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Neuroblastoma is the most common pediatric extracranial solid tumor and is derived from trunk neural crest cells (tNCC) and its progenitor sympathoadrenal (SA) cells. While human pluripotent stem cell (PSC) models of neuroblastoma have been described,the PSC were differentiated using protocols that made neural crest cells,but not specifically the trunk subtype. Here,we compared four recent protocols to differentiate pluripotent stem cells (PSC) toward SA cells and examined their efficiency at generating SA cells along with earlier cell states (neuromesodermal progenitors [NMP],tNCC),as well as generating MYCN-driven tumors. Interestingly,the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two PSC lines of different genders. This protocol also generated tumors with the highest level of PHOX2B,a marker of neuroblastoma. Transcriptionally,however,each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus,we identified a protocol that reliably generates adrenergic neuroblastoma. View Publication

BackgroundPrevious research showed that the intracellular complement system,with CD46 as its central molecule,regulates the Th1 response associated with IFN-γ production and transition to a type 1 regulatory response (Tr1) characterized by IL-10 production. This transition can be influenced by a vitamin D (calcitriol),favouring a shift towards Tr1 cells and increased IL-10 production,as described in some autoimmune diseases.ObjectiveIt is unknown whether calcitriol modulates CD46-induced Th1 response towards regulatory type 1 T cells (Tr1) in allergic eosinophilic asthma and its value in relation to reducing inflammatory response.MethodsCD4+ T cells from 58 patients with allergic eosinophilic asthma (AEA) and 49 healthy donors (HDs) were stimulated with αCD3/αCD46/IL-2 or αCD3/αCD46/IL-2/Calcitriol in vitro for 60 h and analyzed by flow cytometry. IFN-γ and IL-10 levels in cell culture supernatants were measured using ELISA.ResultsCD4+ T cells from patients with AEA demonstrated elevated CD46 expression in both the non-activated state and under stimulation conditions with αCD3/αCD46/IL-2 or αCD3/αCD46/IL-2/Calcitriol. Moreover,CD46 expression in AEA patients fluctuated with the pollen season,showing a significant increase during period of low pollen exposure. Calcitriol further induced CD4+Tr1 cells from in vitro generated CD4+Th1 cells in both HDs and AEA patients. However,in both cohorts were individuals (HDs: 35/49,AEA: 40/58) who responded to calcitriol with a more pronounced regulatory response. The calcitriol-induced regulatory effect manifested by a stronger surface decrease of CD46 on activated CD4+ T cells (by 40% in HDs and by 26% in AEA),accompanied by a significant inhibition of IFN-γ and increased IL-10 production (by 31% in HDs and by 85% in AEA). These individuals were termed as the CD46D group. Contrary to this,calcitriol induced an increase in CD46 expression at the CD4+ T cell surface in a minor group of HDs (14/49),and AEA patients (18/58),who were termed as the CD46I group. In CD46I group,CD4+ T cells produced less IFN-γ in comparison with CD46D group (by 33% in HDs and by 43% in AEA) and were unable to upregulate IL-10 production following stimulation with αCD3/αCD46/IL-2/Calcitriol.ConclusionOur results suggest the potential existence of a key for stratifying individuals suitable for calcitriol treatment in the context of low serum vitamin D levels. After validation in clinical studies,this key could be used as an adjunctive therapy not only for patients with allergic eosinophilic asthma,but also for other diseases. View Publication

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability and endocrine disorder caused by pathogenic variants of plant homeodomain finger gene 6 (PHF6). An understanding of the role of PHF6 in vivo in the development of the mammalian nervous system is required to advance our knowledge of how PHF6 mutations cause BFLS. Here,we show that PHF6 protein levels are greatly reduced in cells derived from a subset of patients with BFLS. We report the phenotypic,anatomical,cellular and molecular characterization of the brain in males and females in two mouse models of BFLS,namely loss of Phf6 in the germline and nervous system-specific deletion of Phf6. We show that loss of PHF6 resulted in spontaneous seizures occurring via a neural intrinsic mechanism. Histological and morphological analysis revealed a significant enlargement of the lateral ventricles in adult Phf6-deficient mice,while other brain structures and cortical lamination were normal. Phf6 deficient neural precursor cells showed a reduced capacity for self-renewal and increased differentiation into neurons. Phf6 deficient cortical neurons commenced spontaneous neuronal activity prematurely suggesting precocious neuronal maturation. We show that loss of PHF6 in the foetal cortex and isolated cortical neurons predominantly caused upregulation of genes,including Reln,Nr4a2,Slc12a5,Phip and ZIC family transcription factor genes,involved in neural development and function,providing insight into the molecular effects of loss of PHF6 in the developing brain. Author summaryThe Börjeson-Forssman-Lehmann Syndrome (BFLS) is an intellectual disability and endocrine disorder. Mutations in the plant homeodomain finger 6 gene (PHF6) cause the disorder. We show here that a subset of BFLS patients lack PHF6 and report the effects of loss of PHF6 in an animal model with complete loss of PHF6 from conception. The cerebral cortex is the site of higher brain functions,including cognition and decision-making. We report here the effects of lack of PHF6 on the developing brain,cerebral cortex and neuronal cells isolated from this structure. Since PHF6 associates with the genetic material in the cell nucleus and has been proposed to regulate gene activity,we also report the effects of lack of PHF6 on gene expression in the cerebral cortex and purified neuronal cells. We observed that loss of PHF6 results in the dysregulation of neuronal development and differentiation genes,including genes involved in disorders such as Parkinson’s disease,epilepsy,neuroblastoma,attention deficit disorder,autism and schizophrenia. Lastly,we report that mice lacking PHF6 mirror BFLS patients in that they also suffer from spontaneous epileptic seizures. Our mouse models and findings will be useful to further investigate BFLS neuronal function and other aspects of the disorder in the adult. View Publication

Iron is an essential biomineral in the human body. Here,we describe a subset of iron-loaded cancer-associated fibroblasts,termed as FerroCAFs,that utilize iron to induce immunosuppression in prostate cancer and predict an unfavorable clinical outcome. FerroCAFs secrete myeloid cell-associated proteins,including CCL2,CSF1 and CXCL1,to recruit immunosuppressive myeloid cells. We report the presence of FerroCAFs in prostate cancer from both mice and human,as well as in human lung and ovarian cancers,and identify a conserved cell surface marker,the poliovirus receptor. Mechanistically,the accumulated iron in FerroCAFs is caused by Hmox1-mediated iron release from heme degradation. The intracellular iron activates the Kdm6b,an iron-dependent epigenetic enzyme,to induce an accessible chromatin state and transcription of myeloid cell-associated protein genes. Targeting the FerroCAFs by inhibiting the Hmox1/iron/Kdm6b signaling axis incurs anti-tumor immunity and tumor suppression. Collectively,we report an iron-loaded FerroCAF cluster that drives immunosuppression through an iron-dependent epigenetic reprogramming mechanism and reveal promising therapeutic targets to boost anti-tumor immunity. Prostate cancers (PCa) are characterized by an immunosuppressive tumor microenvironment. Here the authors identify a subset of iron-loaded cancer-associated fibroblasts secreting myeloid cell-associated proteins and driving immunosuppression in PCa. View Publication

The regeneration of epithelia is crucial for maintaining intestinal homeostasis. Irisin is an exercise-induced hormone originally found to be secreted by skeletal muscles,thereby regulating energy metabolism. Recent studies have revealed that irisin protected against gut inflammation. However,the direct effects of irisin on the intestinal epithelial cells remain to be elucidated. In this study,mouse intestinal organoids were used to assess the effects of irisin on the proliferation of the intestinal epithelial cells. At a concentration of 100 ng/mL irisin significantly increased the growth of the intestinal organoids and upregulated the Wnt/β-catenin and focal adhesion kinase (FAK) signaling pathway genes. Notably,a FAK inhibitor 14 blocked the effects of irisin on the proliferation of the intestinal epithelial cells by inhibiting FAK phosphorylation,as well as the expressions of Wnt target genes. Furthermore,irisin (100 ng/mL) improved the recovery of the intestinal organoids from cellular damages caused by TNF-α,and markedly increased the expression of Wnt target genes in the intestinal epithelial cells. Taken together,irisin activates Wnt/β-catenin and FAK signaling pathways in the intestinal epithelial cells,thereby promoting intestinal epithelial self-renewal under normal homeostatic conditions and intestinal epithelial regeneration upon damages. View Publication

Airway inflammation,a protective response in the human body,can disrupt normal organ function when chronic,as seen in chronic obstructive pulmonary disease (COPD) and asthma. Chronic bronchitis induces goblet cell hyperplasia and metaplasia,obstructing airflow. Traditional animal testing is often replaced by in vitro three-dimensional cultures of human epithelial cells to assess chronic cell responses. However,these cells are cultured horizontally,differing from the tubular structure of the human airway and failing to accurately reproduce airway stenosis. To address this,we developed the Bronchus-on-a-Chip (BoC) system. The BoC uses a novel microfluidic design in a standard laboratory plate,embedding 62 chips in one plate. Human bronchial epithelial cells were cultured against a collagen extracellular matrix for up to 35 days. Characterization included barrier integrity assays,microscopy,and histological examination. Cells successfully cultured in a tubular structure,with the apical side air-lifted. Epithelial cells differentiated into basal,ciliated,and secretory cells,mimicking human bronchial epithelium. Upon exposure to inducers of goblet cell hyperplasia and metaplasia,the BoC system showed mucus hyperproduction,replicating chronic epithelial responses. This BoC system enhances in vitro testing for bronchial inflammation,providing a more human-relevant and high-throughput method.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-77665-3. View Publication

Hereditary spherocytosis (HS) is the most prevalent form of congenital hemolytic anemia,being caused by genetic mutations in genes encoding red blood cell cytoskeletal proteins. Mutations in the ANK1 and SPTB genes are the most common causes of HS.; however,pathogenicity analyses of these mutations remain limited. This study identified three novel heterozygous mutations in 3 HS patients: c.1994 C > A in ANK1,c.5692 C > T,and c.3823delG in SPTB by whole-exome sequencing (WES) and validated by Sanger sequencing. To investigate the functional consequences of these mutations,we studied their pathogenicity using in vitro culture erythroblast derived from CD34 + stem cells. All three mutations lead to the generation of a premature stop codon. Real-time PCR assay revealed that the two SPTB mutations resulted in reduced SPTB mRNA expression,suggesting a potential role for the nonsense-mediated mRNA degradation pathway. For the ANK1 mutation,gene expression was not reduced but was predicted to produce a truncated version of the ANK1 protein. Flow cytometry analysis of red blood cell-derived microparticles (MPs) revealed that HS patients had higher MP levels compared to normal subjects. This study contributes to the current understanding of the molecular mechanisms underlying mutations in the ANK1 and SPTB genes in HS.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-78622-w. View Publication

Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed,the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here,we show that the nuclear localization of the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) ensures mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability,including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces severe methylation defects and impedes correct mitosis completion. MTHFD2 deficient cells display chromosome congression and segregation defects and accumulate chromosomal aberrations. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells,whereas restricting MTHFD2 to the nucleus is sufficient to ensure correct mitotic progression. Our discovery uncovers a nuclear role for MTHFD2,supporting the notion that translocation of metabolic enzymes to the nucleus is required to meet precise chromatin needs. The nuclear localization of metabolic enzymes is fascinating and in most cases remains a mystery. Here,Pardo Lorente and colleagues show that nuclear MTHFD2 is required for successful mitosis by controlling centromeric histone methylation. View Publication

Sodium oligomannate (GV-971),an oligosaccharide drug approved in China for treating mild-to-moderate Alzheimer’s disease (AD),was previously found to recondition the gut microbiota and limit altered peripheral Th1 immunity in AD transgenic mice. As a follow-up study,we here made advances by pinpointing a Lactobacillus murinus (L.m.) strain that highly expressed a gene encoding a putative adhesin containing Rib repeats (Ribhigh-L.m.) particularly enriched in 5XFAD transgenic mice. Mechanistically,Ribhigh-L.m. adherence to the gut epithelia upregulated fecal metabolites,among which lactate ranked as the top candidate. Excess lactate stimulated the epithelial production of serum amyloid A (SAA) in the gut via the GPR81-NFκB axis,contributing to peripheral Th1 activation. Moreover,GV-971 disrupted the adherence of Ribhigh-L.m. to gut epithelia via direct binding to Rib,which corrected the excess lactate,reduced SAA,and alleviated Th1-skewed inflammation. Together,we gained further insights into the molecular link between gut bacteria and AD progression and the mechanism of GV-971 in treating AD. View Publication

We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency,scalability,and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells,HT Smart-seq3 demonstrated higher cell capture efficiency,greater gene detection sensitivity,and lower dropout rates. Additionally,when sufficiently scaled,HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably,through T-cell receptor (TCR) reconstruction,HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments,enabling more comprehensive TCR profiling across a broader range of species. Taken together,HT Smart-seq3 overcomes key technical challenges,offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires,particularly well-suited for low-input,low-RNA content samples.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-024-11036-0. View Publication

The RVd therapy,combining lenalidomide,bortezomib,and dexamethasone,is a mainstay treatment for multiple myeloma. A multiple myeloma patient developed pure red cell aplasia (PRCA) following RVd treatment,despite the absence of common PRCA triggers. In vitro analyses reveal lenalidomide as a pivotal disruptor of erythropoiesis. Single-cell transcriptome analysis unveils hyperactive CD8+ T cells and impaired erythropoiesis in the patient’s bone marrow. Unexpectedly,the patient’s erythroid cells display abnormally high expression of genes in the antigen presentation pathway,particularly those for major histocompatibility class I (MHC-I) molecules. Functional assays demonstrate that lenalidomide treatment further augmented MHC-I expression in the patient’s erythroid cells. Blocking MHC-I or depleting T cells alleviates the defective erythropoiesis of PRCA,suggesting that the interaction between erythroid cells with elevated MHC-I and T cells in the bone marrow might contribute to PRCA. Taken together,our study implicates a mechanism underlying lenalidomide-induced PRCA in treating cancer patients. On rare occasions,some multiple myeloma patients develop pure red cell aplasia (PRCA) following combination therapy for multiple myeloma. Here the authors characterise bone marrow cells from PRCA patients by single cell sequencing showing hyperactive CD8+ T cells,impaired erythropoiesis,increase of MHC-I proteins and antigen presentation associated genes in erythroid cells. View Publication

Chromothripsis is a frequent form of genome instability,whereby a presumably single catastrophic event generates extensive genomic rearrangements of one or multiple chromosome(s). However,little is known about the heterogeneity of chromothripsis across different clones from the same tumour,as well as changes in response to treatment. Here we analyse single-cell genomic and transcriptomic alterations linked with chromothripsis in human p53-deficient medulloblastoma and neural stem cells (n = 9). We reconstruct the order of somatic events,identify early alterations likely linked to chromothripsis and depict the contribution of chromothripsis to malignancy. We characterise subclonal variation of chromothripsis and its effects on extrachromosomal circular DNA,cancer drivers and putatively druggable targets. Furthermore,we highlight the causative role and the fitness consequences of specific rearrangements in neural progenitors. Chromothripsis (CT) is a type of genome instability which is prevalent in medulloblastoma with germline TP53 mutations (Li-Fraumeni syndrome,LFS). Here the authors combine single-cell genomic and transcriptomic analyses to reveal the clonal heterogeneity and functional consequences of CT in LFS medulloblastoma. View Publication