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Peripheral nerve injury (PNI) remains a significant clinical challenge,often leading to long-term functional impairment. Despite advances in therapies,current repair strategies offer unsatisfactory clinical outcomes. Exosomes derived from induced pluripotent stem cells (iPSC-Exos) have emerged as a promising therapeutic approach in regenerative medicine. This study assesses the efficacy and safety of iPSC-Exos in a rat model of sciatic nerve crush injury. Briefly,iPSCs were generated from peripheral blood mononuclear cells (PBMCs) of healthy donors using Sendai virus vectors and validated for pluripotency. iPSC-Exos were characterized and injected at the injury site. Functional recovery was assessed through gait analysis,grip strength,and pain response. Histological and molecular analyses were used to examine axonal regeneration,myelination,Schwann cell (SC) activation,angiogenesis,and changes in gene expression. iPSC-Exos were efficiently internalized by SC,promoting their proliferation. No adverse effects were observed between groups on body weight,organ histology,or hematological parameters. iPSC-Exos injection significantly enhanced nerve regeneration,muscle preservation,and vascularization,with RNA sequencing revealing activation of PI3K-AKT and focal adhesion pathways. These findings support iPSC-Exos as a safe and effective non-cell-based therapy for PNIs,highlighting their potential for clinical applications in regenerative medicine. View Publication

IntroductionRetinal ganglion cells (RGCs) are susceptible to degenerative conditions such as glaucoma and traumatic optic neuropathies,which lead to vision loss. MicroRNA-21-5p has demonstrated potential neuroprotective effects,but its mechanisms in optic nerve injury remain underexplored. This study evaluates the neuroprotective role of microRNA-21-5p derived from induced pluripotent stem cells (iPSCs) in an optic nerve crush (ONC) model.Materials and methodsIn vitro qPCR demonstrated that the expression of microRNA-21-5p was increased in the co-culture medium of RGCs and iPSCs. Subsequently,in the in vivo experiments,we used a microRNA-21-5p agonist to assess its protective effects on RGCs. RNA sequencing was then performed in a mouse ONC model after treatment with a microRNA-21-5p agonist to explore the mechanisms underlying its neuroprotective effects on RGCs.ResultsAs demonstrated in our previous experiments,the RGCs-iPSCs co-culture group led to a higher survival rate of RGCs,as indicated by live/dead cell staining,compared to the RGCs-only group. Quantitative PCR (qPCR) results revealed a significant increase in the expression of microRNA-21-5p in the medium of the RGCs-iPSCs co-culture group. Furthermore,the survival rate of mouse retinal RGCs treated with a microRNA-21-5p agonist was significantly greater than that of the control group. Lastly,RNA sequencing of the retina from microRNA-21-5p agonist-treated mice indicated that microRNA-21-5p plays a protective role in RGCs by downregulating the expression of several genes,including Irf1,Ccl4,Itk,Cxcr2,Dclre1c,Traf1,Traf2,Rbl1,Cxcl5,Cxcl3,Cxcl1,Cxcl9,Il2rg,Cd3e,Cd3d,Cxcl10,Ccl5,Ccl12,Tap1,and Cxcr4.ConclusionMicroRNA-21-5p derived from iPSCs can enhance the survival rate of RGCs in the ONC model. This suggests that microRNA-21-5p may represent a novel and effective strategy for repairing RGC damage. Such a strategy could potentially be realized through the modulation of apoptosis,T-cell regulatory pathways,or TNF-? signaling.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12886-025-04244-z. View Publication

BackgroundSchizophrenia (SCZ) is a severe psychiatric disorder associated with alterations in early brain development. Details of underlying pathomechanisms remain unclear,despite genome and transcriptome studies providing evidence for aberrant cellular phenotypes and pathway deregulation in developing neuronal cells. However,mechanistic insight at the protein level is limited.MethodsHere,we investigate SCZ-specific protein expression signatures of neuronal progenitor cells (NPC) derived from patient iPSC in comparison to healthy controls using high-throughput Western Blotting (DigiWest) in a targeted proteomics approach.ResultsSCZ neural progenitors displayed altered expression and phosphorylation patterns related to Wnt and MAPK signaling,protein synthesis,cell cycle regulation and DNA damage response. Consistent with impaired cell cycle control,SCZ NPCs also showed accumulation in the G2/M cell phase and reduced differentiation capacity. Furthermore,we correlated these findings with elevated p53 expression and phosphorylation levels in SCZ patient-derived cells,indicating a potential implication of p53 in hampering cell cycle progression and efficient neurodevelopment in SCZ.ConclusionsThrough targeted proteomics we demonstrate that SCZ NPC display coherent mechanistic alterations in regulation of DNA damage response,cell cycle control and p53 expression. These findings highlight the suitability of iPSC-based approaches for modeling psychiatric disorders and contribute to a better understanding of the disease mechanisms underlying SCZ,particularly during early development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12888-024-06127-x. View Publication

ABSTRACTGenetic variants in multiple sphingolipid biosynthesis genes cause human brain disorders. A recent study looked at people from 12 unrelated families with variants in the gene SMPD4,a neutral sphingomyelinase that metabolizes sphingomyelin into ceramide at an early stage of the biosynthesis pathway. These individuals have severe developmental brain malformations,including microcephaly and cerebellar hypoplasia. The disease mechanism of SMPD4 was not known and so we pursued a new mouse model. We hypothesized that the role of SMPD4 in producing ceramide is important for making primary cilia,a crucial organelle mediating cellular signaling. We found that the mouse model has cerebellar hypoplasia due to failure of Purkinje cell development. Human induced pluripotent stem cells lacking SMPD4 exhibit neural progenitor cell death and have shortened primary cilia,which is rescued by adding exogenous ceramide. SMPD4 production of ceramide is crucial for human brain development. Summary: Mouse and human stem cell models of SMPD4 loss of function demonstrate that SMPD4 promotes cilia function and neural development. View Publication

The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied,the role of biophysical microenvironments in early lineage commitment remains elusive. Here,we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies,facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation,suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling,DNA histone modifications,and patterned germ layer specification. Notably,altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis,offering valuable insights for material design in tissue engineering and regenerative medicine applications. Graphical abstractImage 1 View Publication

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein,we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors,which transit to late,and further to transient neurogenic progenitors,that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples,we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells,we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs,which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids. Formation of the retina during development involves the coordinated action of retinal progenitor cells and their differentiated cell types,which is key for producing a functioning eye. Here the authors provide a detailed atlas of human retinal development,combining scRNA-seq and spatial transcriptomics,and identify key genetic factors that mediate retinal progenitor cell proliferation and differentiation. View Publication

Dysregulation of TDP-43 as seen in TDP-43 proteinopathies leads to specific RNA splicing dysfunction. While discovery studies have explored novel TDP-43-driven splicing events in induced pluripotent stem cell (iPSC)-derived neurons and TDP-43 negative neuronal nuclei,transcriptome-wide investigations in frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP) brains remain unexplored. Such studies hold promise for identifying widespread novel and relevant splicing alterations in FTLD-TDP patient brains. We conducted the largest differential splicing analysis (DSA) using bulk short-read RNAseq data from frontal cortex (FCX) tissue of 127 FTLD-TDP (A,B,C,GRN and C9orf72 carriers) and 22 control subjects (Mayo Clinic Brain Bank),using Leafcutter. In addition,long-read bulk cDNA sequencing data were generated from FCX of 9 FTLD-TDP and 7 controls and human TARDBP wildtype and knock-down iPSC-derived neurons. Publicly available RNAseq data (MayoRNAseq,MSBB and ROSMAP studies) from Alzheimer’s disease patients (AD) was also analyzed. Our DSA revealed extensive splicing alterations in FTLD-TDP patients with 1881 differentially spliced events,in 892 unique genes. When evaluating differences between FTLD-TDP subtypes,we found that C9orf72 repeat expansion carriers carried the most splicing alterations after accounting for differences in cell-type proportions. Focusing on cryptic splicing events,we identified STMN2 and ARHGAP32 as genes with the most abundant and differentially expressed cryptic exons between FTLD-TDP patients and controls in the brain,and we uncovered a set of 17 cryptic events consistently observed across studies,highlighting their potential relevance as biomarkers for TDP-43 proteinopathies. We also identified 16 cryptic events shared between FTLD-TDP and AD brains,suggesting potential common splicing dysregulation pathways in neurodegenerative diseases. Overall,this study provides a comprehensive map of splicing alterations in FTLD-TDP brains,revealing subtype-specific differences and identifying promising candidates for biomarker development and potential common pathogenic mechanisms between FTLD-TDP and AD.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00401-025-02901-7. View Publication

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A,we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3,HAAO,HNF1A,MAP3K11) and previously unidentified (ABCD3,CDKN2AIP,USH1C,VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs,the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1,GAD2 and HOPX gene expression,potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall,our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function,and set up a framework for gene discovery and functional validation. Here,the authors generated a genome-wide map of the global targets bound by HNF4A and HNF1A in beta cells and hepatic cells,and highlighted notable downstream pathways and target genes that may influence beta cell function. This approach also shed light on a potentially activating effect of a HNF4A type 2 diabetes risk variant. View Publication

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart,lungs,kidney,and brain are all hallmarks of the disease,the most severe symptoms of TSC are often neurological,including seizures,autism,psychiatric disorders,and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented,it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes,we observed aberrant early neurodevelopment in vitro,including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions,including loci associated with key genes in neurodevelopment. Collectively,these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated,with implications for whether and how prenatal treatment should be pursued. View Publication

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease. Primary symptoms of PD arise with the loss of dopaminergic (DA) neurons in the Substantia Nigra Pars Compacta,but PD also affects the hippocampus and cortex,usually in its later stage. Approximately 15% of PD cases are familial with a genetic mutation. Two of the most associated genes with autosomal recessive (AR) early-onset familial PD are PINK1 and PRKN. In vitro studies of these genetic mutations are needed to understand the neurophysiological changes in patients’ neurons that may contribute to neurodegeneration. In this work,we generated and differentiated DA and hippocampal neurons from human induced pluripotent stem cells (hiPSCs) derived from two patients with a double mutation in their PINK1 and PRKN (one homozygous and one heterozygous) genes and assessed their neurophysiology compared to two healthy controls. We showed that the synaptic activity of PD neurons generated from patients with the PINK1 and PRKN mutations is impaired in the hippocampus and dopaminergic neurons. Mutant dopaminergic neurons had enhanced excitatory post-synaptic activity. In addition,DA neurons with the homozygous mutation of PINK1 exhibited more pronounced electrophysiological differences compared to the control neurons. Signaling network analysis of RNA sequencing results revealed that Focal adhesion and ECM receptor pathway were the top two upregulated pathways in the mutant PD neurons. Our findings reveal that the phenotypes linked to PINK1 and PRKN mutations differ from those from other PD mutations,suggesting a unique interplay between these two mutations that drives different PD mechanisms. View Publication

Cerebral organoids co-cultured with patient derived glioma stem cells (GLICOs) are an experimentally tractable research tool useful for investigating the role of the human brain tumor microenvironment in glioblastoma. Here we describe long-term GLICOs,a novel model in which COs are grown from embryonic stem cell cultures containing low levels of GSCs and tumor development is monitored over extended durations (ltGLICOs). Single-cell profiling of ltGLICOs revealed an unexpectedly long latency period prior to GSC expansion,and that normal organoid development was unimpaired by the presence of low numbers of GSCs. However,as organoids age they experience chronic hypoxia and oxidative stress which remodels the tumor microenvironment to promote GSC expansion. Receptor-ligand modelling identified astrocytes,which secreted various pro-tumorigenic ligands including FGF1,as the primary cell type for GSC crosstalk and single-cell multi-omic analysis revealed these astrocytes were under the control of ischemic regulatory networks. Functional validation confirmed hypoxia as a driver of pro-tumorigenic astrocytic ligand secretion and that GSC expansion was accelerated by pharmacological induction of oxidative stress. When controlled for genotype,the close association between glioma aggressiveness and patient age has very few proposed biological explanations. Our findings indicate that age-associated increases in cerebral vascular insufficiency and associated regional chronic cerebral hypoxia may contribute to this phenomenon.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-024-01755-6. View Publication

SummaryMotivated by the cellular heterogeneity in complex tissues,particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models,we developed a complete workflow to reproducibly characterize cell types in complex tissues. Our approach combines a flow cytometry (FC) antibody panel with our computational pipeline CelltypeR,enabling dataset aligning,unsupervised clustering optimization,cell type annotating,and statistical comparisons. Applied to human iPSC derived midbrain organoids,it successfully identified the major brain cell types. We performed fluorescence-activated cell sorting of CelltypeR-defined astrocytes,radial glia,and neurons,exploring transcriptional states by single-cell RNA sequencing. Among the sorted neurons,we identified subgroups of dopamine neurons: one reminiscent of substantia nigra cells most vulnerable in Parkinson’s disease. Finally,we used our workflow to track cell types across a time course of organoid differentiation. Overall,our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets in complex tissues. Graphical abstract Highlights•CelltypeR is a flow cytometry and computational pipeline for cell type quantification•Identified brain cell types in midbrain organoids and measured changes in proportions•Enriched selected populations using FACS and characterized by single-cell RNA sequencing•Identified substantia nigra–like dopaminergic neurons sensitive in Parkinson’s disease Neuroscience; Cell biology; Omics View Publication