技术资料

Human pluripotent stem cells (hPSCs) have the potential to differentiate into various cell types,including pancreatic insulin-producing β cells,which are crucial for developing therapies for diabetes. However,current methods for directing hPSC differentiation towards pancreatic β-like cells are often inefficient and produce cells that do not fully resemble the native counterparts. Here,we report that highly selective tankyrase inhibitors,such as WIKI4,significantly enhances pancreatic differentiation from hPSCs. Our results show that WIKI4 promotes the formation of pancreatic progenitors that give rise to islet-like cells with improved β-like cell frequencies and glucose responsiveness compared to our standard cultures. These findings not only advance our understanding of pancreatic development,but also provide a promising new tool for generating pancreatic cells for research and potential therapeutic applications. Subject terms: Stem-cell differentiation,Organogenesis,Type 1 diabetes View Publication

In Multiple Sclerosis (MS),inflammatory demyelinated lesions in the brain and spinal cord lead to neurodegeneration and progressive disability. Remyelination can restore fast saltatory conduction and neuroprotection but is inefficient in MS especially with increasing age,and is not yet treatable with therapies. Intrinsic and extrinsic inhibition of oligodendrocyte progenitor cell (OPC) function contributes to remyelination failure,and we hypothesised that the transplantation of ‘improved’ OPCs,genetically edited to overcome these obstacles,could improve remyelination. Here,we edit human(h) embryonic stem cell-derived OPCs to be unresponsive to a chemorepellent released from chronic MS lesions,and transplant them into rodent models of chronic lesions. Edited hOPCs display enhanced migration and remyelination compared to controls,regardless of the host age and length of time post-transplant. We show that genetic manipulation and transplantation of hOPCs overcomes the negative environment inhibiting remyelination,with translational implications for therapeutic strategies for people with progressive MS. Subject terms: Multiple sclerosis,Multiple sclerosis,Regeneration 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

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPCs),but they have many outstanding concerns. Specifically,their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging,a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally,this process results in the packaging of complete viral particles only containing rAAV genomes; however,this is not the case. Through this study,we were able to leverage single-stranded virus (SSV) sequencing,a next-generation sequencing-based method to quantify all DNA species present within rAAV preps. From this,it was determined that much of the DNA within some rAAV preps is not vector-genome derived,and there is wide variability in the contamination by DNA across various preps. Furthermore,we demonstrate that transducing CD34 + HSPCs with preps with higher contaminating DNA resulted in decreased clonogenic potential,altered transcriptomic profiles,and decreased genomic editing. Collectively,this study characterized the effects of DNA contamination within rAAV preps on CD34 + HSPC cellular potential. View Publication

Extracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising,natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture,human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However,hiPSC-EVs only have a moderate ability for brain delivery. Herein,we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor. An RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging. Here we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype,express pluripotency markers,and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways,compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency. Taken together,we have established stable,genetically engineered hiPSCs for producing EVs with RVG expression,offering the improved ability for brain-targeted drug delivery. The online version contains supplementary material available at 10.1186/s13287-024-03955-2. View Publication

Aging is characterized by the accumulation of proteins that display amyloid-like behavior. However,the molecular mechanisms by which these proteins arise remain unclear. Here,we demonstrate that amyloid-like proteins are produced in a variety of human cell types,including stem cells,brain organoids and fully differentiated neurons by mistakes that occur in messenger RNA molecules. Some of these mistakes generate mutant proteins already known to cause disease,while others generate proteins that have not been observed before. Moreover,we show that these mistakes increase when cells are exposed to DNA damage,a major hallmark of human aging. When taken together,these experiments suggest a mechanistic link between the normal aging process and age-related diseases. Subject terms: Protein aggregation,Mechanisms of disease,Transcription View Publication

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here,we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data,FN mutations disrupted protein secretion from MSCs,causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP,HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER. Massive amounts of these vesicles were not cleared from the cytosol,and a smaller subset showed the presence of lysosomal markers. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation,reduced chondrogenic markers,and compromised cell proliferation in mutant cells. Moreover,FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGFβ1) expression,crucial for mesenchymal condensation. Exogenous FN or TGFβ1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis. The online version contains supplementary material available at 10.1007/s00018-024-05444-4. View Publication

In calcium imaging studies,Ca 2+ transients are commonly interpreted as neuronal action potentials (APs). However,our findings demonstrate that robust optical Ca 2+ transients primarily stem from complex “AP-Plateaus”,while simple APs lacking underlying depolarization envelopes produce much weaker photonic signatures. Under challenging in vivo conditions,these “AP-Plateaus” are likely to surpass noise levels,thus dominating the Ca 2+ recordings. In spontaneously active neuronal culture,optical Ca 2+ transients (OGB1-AM,GCaMP6f) exhibited approximately tenfold greater amplitude and twofold longer half-width compared to optical voltage transients (ArcLightD). The amplitude of the ArcLightD signal exhibited a strong correlation with the duration of the underlying membrane depolarization,and a weaker correlation with the presence of a fast sodium AP. Specifically,ArcLightD exhibited robust responsiveness to the slow “foot” but not the fast “trunk” of the neuronal AP. Particularly potent stimulators of optical signals in both Ca 2+ and voltage imaging modalities were APs combined with plateau potentials (AP-Plateaus),resembling dendritic Ca 2+ spikes or “UP states” in pyramidal neurons. Interestingly,even the spikeless plateaus (amplitude > 10 mV,duration > 200 ms) could generate conspicuous Ca 2+ optical signals in neurons. Therefore,in certain circumstances,Ca 2+ transients should not be interpreted solely as indicators of neuronal AP firing. Subject terms: Biological techniques,Biophysics,Neuroscience,Physiology 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

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

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance,they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors,which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT),a well-known checkpoint receptor involved in T-cell exhaustion,has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here,we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability,are rapidly and efficiently taken up by cells,and can be easily incorporated into cell product manufacturing. INTASYL PH-804,which targets TIGIT,suppresses TIGIT mRNA and protein expression in NK cells,resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition,thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy,particularly for hematological malignancies. The online version contains supplementary material available at 10.1007/s00262-024-03835-x. View Publication

Mutation of tet methylcytosine dioxygenase 2 (encoded by TET2 ) drives myeloid malignancy initiation and progression 1 – 3 . TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant haematopoietic stem cell self-renewal 4,5 . However,the open chromatin observed in TET2-deficient mouse embryonic stem cells,leukaemic cells and haematopoietic stem and progenitor cells 5 is inconsistent with the designated role of DNA 5-methylcytosine oxidation of TET2. Here we show that chromatin-associated retrotransposon RNA 5-methylcytosine (m 5 C) can be recognized by the methyl-CpG-binding-domain protein MBD6,which guides deubiquitination of nearby monoubiquitinated Lys119 of histone H2A (H2AK119ub) to promote an open chromatin state. TET2 oxidizes m 5 C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. TET2 depletion thereby leads to globally decreased H2AK119ub,more open chromatin and increased transcription in stem cells. TET2- mutant human leukaemia becomes dependent on this gene activation pathway,with MBD6 depletion selectively blocking proliferation of TET2 -mutant leukaemic cells and largely reversing the haematopoiesis defects caused by Tet2 loss in mouse models. Together,our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m 5 C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies. Subject terms: Molecular biology,Chromatin View Publication