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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

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.

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.

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.

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

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.

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

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.