻KI mice. In vitro experiments showed enhanced differentiation capacity and reduced senescence of bone marrow mesenchymal stem cells (BM-MSCs) from p16⁻⁄⁻KI mice. Molecular analyses indicated that p16 knockout partially reversed oxidative stress, DNA damage, and cellular senescence observed in KI mice, as evidenced by upregulated antioxidant enzymes, reduced DNA damage markers, and decreased senescence markers. These findings highlight the critical role of p16-mediated cellular senescence in the premature osteoporosis phenotype of KI mice, suggesting that targeting cellular senescence pathways could offer a promising therapeutic strategy for premature osteoporosis and age-related bone loss. This research provides new insights into the interplay between genetic factors, cellular senescence, and bone metabolism in the context of aging and osteoporosis.

Gut microbiota plays an important role in regulating brain function and adult neurogenesis. Although probiotics have recently been reported as effective against certain psychiatric disorders, the underlying mechanisms remain unclear. In particular, the combination of 3 probiotic strains, Bacillus subtilis TO-A, Enterococcus faecium T-110, and Clostridium butyricum TO-A, hereafter referred to as ProB3, has been reported to potentially alleviate psychiatric symptoms in patients with schizophrenia. Herein, we show that ProB3 promotes adult neurogenesis in mice and restores its dysregulation in germ-free (GF) mice. ProB3 colonization in GF mice enhanced the proliferation of adult neural stem cells compared to specific-pathogen-free and GF mice. Furthermore, ProB3 colonization was sufficient to ameliorate the arrest of newborn neuron maturation and the diminution of quiescent neural stem cells in GF mice. ProB3 colonization in mice increased the levels of several metabolites in the blood, including theanine and 3-hydroxybutyrate, and imidazole peptides, including anserine, which promoted proliferation, neurogenesis, and maturation of newborn neurons in cultured human fetus neural stem cells, as well as mouse adult hippocampal neural stem cells. Collectively, these results indicate that the essential role of the gut microbiota in adult hippocampal neurogenesis can be effectively complemented by the intake of a specific 3-strain probiotic, ProB3, providing novel insights into the brain-gut axis.

Exosomes in the hippocampal dentate gyrus (DG) are essential for modulating the cell signaling controlling the neural differentiation of hippocampal neural stem cells (NSCs), which may determine the level of hippocampal adult neurogenesis. In the present study, we found that exosomes secreted by immature neurons may promote the neuronal differentiation of mouse NSCs in vitro. By miRNA sequencing, we discovered that miR-7a-5p was significantly lower in exosomes from differentiated immature neurons than those from undifferentiated NSCs. By modulating the level of miR-7a-5p, the mimic and inhibitor of miR-7a-5p could either inhibit or promote the neuronal differentiation of NSCs, respectively. Moreover, we confirmed that miR-7a-5p affected neurogenesis by directly targeting Tcf12, a transcription factor responsible for the differentiation of NSCs. The siRNA of Tcf12 inhibited neuronal differentiation of NSCs, while overexpression of Tcf12 promoted NSC differentiation. Thus, we conclude that the miR-7a-5p content in neural exosomes is essential to the fate determination of adult hippocampal neurogenesis and that miR-7a-5p directly targets Tcf12 to regulate adult hippocampal neurogenesis.

Ischemic stroke (IS) is a significant and potentially life-threatening disease with limited treatment options, often resulting in severe disability. Bone marrow stromal cells (BMSCs) transplantation has exhibited promising neuroprotection following cerebral ischemia-reperfusion injury (CIRI). However, the effectiveness is hindered by their low homing rate when administered through the vein. In this study, we aimed to enhance the homing ability of BMSCs through lentivirus transfection to express fucosyltransferase 7. This glycosylation engineered CD44 on BMSCs to express hematopoietic cell E-selectin/L-selectin ligand (HCELL), which is the most potent E-selectin ligand. Following enforced HCELL expression, the transplantation of BMSCs was then evaluated in a middle cerebral artery occlusion model. Results showed that HCELL+BMSCs significantly ameliorated neurological deficits and reduced the volume of cerebral infarction. Furthermore, the transplantation led to a decrease in apoptosis by upregulating BCL-2 and downregulating BAX, also reduced the mRNA levels of inflammatory factors, such as interleukin-1β (IL-1β), IL-2, IL-6, and tumor necrosis factor-alpha (TNF-α) in the ischemic brain tissue. Notably, enforced HCELL expression facilitated the migration of BMSCs toward cerebral ischemic lesions and their subsequent transendothelial migration through the upregulation of PTGS-2, increased production of PGE2 and activation of VLA-4. In summary, our study demonstrates that transplantation of HCELL+BMSCs effectively alleviates CIRI, and that enforced HCELL expression enhances the homing of BMSCs to cerebral ischemic lesions and their transendothelial migration via PTGS-2/PGE2/VLA-4. These findings indicate that enforced expression of HCELL on BMSCs could serve as a promising therapeutic strategy for the treatment of ischemic stroke.

Impaired bone healing following tooth extraction poses a significant challenge for implantation. As a crucial component of the natural immune system, the NLRP3 inflammasome is one of the most extensively studied pattern-recognition receptors, and is involved in multiple diseases. Yet, the role of NLRP3 in bone healing remains to be clarified. Here, to investigate the effect of NLRP3 on bone healing, we established a maxillary first molar extraction model in wild-type and NLRP3KO mice using minimally invasive techniques. We observed that NLRP3 was activated during the bone repair phase, and its depletion enhanced socket bone formation and osteoblast differentiation. Moreover, NLRP3 inflammasome activation was found to inhibit osteogenic differentiation in alveolar bone-derived mesenchymal stem cells (aBMSCs), an effect mitigated by NLRP3 deficiency. Mechanistically, we established that the SMAD2/3-RUNX2 signaling pathway is a downstream target of NLRP3 inflammasome activation, and SMAD2/3 knockdown partially reversed the significant decrease in expression of RUNX2, OSX, and ALP induced by NLRP3. Thus, our findings demonstrate that NLRP3 negatively modulates alveolar socket bone healing and contributes to the understanding of the NLRP3-induced signaling pathways involved in osteogenesis regulation.