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BackgroundThioredoxin-interacting protein (TXNIP) plays a role in regulating endoplasmic reticulum (ER) and oxidative stress,which disrupt glucose homeostasis in diabetes. However,the impact of TXNIP deficiency on the differentiation and functionality of human stem cell-derived somatic metabolic cells remains unclear.MethodsWe used CRISPR-Cas12a genome editing to generate TXNIP-deficient (TXNIP?/?) H1 human embryonic stem cells (H1-hESCs). These cells were differentiated into hepatocyte-like cells (HLCs) and stem-cell-derived insulin-producing islets (SC-islets). The maturation and functionality TXNIP?/? and TXNIP+/+ SC-islets were assessed by implantation under the kidney capsule of male or female NOD-SCID mice.ResultsTXNIP deficiency significantly increased H1-hESC proliferation without affecting pluripotency,viability,or differentiation potential into HLCs and SC-islets. Bulk RNA-sequencing of thapsigargin-treated TXNIP?/? and TXNIP+/+ hESCs revealed differential expression of stress-responsive genes,with enriched apoptosis-related pathways in TXNIP+/+ cells,but minimal transcriptional changes specific to TXNIP deficiency. In HLCs,TXNIP deletion reduced albumin secretion and insulin signalling,as indicated by decreased AKT phosphorylation,while showing no differences in glycolytic activity or lipid metabolism markers. Under thapsigargin-induced ER stress,TXNIP?/? HLCs exhibited transiently reduced eIF2? phosphorylation and lower BiP expression,suggesting compromised adaptive responses to prolonged stress. SC-islets derived from TXNIP?/? hESCs showed comparable viability,endocrine cell composition,and cytokine responses to TXNIP+/+ islets. Following IFN? or IFN? treatment,STAT1 phosphorylation was increased in TXNIP?/? SC-islets,indicating that IFN signalling remained intact despite TXNIP deficiency. Upon implantation into NOD-SCID mice,both TXNIP?/? and TXNIP+/+ SC-islets produced human C-peptide and responded to glucose stimulation. However,TXNIP?/? SC-islets did not demonstrate enhanced glycaemic control or glucose-stimulated insulin secretion compared to controls.ConclusionsOur study demonstrates that TXNIP deficiency does not improve the differentiation or functionality of HLCs and SC-islets. We present the generation and characterisation of TXNIP?/? and TXNIP+/+ H1-hESCs,HLCs,and SC-islets as valuable models for future studies on the role of TXNIP in metabolic cell biology.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04314-5. View Publication

MicroRNAs (miRNAs) are small non-coding RNAs that play crucial roles in post-transcriptional gene regulation. Poly(A) RNA polymerase D5 (PAPD5) catalyzes the addition of adenosine to the 3? end of miRNAs. In this study,we demonstrate that the Yin Yang 1 protein,a transcriptional repressor of PAPD5,is recruited to both RNA foci and protein aggregates,resulting in an upregulation of PAPD5 expression in Huntington’s disease (HD). Additionally,we identify a subset of PAPD5-regulated miRNAs with increased adenylation and reduced expression in our disease model. We focus on miR-7-5p and find that its reduction causes the activation of the TAB2-mediated TAK1–MKK4–JNK pro-apoptotic pathway. This pathway is also activated in induced pluripotent stem cell-derived striatal neurons and post-mortem striatal tissues isolated from HD patients. In addition,we discover that a small molecule PAPD5 inhibitor,BCH001,can mitigate cell death and neurodegeneration in our disease models. This study highlights the importance of PAPD5-mediated miRNA dysfunction in HD pathogenesis and suggests a potential therapeutic direction for the disease. PAPD5 is responsible for adenylation of microRNAs. Here,the authors show that elevated level of PAPD5 enhances the adenylation and reduced expression of miR-7-5p. As a result,expression of TAB2,a target of miR-7-5p,is induced triggering neuronal apoptosis in Huntington’s disease. View Publication

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

BackgroundOP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However,the whole co-culture procedure usually needs 14–18 days,including preparing OP9 cells at least 4 days. Therefore,the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency.MethodsIn the experimental group,we set six different densities of OP9 cells and just cultured them for 24 h before co-culture,and in the control group,OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them.ResultsOP9 cells were randomly assigned into two groups. In the experimental group,six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast,in the control group,OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining,and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day,followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group.ConclusionThe peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency. View Publication

The 22q11.2 deletion syndrome (22qDS) is a human disorder where the majority of clinical manifestations originate during embryonic development. 22qDS is caused by a microdeletion in one chromosome 22,including DGCR8,an essential gene for microRNA (miRNA) production. However,the impact of DGCR8 hemizygosity on human development is still unclear. In this study,we generated two human pluripotent cell models containing a single functional DGCR8 allele to elucidate its role in early development. DGCR8+/? human embryonic stem cells (hESCs) showed increased apoptosis as well as self-renewal and differentiation defects in both the naïve and primed states. The expression of primate-specific miRNAs was largely affected,due to impaired miRNA processing and chromatin accessibility. DGCR8+/? hESCs also displayed a pronounced reduction in human endogenous retrovirus class H (HERVH) expression,a primate-specific retroelement essential for pluripotency maintenance. The reintroduction of miRNAs belonging to the primate-specific C19MC cluster as well as the miR-371-3 cluster rescued the defects of DGCR8+/? cells. Mechanistically,downregulation of HERVH by depletion of primate-specific miRNAs was mediated by KLF4. Altogether,we show that DGCR8 is haploinsufficient in humans and that miRNAs and transposable elements may have co-evolved in primates as part of an essential regulatory network to maintain stem cell identity. View Publication

Biallelic mutations in the DCPS gene disrupting the decapping activity of the scavenger decapping protein DcpS,leads to neurodevelopmental deficiencies and intellectual disability. However,the molecular basis for the neurogenesis defects in these individuals remains unknown. Here we show that cells derived from individuals with a DCPS mutation harbor a creatine deficiency and a corresponding elevation of the creatine precursor,guanidinoacetate (GAA). The altered metabolite levels are a consequence of a reduction in both the mRNA and protein levels for the enzyme that converts GAA into creatine,guanidinoacetate methyltransferase. Importantly,the compromised neurogenesis and neurite outgrowth phenotypes observed during the differentiation of DcpS mutant patient derived induced pluripotent stem cells into neurons was reversed upon supplementation of creatine monohydrate. These findings suggest creatine deficiency as an underlying factor for the neurogenetic defect detected in DcpS mutant cells and a potential driver of the neurological deficiencies in affected individuals. View Publication

Label-free detection of biological events at single-cell resolution in the brain can non-invasively capture brain status for medical diagnosis and basic neuroscience research. NADH is an universal coenzyme that not only plays a central role in cellular metabolism but may also be used as a biomarker to capture metabolic processes in brain cells and structures. We have developed a new label-free,multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H in living cells. The imaging depth of NAD(P)H in tissues with all-optical methods is limited to ~100??m in brain tissue by the strong absorption of the near-ultraviolet fluorescence. Here,acoustic detection of the thermal signature of multi-photon (three-photon) excitation of NAD(P)H,a low quantum yield fluorophore,allows detection at an unprecedented depth while the focused excitation ensures high spatial resolution. We validated the photoacoustic detection of NAD(P)H by monitoring an increase in intracellular NAD(P)H in HEK293T cells and HepG2 cells incubated in NADH solution. We also demonstrated the detection of endogenous NAD(P)H photoacoustic signals in brain slices to 700 ?m depth and in cerebral organoids to 1100 ?m depth. Finally,we developed and demonstrated simultaneous photoacoustic and optical imaging of NAD(P)H in brain cells with a real-time image acquisition and processing pipeline. This approach could open a new door to monitor brain metabolic changes during development and disease,and changes due to neuronal activity,at single-cell level deep in the brains of both humans and animals. Label-free,multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H of neurons in brain slices and cerebral organoids. View Publication

Fabry disease (FD) is a lysosomal storage disorder in which ?-galactosidase (GLA) deficiency leads to a build-up of globo-triaosylceramide (Gb3) in various cell types. Gb3 accumulation leads to the abnormalities of microvascular function associated with FD. Previously,we discovered significant abnormalities in vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells. We then used a cell-based system to screen a group of clinical compounds for candidates capable of rescuing those abnormalities. Lomerizine was one of the most promising candidates because it alleviated a variety of FD-associated phenotypes both in vitro and in vivo. Lomerizine reduced mitochondria Ca2+?levels,ROS generation,and the maximal respiration of FD-VECs in vitro. This led to a suppression of the endothelial-to-mesenchymal transition (EndMT) and rescued FD-VEC function. Furthermore,FD-model mice (Gla?/?/TSP1Tg) treated orally with lomerizine for 6 months showed clear improvement of several FD phenotypes,including left ventricular hypertrophy,renal fibrosis,anhidrosis,and heat intolerance. Thus,our results suggest lomerizine as a novel candidate for FD therapy. 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