技术资料

Human pluripotent stem cells (hPSCs) represent a powerful model system to study early developmental processes. However,lineage specification into trophectoderm (TE) and trophoblast (TB) differentiation remains poorly understood,and access to well-characterized placental cells for biomedical research is limited,largely depending on fetal tissues or cancer cell lines. Here,we developed novel strategies enabling highly efficient TE specification that generates cytotrophoblast (CTB) and multinucleated syncytiotrophoblast (STB),followed by the establishment of trophoblast stem cells (TSCs) capable of differentiating into extravillous trophoblast (EVT) and STB after long-term expansion. We confirmed stepwise and controlled induction of lineage- and cell-type-specific genes consistent with developmental biology principles and benchmarked typical features of placental cells using morphological,biochemical,genomics,epigenomics,and single-cell analyses. Charting a well-defined roadmap from hPSCs to distinct placental phenotypes provides invaluable opportunities for studying early human development,infertility,and pregnancy-associated diseases. Subject areas: Natural sciences,Biological sciences,Cell biology,Stem cells research View Publication

Despite significant progress in the prognosis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) in recent decades,a notable portion of children still confronts challenges such as treatment resistance and recurrence,leading to limited options and a poor prognosis. LIM domain-binding protein 1 (LDB1) has been confirmed to exert a crucial role in various physiological and pathological processes. In our research,we aim to elucidate the underlying function and mechanisms of LDB1 within the background of T-ALL. Employing short hairpin RNA (shRNA) techniques,we delineated the functional impact of LDB1 in T-ALL cell lines. Through the application of RNA-Seq,CUT&Tag,and immunoprecipitation assays,we scrutinized master transcription factors cooperating with LDB1 and identified downstream targets under LDB1 regulation. LDB1 emerges as a critical transcription factor co-activator in cell lines derived from T-ALL. It primarily collaborates with master transcription factors (ERG,ETV6,IRF1) to cooperatively regulate the transcription of downstream target genes. Both in vitro and in vivo experiments affirm the essential fuction of LDB1 in the proliferation and survival of cell lines derived from T-ALL,with MYB identified as a significant downstream target of LDB1. To sum up,our research establishes the pivotal fuction of LDB1 in the tumorigenesis and progression of T-ALL cell lines. Mechanistic insights reveal that LDB1 cooperates with ERG,ETV6,and IRF1 to modulate the expression of downstream effector genes. Furthermore,LDB1 controls MYB through remote enhancer modulation,providing valuable mechanistic insights into its involvement in the progression of T-ALL. The online version contains supplementary material available at 10.1186/s13046-024-03199-1. View Publication

CRISPR-based genome editing of pseudogene-associated disorders,such as p47 phox -deficient chronic granulomatous disease (p47 CGD),is challenged by chromosomal rearrangements due to presence of multiple targets. We report that interactions between highly homologous sequences that are localized on the same chromosome contribute substantially to post-editing chromosomal rearrangements. We successfully employed editing approaches at the NCF1 gene and its pseudogenes,NCF1B and NCF1C,in a human cell line model of p47 CGD and in patient-derived human hematopoietic stem and progenitor cells. Upon genetic engineering,a droplet digital PCR-based method identified cells with altered copy numbers,spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context,such as the presence of homologous regions,whose instability can constitute a risk factor for chromosomal rearrangements upon genome editing. Subject terms: CRISPR-Cas9 genome editing,Targeted gene repair,Haematopoietic stem cells 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

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

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

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