技术资料

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

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

The inflammatory response to wear particles derived from hip prothesis is considered a hallmark of periprosthetic osteolysis,which can ultimately lead to the need for revision surgery. Exosomes (Exos) have been associated with various bone pathologies,and there is increasing recognition in the literature that they actively transport molecules throughout the body. The role of wear particles in osteoblast-derived Exos is unknown,and the potential contribution of Exos to osteoimmune communication and periprosthetic osteolysis niche is still in its infancy. Given this,we investigate how titanium dioxide nanoparticles (TiO 2 NPs),similar in size and composition to prosthetic wear particles,affect Exos biogenesis. Two osteoblastic cell models commonly used to study the response of osteoblasts to wear particles were selected as a proof of concept. The contribution of Exos to periprosthetic osteolysis was assessed by functional assays in which primary human macrophages were stimulated with bone-derived Exos. We demonstrated that TiO 2 NPs enter multivesicular bodies,the nascent of Exos,altering osteoblast-derived Exos secretion and molecular cargo. No significant differences were observed in Exos morphology and size. However,functional assays reveal that Exos cargo enriched in uPA stimulates macrophages to a mixed M1 and M2 phenotype,inducing the release of pro- and anti-inflammatory signals characteristic of periprosthetic osteolysis. In addition,we demonstrated the expression of uPA in exosomes derived from the urine of patients with osteolysis. These results suggest that uPA can be a potential biomarker of osteolysis. In the future,uPa may serve as a possible non-invasive biomarker to identify patients at risk for peri-implant osteolysis. View Publication

Dimethylsulfoxide (DMSO) has conventionally been used for cell cryopreservation both in research and in clinical applications,but has long-term cytotoxic effects. Trehalose,a natural disaccharide,has been proposed as a non-toxic cryoprotectant. However,the lack of specific cell membrane transporter receptors inhibits transmembrane transport and severely limits its cryoprotective capability. This research presents a method to successfully deliver trehalose into mesenchymal stem cells (MSCs) using ultrasound in the presence of microbubbles. The optimised trehalose concentration was shown to be able to not only preserve membrane integrity and cell viability but also the multipotency of MSCs,which are essential for stem cell therapy. Confocal imaging revealed that rhodamine-labelled trehalose was transported into cells rather than simply attached to the membrane. Additionally,the membranes were successfully preserved in lyophilised cells. This study demonstrates that ultrasonication with microbubbles facilitated trehalose delivery,offering promising cryoprotective capability without the cytotoxicity associated with DMSO-based methods. Subject terms: Membrane biophysics,Biomedical engineering View Publication

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes,but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR,which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends,capturing the target region for sequencing on PacBio’s Sequel,Sequel II,or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized,even for repeats in the thousands. Key features • This protocol is adapted from PacBio’s previous “no-amp targeted sequencing utilizing the CRISPR-Cas9 system.” • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type,blood,or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio. View Publication

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD,even before symptom onset. Because these early disease phenotypes remain incompletely understood,we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients,presymptomatic C9ORF72-HRE (C9-HRE) carriers,and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition,single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids,including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further,molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly,organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology,whereas the extent to which more downstream cellular defects,as found in C9-ALS/FTD models,were detected varied for the different presymptomatic C9-HRE cases. Together,these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies. The online version contains supplementary material available at 10.1186/s40478-024-01857-1. View Publication

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (V H ) and light chain germline segment pairs,or parallel lineages,bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct V H lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo. Subject terms: Protein design,Protein design,VDJ recombination,Class switch recombination,Plasma cells View Publication

Poor graft function (PGF),characterized by myelosuppression,represents a significant challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) with human cytomegalovirus (HCMV) being established as a risk factor for PGF. However,the underlying mechanism remains unclear. Bone marrow endothelial progenitor cells (BM-EPCs) play an important role in supporting hematopoiesis and their dysfunction contributes to PGF development. We aim to explore the effects of CMV on BM-EPCs and its underlying mechanism. We investigated the compromised functionality of EPCs derived from individuals diagnosed with HCMV viremia accompanied by PGF,as well as after infected by HCMV AD 169 strain in vitro,characterized by decreased cell proliferation,tube formation,migration and hematopoietic support,and increased apoptosis and secretion of TGF-β1. We demonstrated that HCMV-induced TGF-β1 secretion by BM-EPCs played a dominant role in hematopoiesis suppression in vitro experiment. Moreover,HCMV down-regulates Vitamin D receptor (VDR) and subsequently activates p38 MAPK pathway to promote TGF-β1 secretion by BM-EPCs. HCMV could infect BM-EPCs and lead to their dysfunction. The secretion of TGF-β1 by BM-EPCs is enhanced by CMV through the activation of p38 MAPK via a VDR-dependent mechanism,ultimately leading to compromised support for hematopoietic progenitors by BM EPCs,which May significantly contribute to the pathogenesis of PGF following allo-HSCT and provide innovative therapeutic strategies targeting PGF. View Publication

Addiction is known to occur through the consumption of substances such as pharmaceuticals,illicit drugs,food,alcohol and tobacco. These addictions can be viewed as drug addiction,resulting from the ingestion of chemical substances contained in them. Multiple neural networks,including the reward system,anti‐reward/stress system and central immune system in the brain,are believed to be involved in the onset of drug addiction. Although various compound evaluations using microelectrode array (MEA) as an in vitro testing methods to evaluate neural activities have been conducted,methods for assessing addiction have not been established. In this study,we aimed to develop an in vitro method for assessing the addiction of compounds,as an alternative to animal experiments,using human iPS cell‐derived dopaminergic neurons with MEA measurements. MEA data before and after chronic exposure revealed specific changes in addictive compounds compared to non‐addictive compounds,demonstrating the ability to estimate addiction of compound. Additionally,conducting gene expression analysis on cultured samples after the tests revealed changes in the expression levels of various receptors (nicotine,dopamine and GABA) due to chronic administration of addictive compounds,suggesting the potential interpretation of these expression changes as addiction‐like responses in MEA measurements. The addiction assessment method using MEA measurements in human iPS cell‐derived dopaminergic neurons conducted in this study proves effective in evaluating addiction of compounds on human neural networks. View Publication

Human pluripotent stem cells (hPSCs) represent a powerful model system to study early developmental processes. However,lineage specification into trophectoderm (TE) and trophoblast (TB) differentiation remains poorly understood,and access to well-characterized placental cells for biomedical research is limited,largely depending on fetal tissues or cancer cell lines. Here,we developed novel strategies enabling highly efficient TE specification that generates cytotrophoblast (CTB) and multinucleated syncytiotrophoblast (STB),followed by the establishment of trophoblast stem cells (TSCs) capable of differentiating into extravillous trophoblast (EVT) and STB after long-term expansion. We confirmed stepwise and controlled induction of lineage- and cell-type-specific genes consistent with developmental biology principles and benchmarked typical features of placental cells using morphological,biochemical,genomics,epigenomics,and single-cell analyses. Charting a well-defined roadmap from hPSCs to distinct placental phenotypes provides invaluable opportunities for studying early human development,infertility,and pregnancy-associated diseases. Subject areas: Natural sciences,Biological sciences,Cell biology,Stem cells research View Publication