技术资料

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

Although bone marrow-derived cells with high aldehyde dehydrogenase activity (ALDH br ) have shown therapeutic potential against various diseases in animal studies,clinical trials have failed to show concurrent findings. We aimed to clarify the optimal conditions for the efficacy of ALDH br cells by using a murine bleomycin-induced pulmonary fibrosis model. We intravenously transferred male or female donor C57BL/6 mice-derived ALDH br cells into recipient C57BL/6 mice under various conditions,and used mCherry-expressing mice as a donor to trace the transferred ALDH br cells. Pulmonary fibrosis improved significantly when (1) female-derived,not male-derived,and (2) lineage (Lin)-negative,not lineage-positive,ALDH br cells were transferred during the (3) fibrotic,not inflammatory,phase. Consistent with the RNA-sequencing results,female-derived Lin − /ALDH br cells were more resistant to oxidative stress than male-derived cells in vitro,and transferred female-derived Lin − /ALDH br cells were more viable than male-derived cells in the fibrotic lung. The mechanism underlying the antifibrotic effects of Lin − /ALDH br cells was strongly associated with reduction of oxidative stress. Our results indicated that Lin − /ALDH br cell therapy could ameliorate pulmonary fibrosis by reducing oxidative stress and suggested that their efficacy was mediated by sex-related differences. Thus,sex-awareness strategies may be important for clinical application of bone marrow ALDH br cells as a therapeutic tool. The online version contains supplementary material available at 10.1186/s13287-024-03933-8. View Publication

Hematopoietic stem progenitor cells (HSPCs) undergo phenotypical and functional changes during their emergence and development. Although the molecular programs governing the development of human hematopoietic stem cells (HSCs) have been investigated broadly,the relationships between dynamic metabolic alterations and their functions remain poorly characterized. In this study,we comprehensively described the proteomics of HSPCs in the human fetal liver (FL),umbilical cord blood (UCB),and adult bone marrow (aBM). The metabolic state of human HSPCs was assessed via a Seahorse assay,RT‒PCR,and flow cytometry-based metabolic-related analysis. To investigate whether perturbing glutathione metabolism affects reactive oxygen species (ROS) production,the metabolic state,and the expansion of human HSPCs,HSPCs were treated with buthionine sulfoximine (BSO),an inhibitor of glutathione synthetase,and N-acetyl-L-cysteine (NAC). We investigated the metabolomic landscape of human HSPCs from the fetal,perinatal,and adult developmental stages by in-depth quantitative proteomics and predicted a metabolic switch from the oxidative state to the glycolytic state during human HSPC development. Seahorse assays,mitochondrial activity,ROS level,glucose uptake,and protein synthesis rate analysis supported our findings. In addition,immune-related pathways and antigen presentation were upregulated in UCB or aBM HSPCs,indicating their functional maturation upon development. Glutathione-related metabolic perturbations resulted in distinct responses in human HSPCs and progenitors. Furthermore,the molecular and immunophenotypic differences between human HSPCs at different developmental stages were revealed at the protein level for the first time. The metabolic landscape of human HSPCs at three developmental stages (FL,UCB,and aBM),combined with proteomics and functional validations,substantially extends our understanding of HSC metabolic regulation. These findings provide valuable resources for understanding human HSC function and development during fetal and adult life. The online version contains supplementary material available at 10.1186/s13287-024-03930-x. View Publication

In recent years,Raman spectroscopy has garnered growing interest in the field of biomedical research. It offers a non-invasive and label-free approach to defining the molecular fingerprint of immune cells. We utilized Raman spectroscopy on optically trapped immune cells to investigate their molecular compositions. While numerous immune cell types have been studied in the past,the characterization of living human CD3/CD28-stimulated T cell subsets remains incomplete. In this study,we demonstrate the capability of Raman spectroscopy to readily distinguish between naïve and stimulated CD4 and CD8 cells. Additionally,we compared these cells with monocytes and discovered remarkable similarities between stimulated T cells and monocytes. This paper contributes to expanding our knowledge of Raman spectroscopy of immune cells and serves as a launching point for future clinical applications. View Publication

Intervertebral disc disease (IDD) is a debilitating spine condition that can be caused by intervertebral disc (IVD) damage which progresses towards IVD degeneration and dysfunction. Recently,human pluripotent stem cells (hPSCs) were recognized as a valuable resource for cell-based regenerative medicine in skeletal diseases. Therefore,adult somatic cells reprogrammed into human induced pluripotent stem cells (hiPSCs) represent an attractive cell source for the derivation of notochordal-like cells (NCs) as a first step towards the development of a regenerative therapy for IDD. Utilizing a differentiation method involving treatment with a four-factor cocktail targeting the BMP,FGF,retinoic acid,and Wnt signaling pathways,we differentiate CRISPR/Cas9-generated mCherry-reporter knock-in hiPSCs into notochordal-like cells. Comprehensive analysis of transcriptomic changes throughout the differentiation process identified regulation of histone methylation as a pivotal driver facilitating the differentiation of hiPSCs into notochordal-like cells. We further provide evidence that specific inhibition of histone demethylases KDM2A and KDM7A/B enhanced the lineage commitment of hiPSCs towards notochordal-like cells. Our results suggest that inhibition of KDMs could be leveraged to alter the epigenetic landscape of hiPSCs to control notochord-specific gene expression. Thus,our study highlights the importance of epigenetic regulators in stem cell-based regenerative approaches for the treatment of disc degeneration. View Publication

Monocytes,the circulating macrophage precursors,contribute to diseases like atherosclerosis and asthma. Long non-coding RNAs (lncRNAs) have been shown to modulate the phenotype and inflammatory capacity of monocytes. We previously discovered the lncRNA SMANTIS,which contributes to cellular phenotype expression by controlling BRG1 in mesenchymal cells. Here,we report that SMANTIS is particularly highly expressed in monocytes and lost during differentiation into macrophages. Moreover,different types of myeloid leukemia presented specific SMANTIS expression patterns. Interaction studies revealed that SMANTIS binds RUNX1,a transcription factor frequently mutated in AML,primarily through its Alu-element on the RUNT domain. RNA-seq after CRISPR/Cas9-mediated deletion of SMANTIS or RUNX1 revealed an association with cell adhesion and both limited the monocyte adhesion to endothelial cells. Mechanistically,SMANTIS KO reduced RUNX1 genomic binding and altered the interaction of RUNX1 with EP300 and CBFB. Collectively,SMANTIS interacts with RUNX1 and attenuates monocyte adhesion,which might limit monocyte vascular egress. Subject terms: Long non-coding RNAs,Transcription View Publication

Despite their role as innate sentinels,macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV),a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here,with the use of viral chimeras and evolutionary selection analysis,we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation,we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220,which has undergone positive selection,is indispensable for CHIKV production in macrophages,as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally,we show that the antiviral activity of eIF3k is translation-independent,and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm,where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors,as well as potential targets for therapeutic intervention. View Publication

The roles of the transcriptional factor SIX2 have been identified in several tumors. However,its roles in gastric cancer (GC) progression have not yet been revealed. Our objective is to explore the impact and underlying mechanisms of SIX2 on the stemness of GC cells. Lentivirus infection was employed to establish stable expression SIX2 or PFN2 in GC cells. Gain- and loss-of-function experiments were conducted to detect changes of stemness markers,flow cytometry profiles,tumor spheroid formation,and tumor-initiating ability. ChIP,RNA-sequencing,tissue microarray,and bioinformatics analysis were performed to reveal the correlation between SIX2 and PFN2. The mechanisms underlying the SIX2/PFN2 loop-mediated effects were elucidated through tissue microarray analysis,RNA stability assay,IP-MS,Co-Immunoprecipitation,and inhibition of the JNK signaling pathway. The stemness of GC cells was enhanced by SIX2. Mechanistically,SIX2 directly bound to PFN2’s promoter and promoted PFN2 activity. PFN2,in turn,promoted the mRNA stability of SIX2 by recruiting RNA binding protein YBX-1,subsequently activating the downstream MAPK/JNK pathway. This study unveils the roles of SIX2 in governing GC cell stemness,defining a novel SIX2/PFN2 regulatory loop responsible for this regulation. This suggests the potential of targeting the SIX2/PFN2 loop for GC treatment (Graphical Abstracts). The online version contains supplementary material available at 10.1186/s12967-024-05618-5. 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

Rhodiola species have been utilized as functional foods in Asia and Europe for promoting health. Research has demonstrated that Rhodiola has the potential to alleviate inflammatory bowel disease (IBD) in animal models. However,the specific active components and the underlying mechanism for ameliorating intestinal damage remain unclear. This study aims to explore the relieving effect of Rosavin (Rov),a known active constituent of Rhodiola,in IBD and the regulatory mechanisms. The therapeutic effect of Rov was evaluated using a murine model of acute colitis induced by dextran sulfate sodium salt (DSS). Inflammatory cytokines and neutrophil activation markers were measured by corresponding kits. Immunohistochemistry,immunofluorescence,TUNEL,and EdU assays were applied to investigate the tight conjunction proteins expression,epithelial marker expression,number of apoptotic cells,and epithelial proliferation,respectively. The protection effect of Rov on gut epithelial injury was assessed using TNF-α-induced intestinal organoids. Additinally,RNA sequencing was applied to observe the genetic alteration profile in these intestinal organoids. Oral administration of Rov significantly attenuated weight loss and restored colon length in mice. Notably,Rov treatment led to decreased levels of pro-inflammatory cytokines and neutrophil activation markers while increasing anti-inflammatory factors. Importantly,Rov restored intestinal despair by increasing the number of Lgr5 + stem cells,Lyz1 + Paneth cells and Muc2 + goblet cells in intestines of colitis mice,displaying reduced epithelial apoptosis and recovered barrier function. In TNF-α-induced intestinal organoids,Rov facilitated epithelial cell differentiation and protected against TNF-α-induced damage. RNA sequencing revealed upregulation in the gene expression associated with epithelial cells (including Lgr5 +,Lyz1 + and Muc2 + cells) proliferation and defensin secretion,unveiling the protective mechanisms of Rov on the intestinal epithelial barrier. Rov holds potential as a natural prophylactic agent against IBD,with its protective action on the intestinal epithelium being crucial for its therapeutic efficacy. View Publication

Immune checkpoint blockade (ICB) has made remarkable achievements,but newly identified armored and cold tumors cannot respond to ICB therapy. The high prevalence of concomitant medications has huge impact on immunotherapeutic responses,but the clinical effects on the therapeutic outcome of armored and cold tumors are still unclear. In this research,using large-scale transcriptomics datasets,the expression and potential biological functions of angiotensin II receptor 1 (AGTR1),the target of angiotensin receptor blocker (ARB),were investigated. Next,the roles of ARB in tumor cells and tumor microenvironment cells were defined by a series of in vitro and in vivo assays. In addition,the clinical impacts of ARB on ICB therapy were assessed by multicenter cohorts and meta-analysis. AGTR1 was overexpressed in armored and cold tumors and associated with poor response to ICB therapy. ARB,the inhibitor for AGTR1,only suppressed the aggressiveness of tumor cells with high AGTR1 expression,which accounted for a very small proportion. Further analysis revealed that AGTR1 was always highly expressed in cancer-associated fibroblasts (CAFs) and ARB inhibited type I collagen expression in CAFs by suppressing the RhoA-YAP axis. Moreover,ARB could also drastically reverse the phenotype of armored and cold to soft and hot in vivo,leading to a higher response to ICB therapy. In addition,both our in-house cohorts and meta-analysis further supported the idea that ARB can significantly enhance ICB efficacy. Overall,we identify AGTR1 as a novel target in armored and cold tumors and demonstrate the improved therapeutic efficacy of ICB in combination with ARB. These findings could provide novel clinical insight into how to treat patients with refractory armored and cold tumors. View Publication

Intrahepatic cholangiocarcinoma (iCCA) is a lethal primary liver tumor characterized by clinical aggressiveness,poor prognosis,and scarce therapeutic possibilities. Therefore,new treatments are urgently needed to render this disease curable. Since cumulating evidence supports the oncogenic properties of the Heat Shock Factor 1 (HSF1) transcription factor in various cancer types,we investigated its pathogenetic and therapeutic relevance in iCCA. Levels of HSF1 were evaluated in a vast collection of iCCA specimens. The effects of HSF1 inactivation on iCCA development in vivo were investigated using three established oncogene-driven iCCA mouse models. In addition,the impact of HSF1 suppression on tumor cells and tumor stroma was assessed in iCCA cell lines,human iCCA cancer-associated fibroblasts (hCAFs),and patient-derived organoids. Human preinvasive,invasive,and metastatic iCCAs displayed widespread HSF1 upregulation,which was associated with a dismal prognosis of the patients. In addition,hydrodynamic injection of a dominant-negative form of HSF1 (HSF1dn),which suppresses HSF1 activity,significantly delayed cholangiocarcinogenesis in AKT/NICD,AKT/YAP,and AKT/TAZ mice. In iCCA cell lines,iCCA hCAFs,and patient-derived organoids,administration of the HSF1 inhibitor KRIBB-11 significantly reduced proliferation and induced apoptosis. Cell death was profoundly augmented by concomitant administration of the Bcl-xL/Bcl2/Bcl-w inhibitor ABT-263. Furthermore,KRIBB-11 reduced mitochondrial bioenergetics and glycolysis of iCCA cells. The present data underscore the critical pathogenetic,prognostic,and therapeutic role of HSF1 in cholangiocarcinogenesis. The online version contains supplementary material available at 10.1186/s13046-024-03177-7. View Publication