技术资料

Ribosome profiling,which is based on deep sequencing of ribosome footprints,has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However,the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here,we overcome these hurdles through the development of “Ribo-FilterOut”,which is based on the separation of footprints from ribosome subunits by ultrafiltration,and “Ribo-Calibration”,which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript,the translation initiation rate,and the overall number of translation events before its decay,all in a genome-wide manner. Moreover,our method reveals the allocation of ribosomes under heat shock stress,during aging,and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome. Ribosome profiling faces issues with rRNA contamination and measurements of ribosome numbers on transcripts. Here,the authors develop Ribo-FilterOut and Ribo-Calibration,methods which can be used to estimate kinetic and stoichiometric parameters of translation under various conditions. View Publication

Pancreatic islets are densely packed cellular aggregates containing various hormonal cell types essential for blood glucose regulation. Interactions among these cells markedly affect the glucoregulatory functions of islets along with the surrounding niche and pancreatic tissue-specific geometrical organization. However,stem cell (SC)-derived islets generated in vitro often lack the three-dimensional extracellular microenvironment and peri-vasculature,which leads to the immaturity of SC-derived islets,reducing their ability to detect glucose fluctuations and insulin release. Here,we bioengineer the in vivo-like pancreatic niches by optimizing the combination of pancreatic tissue-specific extracellular matrix and basement membrane proteins and utilizing bioprinting-based geometrical guidance to recreate the spatial pattern of islet peripheries. The bioprinted islet-specific niche promotes coordinated interactions between islets and vasculature,supporting structural and functional features resembling native islets. Our strategy not only improves SC-derived islet functionality but also offers significant potential for advancing research on islet development,maturation,and diabetic disease modeling,with future implications for translational applications. The glucoregulatory functions of pancreatic islets are affected by their surrounding niche and spatial organization. Here,bioengineered stem-cell derived islet niches use bioprinting-based geometrical guidance to promote islet maturation for improved functionality and diabetes research. View Publication

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here,using a candidate-based shRNA screen,we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors,or FMR1- SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells,neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2,for which an FDA-approved small molecule inhibitor,EPZ6438 (also known as tazemetostat),is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately,EZH2 inhibitors do not efficiently cross the blood-brain barrier,limiting their therapeutic use for FXS. Recently,antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons,and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively,our results establish EZH2 inhibition in general,and EZH2 ASOs in particular,as a therapeutic approach for FXS. View Publication

BackgroundOrganoids,as near-physiological 3D culture systems,offer new opportunities to study the pathogenesis of various organs in mimicking the cellular complexity and functionality of human organs.MethodHere we used a quite simple and very practicable method to successfully generate induced pluripotent stem cell (iPSC)-derived human lung organoids (LuOrg) in a matrix-free manner as an alternative to the widely used preclinical mouse models in order to investigate normal lung damage in detail and as close as possible to the patient. We performed detailed morphological and molecular analyses,including bulk and single cell RNA sequencing,of generated lung organoids and evaluated the quality and robustness of our model as a potential in vitro platform for lung diseases,namely radiation-induced lung injury.ResultsA matrix-free method for differentiation of iPSCs can be used to obtain lung organoids that morphologically reflect the target tissue of the human lung very well,especially with regard to the cellular composition. The different cellular fates were investigated following the genotoxic stress induced by radiation and revealed further insights in the radiation-sensitivity of the different lung cells. Finally,we provide cellular gene sets found to be induced in the different lung organoid cellular subsets after irradiation,which could be used as additional RT response and particularly senescence gene sets in future studies.ConclusionBy establishing these free-floating LuOrgs for the investigation of cancer therapeutic approaches as a new and patient-oriented in vitro platform particularly in experimental radiooncology,not only a reduction in the number of experimental animals,but also an adequately and meaningfully replacement of corresponding animal experiments can be achieved. View Publication

Polycomb repressive complex 1 (PRC1) modifies chromatin through catalysis of histone H2A lysine 119 monoubiquitination (H2AK119ub1). RING1 and RNF2 interchangeably serve as the catalytic subunit within PRC1. Pathogenic missense variants in PRC1 core components reveal functions of these proteins that are obscured in knockout models. While Ring1a knockout models remain healthy,the microcephaly and neuropsychiatric phenotypes associated with a pathogenic RING1 missense variant implicate unappreciated functions. Using an in vitro model of neurodevelopment,we observe that RING1 contributes to the broad placement of H2AK119ub1,and that its targets overlap with those of RNF2. PRC1 complexes harboring hypomorphic RING1 bind target loci but do not catalyze H2AK119ub1,reducing H2AK119ub1 by preventing catalytically active complexes from accessing the locus. This results in delayed DNA damage repair and cell cycle progression in neural progenitor cells (NPCs). Conversely,reduced H2AK119ub1 due to hypomorphic RING1 does not generate differential expression that impacts NPC differentiation. In contrast,hypomorphic RNF2 generates a greater reduction in H2AK119ub1 that results in both delayed DNA repair and widespread transcriptional changes. These findings suggest that the DNA damage response is more sensitive to H2AK119ub1 dosage change than is regulation of gene expression. Here,the authors establish a human in vitro model of neurodevelopment to investigate an allelic series of clinically relevant RING1 and RNF2 missense variants. The observations reveal that missense variants function according to a dominant-negative genetic mechanism. View Publication

The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand,FasL. Although Fas is highly expressed in cancer cells,insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here,we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor,fasudil,that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL),fasudil promoted cancer cell apoptosis,but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis. View Publication

Background/Objectives: High-altitude environments have a significant detrimental impact on the cognitive functions of the brain. Qi Jing Wan (QJW),a traditional herbal formula composed of Angelica sinensis,Astragalus membranaceus,and Rhizoma Polygonati Odorati,has demonstrated potential efficacy in treating cognitive disorders. However,its effects on cognitive dysfunction in plateau hypoxic environments remain unclear. Methods: In this study,acute and chronic plateau cognitive impairment mouse models were constructed to investigate the preventive and therapeutic effects of QJW and its significant active ingredient,diosgenin (Dio). Behavioral experiments were conducted to assess learning and memory in mice. Morphological changes in hippocampal neurons and synapses were assessed,and microglial activation and inflammatory factor levels were measured to evaluate brain damage. Potential active ingredients capable of crossing the blood–brain barrier were identified through chemical composition analysis and network database screening,followed by validation in animal and brain organoid experiments. Transcriptomics analysis,immunofluorescence staining,and molecular docking techniques were employed to explore the underlying mechanisms. Results: QJW significantly enhanced learning and memory abilities in plateau model mice,reduced structural damage to hippocampal neurons,restored NeuN expression,inhibited inflammatory factor levels and microglial activation,and improved hippocampal synaptic damage. Transcriptomics analysis revealed that Dio alleviated hypoxic brain damage and protected cognitive function by regulating the expression of PDE4C. Conclusions: These findings indicate that QJW and its significant active ingredient Dio effectively mitigate hypoxic brain injury and prevent cognitive impairment in high-altitude environments. View Publication

During the first month of pregnancy,the brain and spinal cord are formed through a process called neurulation. However,this process can be altered by low serum levels of folic acid,environmental factors,or genetic predispositions. In 2018,a surveillance study in Botswana,a country with a high incidence of human immunodeficiency virus (HIV) and lacking mandatory food folate fortification programs,found that newborns whose mothers were taking dolutegravir (DTG) during the first trimester of pregnancy had an increased risk of neural tube defects (NTDs). As a result,the World Health Organization and the U.S. Food and Drug Administration have issued guidelines emphasizing the potential risks associated with the use of DTG-based antiretroviral therapies during pregnancy. To elucidate the potential mechanisms underlying the DTG-induced NTDs,we sought to assess the potential neurotoxicity of DTG in stem cell-derived brain organoids. The gene expression of brain organoids developed in the presence of DTG was analyzed by RNA sequencing,Optical Coherence Tomography (OCT),Optical Coherence Elastography (OCE),and Brillouin microscopy. The sequencing data shows that DTG induces the expression of the folate receptor (FOLR1) and modifies the expression of genes required for neurogenesis. The Brillouin frequency shift observed at the surface of DTG-exposed brain organoids indicates an increase in superficial tissue stiffness. In contrast,reverberant OCE measurements indicate decreased organoid volumes and internal stiffness. View Publication

ABSTRACTExtracellular vesicles (EVs) and secretory factors play crucial roles in intercellular communication,but the molecular mechanisms and dynamics governing their interplay in human pluripotent stem cells (hPSCs) are poorly understood. Here,we demonstrate that hPSC?secreted milk fat globule?EGF factor 8 (MFGE?8) is the principal corona protein at the periphery of EVs,playing an essential role in controlling hPSC stemness. MFGE?8 depletion reduced EV?mediated self?renewal and survival in hPSC cultures. MFGE?8 in the EV corona bound to integrin ?v?5 expressed in the peripheral zone of hPSC colonies. It activated cyclin D1 and dynamin?1 via the AKT/GSK3? axis,promoting the growth of hPSCs and facilitating the endocytosis of EVs. Internalization of EVs alleviated oxidative stress and cell death by transporting redox and stress response proteins that increased GSH levels. Our findings demonstrate the critical role of the extracellular association of MFGE?8 and EVs in modulating the self?renewal and survival of hPSCs. View Publication

Central nervous system (CNS) cancers are responsible for high rates of morbidity and mortality worldwide. Malignant CNS tumors such as adult Glioblastoma (GBM) and pediatric embryonal CNS tumors such as medulloblastoma (MED) and atypical teratoid rhabdoid tumors (ATRT) present relevant therapeutic challenges due to the lack of response to classic treatment regimens with radio and chemotherapy. Recent findings on the Zika virus’ (ZIKV) ability to infect and kill CNS neoplastic cells draw attention to the virus’ oncolytic potential. Studies demonstrating the safety of using ZIKV for treating malignant CNS tumors,enabling the translation of this approach to clinical trials,are scarce in the literature. Here we developed a co-culture model of mature human cerebral organoids assembled with GBM,MED or ATRT tumor cells and used these assembloids to test ZIKV oncolytic effect,replication potential and preferential targeting between normal and cancer cells. Our hybrid co-culture models allowed the tracking of tumor cell growth and invasion in cerebral organoids. ZIKV replication and ensuing accumulation in the culture medium was higher in organoids co-cultured with tumor cells than in isolated control organoids without tumor cells. ZIKV infection led to a significant reduction in tumor cell proportion in organoids with GBM and MED cells,but not with ATRT. Tumoroids (3D cultures of tumor cells alone) were efficiently infected by ZIKV. Interestingly,ZIKV rapidly replicated in GBM,MED,and ATRT tumoroids reaching significantly higher viral RNA accumulation levels than co-cultures. Moreover,ZIKV infection reduced viable cells number in MED and ATRT tumoroids but not in GBM tumoroids. Altogether,our findings indicate that ZIKV has greater replication rates in aggressive CNS tumor cells than in normal human cells comprising cerebral organoids. However,such higher ZIKV replication in tumor cells does not necessarily parallels oncolytic effects,suggesting cellular intrinsic and extrinsic factors mediating tumor cell death by ZIKV. View Publication

Mutations in GBA1 encoding the lysosomal enzyme ?-glucocerebrosidase (GCase) are among the most prevalent genetic susceptibility factors for Parkinson’s disease (PD),with 10–30% of carriers developing the disease. To identify genetic modifiers contributing to the incomplete penetrance,we examined the effect of 1634 human transcription factors (TFs) on GCase activity in lysates of an engineered human glioblastoma line homozygous for the pathogenic GBA1 L444P variant. Using an arrayed CRISPR activation library,we uncovered 11 TFs as regulators of GCase activity. Among these,activation of MITF and TFEC increased lysosomal GCase activity in live cells,while activation of ONECUT2 and USF2 decreased it. While MITF,TFEC,and USF2 affected GBA1 transcription,ONECUT2 might control GCase trafficking. The effects of MITF,TFEC,and USF2 on lysosomal GCase activity were reproducible in iPSC-derived neurons from PD patients. Our study provides a systematic approach to identifying modulators of GCase activity and deepens our understanding of the mechanisms regulating GCase. View Publication

Aging of the brain vasculature plays a key role in the development of neurovascular and neurodegenerative diseases,thereby contributing to cognitive impairment. Among other factors,DNA damage strongly promotes cellular aging,however,the role of genomic instability in brain endothelial cells (EC) and its potential effect on brain homeostasis is still largely unclear. We here investigated how endothelial aging impacts blood-brain barrier (BBB) function by using excision repair cross complementation group 1 (ERCC1)-deficient human brain ECs and an EC-specific Ercc1 knock out (EC-KO) mouse model. In vitro,ERCC1-deficient brain ECs displayed increased senescence-associated secretory phenotype expression,reduced BBB integrity,and higher sprouting capacities due to an underlying dysregulation of the Dll4-Notch pathway. In line,EC-KO mice showed more P21+ cells,augmented expression of angiogenic markers,and a concomitant increase in the number of brain ECs and pericytes. Moreover,EC-KO mice displayed BBB leakage and enhanced cell adhesion molecule expression accompanied by peripheral immune cell infiltration into the brain. These findings were confined to the white matter,suggesting a regional susceptibility. Collectively,our results underline the role of endothelial aging as a driver of impaired BBB function,endothelial sprouting,and increased immune cell migration into the brain,thereby contributing to impaired brain homeostasis as observed during the aging process. View Publication