技术资料

In Finland,the frequency of isolated cleft palate (CP) is higher than that of isolated cleft lip with or without cleft palate (CL/P). This trend contrasts to that in other European countries but its genetic underpinnings are unknown. We conducted a genome-wide association study in the Finnish population and identified rs570516915,a single nucleotide polymorphism highly enriched in Finns,as strongly associated with CP (P?=?5.25 × 10?34,OR?=?8.65,95% CI 6.11–12.25),but not with CL/P (P?=?7.2 × 10?5),with genome-wide significance. The risk allele frequency of rs570516915 parallels the regional variation of CP prevalence in Finland,and the association was replicated in independent cohorts of CP cases from Finland (P?=?8.82 × 10?28) and Estonia (P?=?1.25 × 10?5). The risk allele of rs570516915 alters a conserved binding site for the transcription factor IRF6 within an enhancer (MCS-9.7) upstream of the IRF6 gene and diminishes the enhancer activity. Oral epithelial cells derived from CRISPR-Cas9 edited induced pluripotent stem cells demonstrate that the CP-associated allele of rs570516915 concomitantly decreases the binding of IRF6 and the expression level of IRF6,suggesting impaired IRF6 autoregulation as a molecular mechanism underlying the risk for CP. Here,the authors perform a genome-wide study and identify a genetic variant enriched in the Finnish population that is strongly associated with isolated cleft palate. This finding suggests a genetic basis for the high prevalence of cleft palate in Finland. View Publication

Rationale: Brain pericytes can acquire multipotency to produce multi-lineage cells following injury. However,pericytes are a heterogenous population and it remains unknown whether there are different potencies from different subsets of pericytes in response to injury.Methods: We used an ischemic stroke model combined with pericyte lineage tracing animal models to investigate brain pericyte heterogeneity under both naïve and brain injury conditions via single-cell RNA-sequencing and immunohistochemistry analysis. In addition,we developed an NG2+ pericyte neural reprogramming culture model from both murine and humans to unveil the role of energy sensor,AMP-dependent kinase (AMPK),activity in modulating the reprogramming/differentiation process to convert pericytes to functional neurons by targeting a Ser 436 phosphorylation on CREB-binding protein (CBP),a histone acetyltransferase.Results: We showed that two distinct pericyte subpopulations,marked by NG2+ and Tbx18+,had different potency following brain injury. NG2+ pericytes expressed dominant neural reprogramming potential to produce newborn neurons,while Tbx18+ pericytes displayed dominant multipotency to produce endothelial cells,fibroblasts,and microglia following ischemic stroke. In addition,we discovered that AMPK modulators facilitated pericyte-to-neuron conversion by modulating Ser436 phosphorylation status of CBP,to coordinate an acetylation shift between Sox2 and histone H2B,and to regulate Sox2 nuclear-cytoplasmic trafficking during the reprogramming/differentiation process. Finally,we showed that sequential treatment of compound C (CpdC) and metformin,AMPK inhibitor and activator respectively,robustly facilitated the conversion of human pericytes into functional neurons.Conclusion: We revealed that two distinct subtypes of pericytes possess different reprogramming potencies in response to physical and ischemic injuries. We also developed a genomic integration-free methodology to reprogram human pericytes into functional neurons by targeting NG2+ pericytes. View Publication

Extraembryonic mesoderm (ExM) is crucial for human development,yet its specification is poorly understood. Human embryonic stem cell (hESC)-based models,including embryoids and differentiated derivatives,are emerging as promising tools for studying ExM development. Despite this,the signaling mechanisms and developmental dynamics that underlie ExM specification from hESCs remain challenging to study. Here,we report that the modulation of BMP,WNT,and Nodal signaling pathways can rapidly (4-5 days) and efficiently (?~90%) induce the differentiation of both naive and primed hESCs into ExM-like cells (ExMs). We reveal that ExM specification from hESCs predominantly proceeds through intermediates exhibiting a primitive streak (PS)-like gene expression pattern and delineate the regulatory roles of WNT and Nodal signaling in this process. Furthermore,we find that the initial pluripotent state governs hESC-based ExM specification by influencing signal response,cellular composition,developmental progression,and transcriptional characteristics of the resulting ExMs. Our study provides promising models for dissecting human ExM development and sheds light on the signaling principles,developmental dynamics,and influences of pluripotency states underlying ExM specification from hESCs. Extraembryonic mesoderm (ExM) is crucial but its formation is unclear. Here,authors develop efficient systems to specify ExM from hESCs and dissect the signaling mechanisms,specification dynamics,and impact of pluripotent states in ExM formation. View Publication

Proteomic analysis of human cerebral organoids may reveal how psychedelics regulate biological processes,shedding light on drug-induced changes in the brain. This study elucidates the proteomic alterations induced by lysergic acid diethylamide (LSD) in human cerebral organoids. By employing high-resolution mass spectrometry-based proteomics,we quantitatively analyzed the differential abundance of proteins in cerebral organoids exposed to LSD. Our findings indicate changes in proteostasis,energy metabolism,and neuroplasticity-related pathways. Specifically,LSD exposure led to alterations in protein synthesis,folding,autophagy,and proteasomal degradation,suggesting a complex interplay in the regulation of neural cell function. Additionally,we observed modulation in glycolysis and oxidative phosphorylation,crucial for cellular energy management and synaptic function. In support of the proteomic data,complementary experiments demonstrated LSD’s potential to enhance neurite outgrowth in vitro,confirming its impact on neuroplasticity. Collectively,our results provide a comprehensive insight into the molecular mechanisms through which LSD may affect neuroplasticity and potentially contribute to therapeutic effects for neuropsychiatric disorders. View Publication

Oral facial cleft (OFC) comprises cleft lip with or without cleft palate (CL/P) or cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) in many genomic loci where the presumed effector gene (for example,IRF6 in the 1q32 locus) is expressed in embryonic oral epithelium. To identify candidates for functional SNPs at eight such loci we conduct a massively parallel reporter assay in a fetal oral epithelial cell line,revealing SNPs with allele-specific effects on enhancer activity. We filter these SNPs against chromatin-mark evidence of enhancers and test a subset in traditional reporter assays,which support the candidacy of SNPs at loci containing FOXE1, IRF6, MAFB, TFAP2A,and TP63. For two SNPs near IRF6 and one near FOXE1,we engineer the genome of induced pluripotent stem cells,differentiate the cells into embryonic oral epithelium,and discover allele-specific effects on the levels of effector gene expression,and,in two cases,the binding affinity of transcription factors FOXE1 or ETS2. Conditional analyses of GWAS data suggest the two functional SNPs near IRF6 account for the majority of risk for CL/P at this locus. This study connects genetic variation associated with OFC to mechanisms of pathogenesis. Non-syndromic orofacial cleft is a relatively common congenital anomaly. Many non-coding genetic variants are associated with this disorder but only a subset is functional. Here the authors use reporter assays and stem cells to reveal members of this subset. View Publication

AbstractThe hippocampus is a brain area linked to cognition. The mechanisms that maintain cognitive activity in humans are poorly understood. Centenarians display extreme longevity which is generally accompanied by better quality of life,lower cognitive impairment,and reduced incidence of pathologies including neurodegenerative diseases. We performed transcriptomic studies in hippocampus samples from individuals of different ages (centenarians [?97 years],old,and young) and identified a differential gene expression pattern in centenarians compared to the other two groups. In particular,several isoforms of metallothioneins (MTs) were highly expressed in centenarians. Moreover,we identified that MTs were mainly expressed in astrocytes. Functional studies in human primary astrocytes revealed that MT1 and MT3 are necessary for their homeostasis maintenance. Overall,these results indicate that the expression of MTs specifically in astrocytes is a mechanism for protection during aging. Higher levels of MT1 and MT3 are detected in hippocampus of very old individuals (over 90) compared with old and young individuals. MTs colocalize with astrocytic markers. View Publication

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches,we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis,the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture,accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development,we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach,we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases. The ability to differentiate human pluripotent stem cells (hPSCs) into insulin producing cells holds potential for diabetes treatments,but many of these approaches lack the complexity needed for in vitro disease modeling. Here they develop an hPSC-derived islet spheroid system,offering an experimental model to study pancreatic budding and islet morphogenesis with human cells. View Publication

SummaryThe human brain microenvironment undergoes dynamic changes during development,which have been incompletely characterized in in vitro models including neural organoids. Here,we used liquid chromatography-mass spectrometry to investigate proteome and secretome changes in human dorsal forebrain organoids derived from three hiPSC lines at days 20,35,and 50 of differentiation. Proteome and immunohistochemical analysis revealed reduced proliferation and increased differentiation of progenitor cells gradually over time. In contrast,secretome analysis showed distinct characteristics at each timepoint — notably,at day 35,the numbers of cell adhesion molecules,synaptic proteins,and proteases were increased. Taken together,we present a resource describing the dynamic features of a neural organoid proteome and secretome across different genetic backgrounds. We describe the unique niche composition of neural organoids during the period of neurogenesis and suggest that synaptic proteins may play a role in guiding neurogenesis. Graphical abstract Highlights•Proteomic analysis of DFOs on three time points shows neural differentiation•Protein secretion increases during peak neurogenesis at D35 and D50•Cell adhesion molecules,synapse proteins,and metalloproteases are mainly secreted at D35•Extracellular matrix proteins are predominantly secreted at D50 Natural sciences; Biological sciences; Neuroscience; Tissue Engineering View Publication

Background and PurposeEsophageal cancer-related gene-4 (ECRG4) participate in inflammation process and can interact with the innate immunity complex TLR4-MD2-CD14 on human granulocytes. In addition,ECRG4 participate in modulation of ion channel function and electrical activity of cardiomyocytes. However,the exact mechanism is unknown. This study aimed to test our hypothesis that ECRG4 contributes to inflammation-induced ion channel dysfunctions in cardiomyocytes.MethodsHuman-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from three donors were treated with lipopolysaccharide (LPS) to establish an endotoxin-induced inflammatory model. Immunostaining,real-time PCR,and patch-clamp techniques were used for the study.ResultsECRG4 was detected in hiPSC-CMs at different differentiation time. LPS treatment increased ECRG4 expression in hiPSC-CMs. Knockdown of ECRG4 decreased the expression level of Toll-Like-Receptor 4 (TLR4,a LPS receptor) and its associated genes and inflammatory cytokines. Furthermore,ECRG4 knockdown shortened the action potential duration (APD) and intercepted LPS-induced APD prolongation by enhancing ISK (small conductance calcium-activated K channel current) and attenuating INCX (Na/Ca exchanger current). Overexpression of ECRG4 mimicked LPS effects on ISK and INCX,which could be prevented by NF?B signaling blockers.ConclusionThis study demonstrated that LPS effects on cardiac ion channel function were mediated by the upregulation of ECRG4,which affects NF?B signaling. Our findings support the roles of ECRG4 in inflammatory responses and the ion channel dysfunctions induced by LPS challenge. View Publication

BackgroundStem-cell-derived therapy is a promising option for tissue regeneration. Human iPSC-derived progenitors of smooth muscle cells (pSMCs) exhibit limited proliferation and differentiation,which minimizes the risk of tumor formation while restoring smooth muscle cells (SMCs). Up to 29% of women suffer from recurrence of vaginal prolapse after prolapse surgery. Therefore,there is a need for therapies that can restore vaginal function. SMCs contribute to vaginal tone and contractility. We sought to examine whether human pSMCs can restore vaginal function in a rat model.MethodsFemale immunocompromised RNU rats were divided into 5 groups: intact controls (n?=?12),VSHAM (surgery?+?saline injection,n?=?35),and three cell-injection groups (surgery?+?cell injection using pSMCs from three patients,n?=?14/cell line). The surgery to induce vaginal injury was analogous to prolapse surgery. Menopause was induced by surgical ovariectomy. The vagina,urethra,bladder were harvested 10 weeks after surgery (5 weeks after cell injection). Organ bath myography was performed to evaluate the contractile function of the vagina,and smooth muscle thickness was examined by tissue immunohistochemistry. Collagen I,collagen III,and elastin mRNA and protein expressions in tissues were assessed.ResultsVaginal smooth muscle contractions induced by carbachol and KCl in the cell-injection groups were significantly greater than those in the VSHAM group. Collagen I protein expression in the vagina of the cell-injections groups was significantly higher than in the VSHAM group. Vaginal elastin protein expression was similar between the cell-injection and VSHAM groups. In the urethra,gene expression levels of collagen I,III,and elastin were all significantly greater in the cell-injection groups than in the VSHAM group. Collagen I,III,and elastin protein expression of the urethra did not show a consistent trend between cell-injection groups and the VSHAM group.ConclusionsHuman iPSC-derived pSMCs transplantation appears to be associated with improved contractile function of the surgically injured vagina in a rat model. This is accompanied by changes in extracellular protein expression the vagina and urethra. These observations support further efforts in the translation of pSMCs into a treatment for regenerating the surgically injured vagina in women who suffer recurrent prolapse after surgery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03900-3. View Publication

In mammals,early embryonic gastrulation process is high energy demanding. Previous studies showed that,unlike endoderm and mesoderm cells,neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process,which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/?-catenin signaling during neuroectoderm differentiation. Notably,histone lactylation,especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments,we further identified that four genes,including PAX6,were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05510-x. View Publication

BackgroundAbnormalities in T cell activation play an important role in the pathogenesis of myocarditis,and persistent T cell responses can lead to autoimmunity and chronic cardiac inflammation,as well as even dilated cardiomyopathy. Although previous work has examined the role of T cells in myocarditis in animal models,the specific mechanism for human cardiomyocytes has not been investigated.MethodsIn this study,we constructed the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and established the T cell-mediated cardiac injury model by co-culturing with activated CD4 + T or CD8 + T cells that were isolated from peripheral mononuclear blood to elucidate the pathogenesis of myocardial cell injury caused by inflammation.ResultsBy combination of quantitative proteomics with tissue and cell immunofluorescence examination,we established a proteome profile of inflammatory myocardia from hiPSC-CMs with obvious cardiomyocyte injury and increased levels of lactate dehydrogenase content,creatine kinase isoenzyme MB and cardiac troponin. A series of molecular dysfunctions of hiPSC-CMs was observed and indicated that CD4 + cells could produce direct cardiomyocyte injury by activating the NOD-like receptor signals pathway.ConclusionsThe data presented in our study established a proteome map of inflammatory myocardial based on hiPSC-CMs injury model. These results can provide guidance in the discovery of improved clinical treatments for myocarditis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03791-4. View Publication