技术资料

Inherited non-hemolytic anemia is a group of rare bone marrow disorders characterized by erythroid defects. Although concerted efforts have been made to explore the underlying pathogenetic mechanisms of these diseases,the understanding of the causative mutations are still incomplete. Here we identify in a diseased pedigree that a gain-of-function mutation in toll-like receptor 8 (TLR8) is implicated in inherited non-hemolytic anemia. TLR8 is expressed in erythroid lineage and erythropoiesis is impaired by TLR8 activation whereas enhanced by TLR8 inhibition from erythroid progenitor stage. Mechanistically,TLR8 activation blocks annexin A2 (ANXA2)-mediated plasma membrane localization of STAT5 and disrupts EPO signaling in HuDEP2 cells. TLR8 inhibition improves erythropoiesis in RPS19+/? HuDEP2 cells and CD34+ cells from healthy donors and inherited non-hemolytic anemic patients. Collectively,we identify a gene implicated in inherited anemia and a previously undescribed role for TLR8 in erythropoiesis,which could potentially be explored for therapeutic benefit in inherited anemia. Decoding the pathogenic genes of the inherited anemia could provide us with novel regulators of pathological and physiological erythropoiesis. Here,the authors show TLR8 is expressed by erythroid cells and regulates erythropoiesis through interacting with EPO signaling. View Publication

Stem cell-derived ?-cells (SC-BCs) represent a potential source for curing diabetes. To date,in vitro generated SC-BCs display an immature phenotype and lack important features in comparison to their bona-fide counterparts. Transplantation into a living animal promotes SC-BCs maturation,indicating that components of the in vivo microenvironment trigger final SC-BCs development. Here,we investigated whether cues of the pancreas specific extracellular matrix (ECM) can improve the differentiation of human induced pluripotent stem cells (hiPSCs) towards ?-cells in vitro. To this aim,a pancreas specific ECM (PanMa) hydrogel was generated from decellularized porcine pancreas and its effect on the differentiation of hiPSC-derived pancreatic hormone expressing cells (HECs) was tested. The hydrogel solidified upon neutralization at 37 °C with gelation kinetics similar to Matrigel. Cytocompatibility of the PanMa hydrogel was demonstrated for a culture duration of 21 days. Encapsulation and culture of HECs in the PanMa hydrogel over 7 days resulted in a stable gene and protein expression of most ?-cell markers,but did not improve ?-cell identity. In conclusion,the study describes the production of a PanMa hydrogel,which provides the basis for the development of ECM hydrogels that are more adapted to the demands of SC-BCs. View Publication

The primed epiblast acts as a transitional stage between the relatively homogeneous naïve epiblast and the gastrulating embryo. Its formation entails coordinated changes in regulatory circuits driven by transcription factors and epigenetic modifications. Using a multi-omic approach in human embryonic stem cell models across the spectrum of peri-implantation development,we demonstrate that the transcription factors ZIC2 and ZIC3 have overlapping but essential roles in opening primed-specific enhancers. Together,they are essential to facilitate progression to and maintain primed pluripotency. ZIC2/3 accomplish this by recruiting SWI/SNF to chromatin and loss of ZIC2/3 or degradation of SWI/SNF both prevent enhancer activation. Loss of ZIC2/3 also results in transcriptome changes consistent with perturbed Polycomb activity and a shift towards the expression of genes linked to differentiation towards the mesendoderm. Additionally,we find an intriguing dependency on the transcriptional machinery for sustained recruitment of ZIC2/3 over a subset of primed-hESC specific enhancers. Taken together,ZIC2 and ZIC3 regulate highly dynamic lineage-specific enhancers and collectively act as key regulators of human primed pluripotency. Here the authors identify ZIC2 and ZIC3 as key regulators of human primed pluripotency which recruit BRG1 to open primed hESC-specific enhancers. 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

IntroductionNeuroinflammation is a key contributor to the pathogenesis of Alzheimer's disease (AD),and impaired clearance of amyloid-? (A?) by microglia is closely associated with disease progression. Oxytocin (OXT),a hypothalamic neuropeptide,has recently been reported to exert anti-inflammatory effects on microglia; however,its therapeutic potential in the human brain remains unclear.MethodsWe generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) to model early AD-like pathology. A? toxicity was induced by applying 3 ?M A?1–42 for 48 h. The protective effects of OXT were evaluated through immunohistochemistry,RT-qPCR,calcium imaging,and multielectrode array (MEA) recordings. The involvement of microglia in A? clearance was assessed by immunostaining and gene expression analysis of TREM2.ResultsA? exposure led to significant deposition of A? in the outer layers of hCOs,accompanied by suppressed neural activity and increased apoptotic signaling. Pretreatment with OXT attenuated A? deposition and caspase-3-mediated apoptosis in a concentration-dependent manner. OXT also restored calcium oscillations and neuronal network activity as measured by MEA. Notably,OXT enhanced the recruitment of microglia to A? deposits and upregulated the expression of TREM2,a key regulator of microglial phagocytosis. Co-expression of oxytocin receptors (OXTR) on Iba1-positive microglia suggests that OXT directly modulates microglial activation and A? clearance.ConclusionsOXT has neuroprotective effects on human cortical organoids by preserving their neuronal activity and promoting microglial-mediated A? clearance. This study provides novel insights into the therapeutic potential of OXT for targeting neuroinflammation and A? pathology in patients with AD. Graphical abstractImage 1 Highlights•Oxytocin reduces A? deposition and apoptosis in human cerebral organoids.•A? impairs neuronal activity,rescued by oxytocin preconditioning.•Oxytocin enhances microglial phagocytosis via OXTR and TREM2 upregulation.•Human iPSC-derived organoids model early A? pathology and oxytocin response. 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

Background: Phenotypically unstable Schwann cell-like cells (SCLCs),derived from mesenchymal stem cells (MSCs) require intercellular contact-mediated cues for Schwann cell (SCs)-fate commitment. Although rat dorsal root ganglion (DRG) neurons provide contact-mediated signals for the conversion of SCLCs into fate-committed SCs,the use of animal cells is clinically unacceptable. To overcome this problem,we previously acquired human induced pluripotent stem cell-derived sensory neurons (hiPSC-dSNs) as surrogates of rat DRG neurons that committed rat bone marrow SCLCs to the SC fate. In this study,we explored whether hiPSC-dSNs could mimic rat DRG neuron effects to obtain fate-committed SCs from hBMSC-derived SCLCs. Methods: hiPSCs were induced into hiPSC-dSNs using a specific chemical small molecule combination. hBMSCs were induced into hBMSC-derived SCLCs in a specific culture medium and then co-cultured with hiPSC-dSNs to generate SCs. The identity of hBMSC-derived SCs (hBMSC-dSCs) was examined by immunofluorescence,western bolt,electronic microscopy,and RNA-seq. Immunofluorescence was also used to detect the myelination capacity. Enzyme-linked immunosorbent assay and neurite outgrowth analysis were used to test the secretion of neurotrophic factors. Results: The hBMSC-dSCs exhibited bi-/tri-polar morphology of SCs and maintained the expression of the SC markers S100,p75NTR,p0,GFAP,and Sox10,even after withdrawing the glia-inducing factors or hiPSC-dSNs. Electronic microscopy and RNA-seq analysis provided evidence that hBMSC-dSCs were similar to the original human SCs in terms of their function and a variety of characteristics. Furthermore,these cells formed MBP-positive segments and secreted neurotrophic factors to facilitate the neurite outgrowth of Neuro2A. Conclusions: These results demonstrated that phenotypically stable and functionally mature hBMSC-dSCs were generated efficiently via the co-culture of hiPSC-dSNs and hBMSC-derived SCLCs. Our findings may provide a promising protocol through which stable and fully developed hBMSC-dSCs can be used for transplantation to regenerate myelin sheath. 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

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here,to understand the pathological mechanisms of Kd,we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis,astrogenesis,and oligodendrogenesis but manifest early myelination defects. Specifically,Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly,myelin is affected in the absence of autophagy and mTOR pathway dysregulation,suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions,Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding,Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether,this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd. 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

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

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