技术资料

Quantification of subcellular structures such as nuclei and cytoplasmic proteins using staining methods based on fluorescent dyes or fluorescently tagged antibodies are widely used in scientific research. Accurate high-throughput quantitation of these assays can be time consuming and challenging. Here,we present our FIJI based Semi-Automated counting Macro termed SAM,and we validate its accuracy against manual counting and other automated counting methods. By introducing this automated quantification tool,we aim to contribute to the ongoing efforts to enhance the reliability,efficiency,and standardization of immunostaining analysis in the field of diabetes research and beyond.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-12144-x. View Publication

BackgroundGenome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool,but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore,genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations,but not in single cells. However,systematic profiling of genome editing outcomes in single iPS cells was lacking. Due to the high mortality of human iPS cells as isolated single cells,it has been difficult to analyze genome-edited iPS cell clones in a high-throughput manner.MethodsIn this study,we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We first introduced point mutations into human iPS cell pools by CRISPR-Cas9. These genome-edited human iPS cells were dissociated and cultured as single cells in extracellular matrices to form cell clumps,which were then isolated using a cell-handling robot to establish genome-edited human iPS cell clones. Genome editing outcomes in these clones were analyzed by amplicon sequencing to determine the genotypes of individual iPS cell clones. We identified and distinguished the sequences of different insertions and deletions induced by CRISPR-Cas9 while determining their genotypes. We also cryopreserved the established iPS cell clones and recovered them after determining their genotypes.ResultsWe analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations,including insertions and deletions,such as 1-bp insertions and 8-bp deletions. Moreover,we successfully cryopreserved and then recovered genome-edited iPS cell clones,demonstrating that our cell-handling robot-based method is valuable in establishing genome-edited iPS cell clones.ConclusionsThis study revealed a previously unknown property of genome editing in human iPS cells that identical sequence manipulations tend to be induced in both copies of the target sequence in individual cells. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04414-2. View Publication

The neuromuscular junction (NMJ) is essential for transmitting signals from motor neurons (MNs) to skeletal muscles (SKMs),and its dysfunction can lead to severe motor disorders. However,our understanding of the NMJ is limited by the absence of accurate human models. Although human induced pluripotent stem cell (iPSC)-derived models have advanced NMJ research,their application is constrained by challenges such as limited differentiation efficiency,lengthy generation times,and cryopreservation difficulties. To overcome these limitations,we developed a rapid human NMJ model using cryopreserved MNs and SKMs derived from iPSCs. Within 12 days of coculture,we successfully recreated NMJ-specific connectivity that closely mirrors in vivo synapse formation. Using this model,we investigated amyotrophic lateral sclerosis (ALS) and replicated ALS-specific NMJ cytopathies with SOD1 mutant and corrected isogenic iPSC lines. Quantitative analysis of 3D confocal microscopy images revealed a critical role of MNs in initiating ALS-related NMJ cytopathies,characterized by alterations in the volume,number,intensity,and distribution of acetylcholine receptors,ultimately leading to impaired muscle contractions. Our rapid and precise in vitro NMJ model offers significant potential for advancing research on NMJ physiology and pathology,as well as for developing treatments for NMJ-related diseases. View Publication

In this study,we developed a cell system to study TCF4 in human oligodendrocyte differentiation,showed that TCF4 regulates human oligodendroglial differentiation in a dose-dependent manner,and established a system to dissect TCF4 function in a human tissue–like context. Heterozygous mutations of TCF4 in humans cause Pitt–Hopkins syndrome,a neurodevelopmental disease associated with intellectual disability and brain malformations. Although most studies focus on the role of TCF4 in neural stem cells and neurons,we here set out to assess the implication of TCF4 for oligodendroglial differentiation. We discovered that both monoallelic and biallelic mutations in TCF4 result in a diminished capacity to differentiate human neural progenitor cells toward myelinating oligodendrocytes through the forced expression of the transcription factors SOX10,OLIG2,and NKX6.2. Using this experimental strategy,we established a novel organoid model,which generates oligodendroglial cells within a human neurogenic tissue–like context. Also,here we found a reduced ability of TCF4 heterozygous cells to differentiate toward oligodendroglial cells. In sum,we establish a role of human TCF4 in oligodendrocyte differentiation and provide a model system,which allows to dissect the disease etiology in a human tissue–like context. View Publication

Stem cell-derived islet cell therapy can effectively treat type 1 diabetes,but its efficacy is hindered by low oxygen supply post-transplantation,particularly in subcutaneous spaces and encapsulation devices,leading to cell dysfunction. The response to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. Here,we show that ? cells within stem cell-derived islets gradually undergo a decline in cell identity and metabolic function in hypoxia. This is linked to reduced expression of immediate early genes (EGR1,FOS,and JUN),which downregulates key ? cell transcription factors. We further identified genes important for maintaining ? cell fitness in hypoxia,with EDN3 as a potent player. Elevated EDN3 expression preserves ? cell identity and function in hypoxia by modulating genes involved in ? cell maturation,glucose sensing and regulation. These insights improve the understanding of hypoxia’s impact on stem cell-derived islets,offering a potential intervention for clinical applications. Hypoxia impairs the efficacy of stem cell-derived islet cell therapy,making it a potential barrier for treatment of type 1 diabetes. Wang et al. identify EDN3 as a key factor that preserves ? cell identity and function in hypoxia,offering possible strategies to improve therapeutic outcomes. View Publication

Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present with skeletal abnormalities and 75% of them have sex reversal. In this study,we use CRISPR/Cas9 to generate a human induced pluripotent stem cell (hiPSC) model from a heathy male donor,based on a previously reported SOX9 splice site mutation in a CD patients. This hiPSCs-derived chondrocytes from heterozygotes (HT) and homozygotes (HM) SOX9 mutation carriers showed significant defects in chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway,ribosome-related,and chromosome segregation-related gene sets were altered in the HT chondrocytes. The profile also showed significant noggin upregulation in CD chondrocytes,with ChIP-qPCR confirming that SOX9 binds to the distal regulatory element of noggin. This suggests SOX9 plays a feedback role in the BMP signaling pathway by modulating noggin expression rather than acting solely as a downstream regulator. This provides further insights into its dosage sensitivity in chondrogenesis. Overexpression of SOX9 showed promising results with improved sulfated glycosaminoglycans (GAGs) aggregation and COL2A1 expression following differentiation. We hope this finding could provide a better understanding of the dosage-dependent role of SOX9 in chondrogenesis and contribute to the development of improved therapeutic targets for CD patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05622-y. View Publication

Angelman syndrome is a neurodevelopmental disorder caused by (epi)genetic lesions of maternal UBE3A. Research has focused largely on the role of UBE3A in neurons due to its imprinting in that cell type. Yet,evidence suggests there may be broader neurodevelopmental impacts of UBE3A dysregulation. Human cerebral organoids might reveal these understudied aspects of UBE3A as they recapitulate diverse cell types of the developing human brain. In this study,scRNAseq on organoids reveals the effects of UBE3A disruption on cell type-specific compositions and transcriptomic alterations. In the absence of UBE3A,progenitor proliferation and structures are disrupted while organoid composition shifts away from proliferative cell types. We observe impacts on non-neuronal cells,including choroid plexus enrichment. Furthermore,EMX1+ cortical progenitors are negatively impacted; potentially disrupting corticogenesis and delaying excitatory neuron maturation. This work reveals impacts of UBE3A on understudied cell types and related neurodevelopmental processes and elucidates potential therapeutic targets. Human cerebral organoids exhibit compositional and transcriptomic alterations in both neuronal and non-neuronal cells in the absence of UBE3A. View Publication