技术资料

During embryogenesis,human hematopoietic stem cells (HSCs) first emerge in the aorta-gonad-mesonephros (AGM) region via transformation of specialized hemogenic endothelial (HE) cells into premature HSC precursors. This process is termed endothelial-to-hematopoietic transition (EHT),in which the HE cells undergo drastic functional and morphological changes from flat,anchorage-dependent endothelial cells to free-floating round hematopoietic cells. Despite its essential role in human HSC development,molecular mechanisms underlying the EHT are largely unknown. This is due to lack of methods to visualize the emergence of human HSC precursors in real time in contrast to mouse and other model organisms. In this study,by inducing HE from human pluripotent stem cells in feeder-free monolayer cultures,we achieved real-time observation of the human EHT in vitro . By continuous observation and single-cell tracking in the culture,it was possible to visualize a process that a single endothelial cell gives rise to a hematopoietic cell and subsequently form a hematopoietic-cell cluster. The EHT was also confirmed by a drastic HE-to-HSC switching in molecular marker expressions. Notably,HSC precursor emergence was not linked to asymmetric cell division,whereas the hematopoietic cell cluster was formed through proliferation and assembling of the floating cells after the EHT. These results reveal unappreciated dynamics in the human EHT,and we anticipate that our human EHT model in vitro will provide an opportunity to improve our understanding of the human HSC development. View Publication

Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington’s disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT,we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2),a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence,mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2,which could then serve as an early interventional target for HD. Subject terms: Huntington's disease,Mechanisms of disease,Stem cells View Publication

Mesenchymal stem cells (MSCs) have long been postulated as an important source cell in regenerative medicine. During subculture expansion,mesenchymal stem cell (MSC) senescence diminishes their multi-differentiation capabilities,leading to a loss of therapeutic potential. Up to date,the extrachromosomal circular DNAs (eccDNAs) have been demonstrated to be involved in senescence but the roles of eccDNAs during MSC. Here we explored eccDNA profiles in human bone marrow MSCs (BM-MSCs). EccDNA and mRNA was purified and sequenced,followed by quantification and functional annotation. Moreover,we mapped our datasets with the downloading enhancer and transcription factor-regulated genes to explore the potential role of eccDNAs. Sequentially,gene annotation analysis revealed that the majority of eccDNA were mapped in the intron regions with limited BM-MSC enhancer overlaps. We discovered that these eccDNA motifs in senescent BMSCs acted as motifs for binding transcription factors (TFs) of senescence-related genes. These findings are highly significant for identifying biomarkers of senescence and therapeutic targets in mesenchymal stem cells (MSCs) for future clinical applications. The potential of eccDNA as a stable therapeutic target for senescence-related disorders warrants further investigation,particularly exploring chemically synthesized eccDNAs as transcription factor regulatory elements to reverse cellular senescence. View Publication

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these embryonically derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic endothelial-macrophage (EndoMac) progenitor cells in the adventitia of embryonic and postnatal mouse aorta,that are independent of Flt3-mediated bone marrow hematopoiesis and derive from an early embryonic CX 3 CR1 + and CSF1R + source. These bipotent progenitors are proliferative and vasculogenic,contributing to adventitial neovascularization and formation of perfused blood vessels after transfer into ischemic tissue. We establish a regulatory role for angiotensin II,which enhances their clonogenic and differentiation properties and rapidly stimulates their proliferative expansion in vivo. Our findings demonstrate that embryonically derived EndoMac progenitors participate in local vasculogenic responses in the aortic wall by contributing to the expansion of endothelial cells and macrophages postnatally. Subject terms: Angiogenesis,Myelopoiesis,Haematopoietic stem cells View Publication

During the metastatic cascade,cancer cells travel through the bloodstream as circulating tumor cells (CTCs) to a secondary site. Clustered CTCs have greater shear stress and treatment resistance,yet their biology remains poorly understood. We therefore engineered a tunable superhydrophobic array device (SHArD). The SHArD-C was applied to culture a clinically relevant model of CTC clusters. Using our device,we cultured a model of cancer cell aggregates of various sizes with immortalized cancer cell lines. These exhibited higher E-cadherin expression and are significantly more capable of surviving high fluid shear stress-related forces compared to single cells and model clusters grown using the control method,helping to explain why clustering may provide a metastatic advantage. Additionally,the SHArD-S,when compared with the AggreWell 800 method,provides a more consistent spheroid-forming device culturing reproducible sizes of spheroids for multiple cancer cell lines. Overall,we designed,fabricated,and validated an easily tunable engineered device which grows physiologically relevant three-dimensional (3D) cancer models containing tens to thousands of cells. View Publication

More than 100 genes have been associated with significantly increased risks of autism spectrum disorders (ASD) with an estimate of ∼1000 genes that may contribute. The new challenge is to investigate the molecular and cellular functions of these genes during neural and brain development,and then even more challenging,to link the altered molecular and cellular phenotypes to the ASD clinical manifestations. In this study,we used single-cell RNA-seq analysis to study one of the top risk genes,CHD8,in cerebral organoids,which models early neural development. We identified 21 cell clusters in the organoid samples,representing non-neuronal cells,neural progenitors,and early differentiating neurons at the start of neural cell fate commitment. Comparisons of the cells with one copy of a CHD8 knockout allele,generated by CRISPR/Cas9 editing,and their isogenic controls uncovered thousands of differentially expressed genes,which were enriched with functions related to neural and brain development,cilium organization,and extracellular matrix organization. The affected genes were also enriched with genes and pathways previously implicated in ASD,but surprisingly not for schizophrenia and intellectual disability risk genes. The comparisons also uncovered cell composition changes,indicating potentially altered neural differential trajectories upon CHD8 reduction. Moreover,we found that cell-cell communications were affected in the CHD8 knockout organoids,including the interactions between neural and glial cells. Taken together,our results provide new data and information for understanding CHD8 functions in the early stages of neural lineage development and interaction. View Publication

Besides neutralizing antibodies,which are considered an important measure for vaccine immunogenicity,Fc-mediated antibody functions can contribute to antibody-mediated protection. They are strongly influenced by structural antibody properties such as subclass and Fc glycan composition. We here applied a systems serology approach to dissect humoral immune responses induced by MVA-MERS-S,an MVA-vectored vaccine against the Middle East respiratory syndrome coronavirus (MERS-CoV). Building on preceding studies reporting the safety and immunogenicity of MVA-MERS-S,our study highlights the potential of a late boost,administered one year after prime,to enhance both neutralizing and Fc-mediated antibody functionality compared to the primary vaccination series. Distinct characteristics were observed for antibodies specific to the MERS-CoV spike protein S1 and S2 subunits,regarding subclass and glycan compositions as well as Fc functionality. These findings highlight the benefit of a late homologous booster vaccination with MVA-MERS-S and may be of interest for the design of future coronavirus vaccines. Subject areas: Cell biology,Immune response,Immunology,Virology View Publication

Proper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance,the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately,the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation. We utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4,a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis,and confirmed the targeting by dual-luciferase reporter assay,western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance. In this study,overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation,respectively. Accordingly,we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression,as well as improved megakaryocytic differentiation,similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p. miR-1915-3p acts as a negative regulator of erythropoiesis,and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs. The online version contains supplementary material available at 10.1186/s12959-024-00615-6. View Publication

CD8 + cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide–MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell–cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell–cell contact,promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux,providing the necessary energy through oxidative phosphorylation,and its own protein translation on 55S ribosomes. However,the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here,we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton,mitochondria,and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. View Publication

Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters,RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide),which are involved in different redox reactions,including the energetic metabolism processes occurring in mitochondria. To date,human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons,which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far,no attention has been dedicated to astrocytes. Here,we demonstrate that in vitro differentiation of astrocytes,which guarantee trophic and metabolic support to neurons,from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons,RTD2 does not compromise the morpho-functional features of astrocytes. View Publication

PU.1 ( SPI1 ) is pivotal in hematopoiesis,yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Comparing human in vivo and in vitro EHT transcriptomes revealed SPI1 ’s regulatory role. Knocking down SPI1 during in vitro EHT led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis,we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells,respectively - as downstream targets of SPI1 . Overexpressing KLF1 or LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically,KLF1 overexpression restores myeloid lineage potential,while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1 - LYL1 axis in the regulatory network in in vivo EHT. Taken together,our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT,potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs). Subject areas: Biological sciences,Molecular biology,Molecular interaction,Cell biology; View Publication

Most studies used animal serum-containing medium for bioengineered-root regeneration,but ethical and safety issues raised by animal serum are a potentially significant risk for clinical use. Thus,this study aimed to find a safer method for bioengineered-root regeneration. The biological properties of human dental pulp stem cells (hDPSCs) cultured in animal component-free (ACF) medium or serum-containing medium (5%,10% serum-containing medium,SCM) were compared in vitro . hDPSCs were cultured in a three-dimensional (3D) environment with human-treated dentin matrix (hTDM). The capacity for odontogenesis was compared using quantitative real-time PCR (qPCR) and Western blot. Subsequently,the hDPSCs/hTDM complexes were transplanted into nude mice subcutaneously. Histological staining was then used to verify the regeneration effect in vivo . ACF medium promoted the migration of hDPSCs,but slightly inhibited the proliferation of hDPSCs in the first three days of culture compared to SCM. However,it had no significant effect on cell aging and apoptosis. After 7 days of 3D culture in ACF medium with hTDM,qPCR showed that DMP1,DSPP,OCN,RUNX2,and β-tubulin III were highly expressed in hDPSCs. In addition,3D cultured hDPSCs/hTDM complexes in ACF medium regenerated dentin,pulp,and periodontal ligament-like tissues similar to SCM groups in vivo . ACF medium was proved to be an alternative medium for bioengineered-root regeneration. The strategy of using ACF medium to regenerate bioengineered-root can improve clinical safety for tooth tissue engineering. View Publication