技术资料

Rett syndrome (RTT) is a neurodevelopmental disorder caused by de novo mutations in the MECP2 gene. Although miRNAs in extracellular vesicles (EVs) have been suggested to play an essential role in several neurological conditions,no prior study has utilized brain organoids to profile EV-derived miRNAs during normal and RTT-affected neuronal development. Here we report the spatiotemporal expression pattern of EV-derived miRNAs in region-specific forebrain organoids generated from female hiPSCs with a MeCP2:R255X mutation and the corresponding isogenic control. EV miRNA and protein expression profiles were characterized at day 0,day 13,day 40,and day 75. Several members of the hsa-miR-302/367 cluster were identified as having a time-dependent expression profile with RTT-specific alterations at the latest developmental stage. Moreover,the miRNA species of the chromosome 14 miRNA cluster (C14MC) exhibited strong upregulation in RTT forebrain organoids irrespective of their spatiotemporal location. Together,our results suggest essential roles of the C14MC and hsa-miR-302/367 clusters in EVs during normal and RTT-associated neurodevelopment,displaying promising prospects as biomarkers for monitoring RTT progression. The online version contains supplementary material available at 10.1007/s00018-024-05409-7. View Publication

KRAS is among the most commonly mutated oncogenes in human malignancies. Although the advent of sotorasib and adagrasib,has lifted the “undruggable” stigma of KRAS,the resistance to KRAS inhibitors quickly becomes a major issue. Here,we reported that aldehyde dehydrogenase 1 family member A1 (ALDH1A1),an enzyme in retinoic acid biosynthesis and redox balance,increases in response to KRAS inhibitors and confers resistance in a range of cancer types. KRAS inhibitors' efficacy is significantly improved in sensitive or drug-resistant cells,patient-derived organoids (PDO),and xenograft models by ALDH1A1 knockout,loss of enzyme function,or inhibitor. Furthermore,we discovered that ALDH1A1 suppresses the efficacy of KRAS inhibitors by counteracting ferroptosis. ALDH1A1 detoxicates deleterious aldehydes,boosts the synthesis of NADH and retinoic acid (RA),and improves RARA function. ALDH1A1 also activates the CREB1/GPX4 pathway,stimulates the production of lipid droplets in a pH-dependent manner,and subsequently prevents ferroptosis induced by KRAS inhibitors. Meanwhile,we established that GTF2I is dephosphorylated at S784 via ERK by KRAS inhibitors,which hinders its nuclear translocation and mediates ALDH1A1's upregulation in response to KRAS inhibitors. In summary,the results offer valuable insights into targeting ALDH1A1 to enhance the effectiveness of KRAS-targeted therapy through ferroptosis in cancer treatment. View Publication

Thyroid hormone receptor alpha (THRα) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development,metabolism,and reproduction. In mammals,THRα has two major splicing isoforms,THRα1 and THRα2. The better-characterized isoform,THRα1,is a transcriptional stimulator of genes involved in cell metabolism and growth. The less-well-characterized isoform,THRα2,lacks the ligand-binding domain (LBD) and is thought to act as an inhibitor of THRα1 activity. The ratio of THRα1 to THRα2 splicing isoforms is therefore critical for transcriptional regulation in different tissues and during development. However,the expression patterns of both isoforms have not been studied in healthy human tissues or in the developing brain. Given the lack of commercially available isoform-specific antibodies,we addressed this question by analyzing four bulk RNA-sequencing datasets and two scRNA-sequencing datasets to determine the RNA expression levels of human THRA1 and THRA2 transcripts in healthy adult tissues and in the developing brain. We demonstrate how 10X Chromium scRNA-seq datasets can be used to perform splicing-sensitive analyses of isoforms that differ at the 3′-end. In all datasets,we found a strong predominance of THRA2 transcripts at all examined stages of human brain development and in the central nervous system of healthy human adults. View Publication

Head and neck squamous cell carcinoma (HNSCC) presents a significant challenge worldwide due to its aggressiveness and high recurrence rates post-treatment,often linked to cancer stem cells (CSCs). Melatonin shows promise as a potent tumor suppressor; however,the effects of melatonin on CSCs remain unclear,and the development of models that closely resemble tumor heterogeneity could help to better understand the effects of this molecule. This study developed a tumor scaffold based on patient fibroblast-derived decellularized extracellular matrix that mimics the HNSCC microenvironment. Our study investigates the antitumoral effects of melatonin within this context. We validated its strong antiproliferative effect on HNSCC CSCs and the reduction of tumor invasion and migration markers,even in a strongly chemoprotective environment,as it is required to increase the minimum doses necessary to impact tumor viability compared to the non-scaffolded tumorspheres culture. Moreover,melatonin exhibited no cytotoxic effects on healthy cells co-cultured in the tumor hydrogel. This scaffold-based platform allows an in vitro study closer to HNSCC tumor reality,including CSCs,stromal component,and a biomimetic matrix,providing a new valuable research tool in precision oncology. View Publication

The emerging field of synthetic morphogenesis implements synthetic biology tools to investigate the minimal cellular processes sufficient for orchestrating key developmental events. As the field continues to grow,there is a need for new tools that enable scientists to uncover nuances in the molecular mechanisms driving cell fate patterning that emerge during morphogenesis. Here,we present a platform that combines cell engineering with biomaterial design to potentiate artificial signaling in pluripotent stem cells (PSCs). This platform,referred to as PSC-MATRIX,extends the use of programmable biomaterials to PSCs competent to activate morphogen production through orthogonal signaling,giving rise to the opportunity to probe developmental events by initiating morphogenetic programs in a spatially constrained manner through non-native signaling channels. We show that the PSC-MATRIX platform enables temporal and spatial control of transgene expression in response to bulk,soluble inputs in synthetic Notch (synNotch)-engineered human PSCs for an extended culture of up to 11 days. Furthermore,we used PSC-MATRIX to regulate multiple differentiation events via material-mediated artificial signaling in engineered PSCs using the orthogonal ligand green fluorescent protein,highlighting the potential of this platform for probing and guiding fate acquisition. Overall,this platform offers a synthetic approach to interrogate the molecular mechanisms driving PSC differentiation that could be applied to a variety of differentiation protocols. View Publication

Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here,we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD),a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin,essential for peptide loading onto MHC-I,as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance. Subject terms: Endoplasmic reticulum,ER-associated degradation,MHC class I,Antigen-presenting cells View Publication

Radiotherapy is widely regarded as the primary therapeutic modality for nasopharyngeal cancer (NPC). Studies have shown that cancer cells with high resistance to radiation,known as radioresistant cancer cells,may cause residual illness,which in turn might contribute to the occurrence of cancer recurrence and metastasis. It has been shown that cancer stem-like cells (CSCs) exhibit resistance to radiation therapy. In the present study,fractionated doses of radiation-induced epithelial-mesenchymal transition (EMT) and ALDH+ CSCs phenotype of NPC tumor spheroids. Furthermore,it has been shown that cells with elevated ALDH activity have increased resistance to the effects of fractionated irradiation. Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular antioxidant systems. A large body of evidence suggests that Nrf2 plays a significant role in the development of radioresistance in cancer. The authors’ research revealed that the application of fractionated irradiation resulted in a decline in Nrf2-dependent reactive oxygen species (ROS) levels,thereby mitigating DNA damage in ALDH+ stem-like NPC cells. In addition,immunofluorescence analysis revealed that subsequent to the process of fractionated irradiation of ALDH+ cells,activated Nrf2 was predominantly localized inside the nucleus. Immunofluorescent analysis also revealed that the presence of the nuclear Nrf2+/NQO1+/ALDH1+ axis might potentially serve as an indicator of poor prognosis and resistance to radiotherapy in patients with NPC. Thus,the authors’ findings strongly suggest that the radioresistance of ALDH-positive NPC CSCs to fractionated irradiation is regulated by nuclear Nrf2 accumulation. Nrf2 exerts its effects through the downstream effector NQO1/ALDH1,which depends on ROS attenuation. View Publication

The increasing scarcity of organs and the significant morbidity linked to dialysis require the development of engineered kidney tissues from human-induced pluripotent stem cells. Integrative approaches that synergize scalable kidney organoid differentiation,tissue biomanufacturing,and comprehensive assessment of their immune response and host integration are essential to accomplish this. Here,we create engineered human kidney tissues composed of organoid building blocks (OBBs) and transplant them into mice reconstituted with allogeneic human immune cells. Tissue-infiltrating human immune cells are composed of effector T cells and innate cells. This immune infiltration leads to kidney tissue injury characterized by reduced microvasculature,enhanced kidney cell apoptosis,and an inflammatory gene signature comparable to kidney organ transplant rejection in humans. Upon treatment with the immunosuppressive agent rapamycin,the induced immune response is greatly suppressed. Our model is a translational platform to study engineered kidney tissue immunogenicity and develop therapeutic targets for kidney rejection. Subject areas: Health sciences,Immunology,Bioengineering,Tissue engineering View Publication

Isobaric labeling-based mass spectrometry (ILMS) has been widely used to quantify,on a proteome-wide scale,the relative protein abundance in different biological conditions. However,large-scale ILMS data sets typically involve multiple runs of mass spectrometry,bringing great computational difficulty to the integration of ILMS samples. We present zMAP,a toolset that makes ILMS intensities comparable across mass spectrometry runs by modeling the associated mean-variance dependence and accordingly applying a variance stabilizing z-transformation. The practical utility of zMAP is demonstrated in several case studies involving the dynamics of cell differentiation and the heterogeneity across cancer patients. The online version contains supplementary material available at 10.1186/s13059-024-03382-9. View Publication

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal,chondrogenic,and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However,there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study,we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently,a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media,MesenCult™-ACF (ACF),and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology,surface markers,senescence status,differentiation capacity,and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. View Publication

Despite a record number of clinical studies investigating various anti-myeloma treatments,the 5-year survival rate for multiple myeloma (MM) patients in the US is only 55%,and almost all patients relapse. Poor patient outcomes demonstrate that myeloma cells are “born to survive” which means they can adapt and evolve following treatment. Thus,new therapeutic approaches to combat survival mechanisms and target treatment resistance are required. Importantly,Mcl-1,anti-apoptotic protein,is required for the development of MM and treatment resistance. This study looks at the possibility of KS18,a selective Mcl-1 inhibitor,to treat MM and overcome resistance. Our investigation demonstrates that KS18 effectively induces cell death in MM by dual regulatory mechanisms targeting the Mcl-1 protein at both transcriptional and post-translational levels. Specifically,KS18 suppresses Mcl-1 activation via STAT-3 pathway and promotes Mcl-1 phosphorylation/ubiquitination/proteasome-dependent protein degradation (UPS). Significantly,KS18 triggered caspase-dependent apoptosis in MM patient samples and bortezomib-resistant cells,synergizing with venetoclax to boost apoptosis. KS18 promises to overcome bortezomib and venetoclax resistance and re-sensitize myeloma cells to chemotherapy. Furthermore,the study shows the tremendous impact of KS18 in inhibiting colony formation in bortezomib-resistant cells and demonstrates significant tumor shrinkage in KS18-treated NSG mice without notable toxicity signs after 4 weeks of therapy with a single acceptable dose each week,indicating its powerful anti-neoplastic and anti-resistance characteristics. This study strongly implies that KS18 may treat MM and provide new hope to patients who are experiencing recurrence or resistance. View Publication

Autism Spectrum Disorder (ASD) is a developmental disorder characterized by impaired social communication and repetitive behaviors. In recent years,a pharmacological mouse model of ASD involving maternal administration of valproic acid (VPA) has become widely used. Newborn pups in this model show an abnormal balance between excitatory and inhibitory (E/I) signaling in neurons and exhibit ASD-like behavior. However,the molecular basis of this model and its implications for the pathogenesis of ASD in humans remain unknown. Using quantitative secretome analysis,we found that the level of leucine-rich repeat and immunoglobulin domain-containing protein 2 (Lingo2) was upregulated in the conditioned medium of VPA model neurons. This upregulation was associated with excitatory synaptic organizer activity. The secreted form of the extracellular domain of Lingo2 (sLingo2) is produced by the transmembrane metalloprotease ADAM10 through proteolytic processing. sLingo2 was found to induce the formation of excitatory synapses in both mouse and human neurons,and treatment with sLingo2 resulted in an increased frequency of miniature excitatory postsynaptic currents in human neurons. These findings suggest that sLingo2 is an excitatory synapse organizer involved in ASD,and further understanding of the mechanisms by which sLingo2 induces excitatory synaptogenesis is expected to advance our understanding of the pathogenesis of ASD. Subject terms: Autism spectrum disorders,Neuroscience View Publication