技术资料

Human-induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) represent a promising and renewable cell source for therapeutic applications. A systematic evaluation of the immunological properties and engraftment potential of iMSCs generated from urine-derived iPSCs is lacking,which has impeded their broader application. In this study,we differentiated urine-derived iPSCs into iMSCs and assessed their fundamental MSC characteristics,immunogenicity,immunomodulatory capacity and in vivo engraftment. Compared to umbilical cord-derived MSCs (UCMSCs),iMSCs demonstrated an enhanced proliferative capacity,a higher level of regenerative gene expression,and lower immunogenicity,strengthening resistance to apoptosis induced by allogeneic peripheral blood mononuclear cells (PBMCs) and the NK-92 cell line. In addition,iMSCs exhibited a diminished ability to inhibit T cell proliferation and activation compared with UCMSCs. Transcriptomic analyses further revealed the decreased expression of immune regulatory factors in iMSCs. After transfusion into mouse models,iMSCs engrafted in the lungs,liver,and spleen and exhibited the ability to migrate to tumor tissues. Our results indicated that iMSCs generated from urine-derived iPSCs have a significant replicative capacity,low immunogenicity and unique immunomodulatory properties,and hence offer obvious advantages in immune privilege and allogenic therapeutic application prospects. View Publication

ABSTRACTDuchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder affecting 1:3500 male births and is associated with myofiber degeneration,regeneration,and inflammation. Glucocorticoid treatments have been the standard of care due to immunomodulatory/immunosuppressive properties but novel genetic approaches,including exon skipping and gene replacement therapy,are currently being developed. The identification of additional biomarkers to assess DMD-related inflammatory responses and the potential efficacy of these therapeutic approaches are thus of critical importance. The current study uses RNA sequencing of skeletal muscle from two mdx mouse models to identify high mobility group box 1 (HMGB1) as a candidate biomarker potentially contributing to DMD-related inflammation. HMGB1 protein content was increased in a human iPSC-derived skeletal myocyte model of DMD and microdystrophin treatment decreased HMGB1 back to control levels. In vivo,HMGB1 protein levels were increased in vehicle treated B10-mdx skeletal muscle compared to B10-WT and significantly decreased in B10-mdx animals treated with adeno-associated virus (AAV)-microdystrophin. However,HMGB1 protein levels were not increased in D2-mdx skeletal muscle compared to D2-WT,demonstrating a strain-specific difference in DMD-related immunopathology. Summary: Duchenne muscular dystrophy is a devastating that currently has limited treatment options. RNA sequencing and downstream analysis in iSkM and mdx samples revealed HMGB1 may be a relevant treatment biomarker. View Publication

Developmental and epileptic encephalopathies (DEEs) feature altered brain development,developmental delay and seizures,with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A,encoding a membrane trafficking protein,and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1,components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles,biasing daughter cells towards a more fate-committed state,ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families. Developmental and epileptic encephalopathies are devastating neurological disorders. Here,the authors establish a cohort of patients with variants in the gene DENND5A and use human stem cells to discover a disease mechanism involving altered cell division. View Publication

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study,we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg,HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV,thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection,we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients,we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. View Publication

Angelman syndrome (AS) and Prader-Willi syndrome (PWS),two distinct neurodevelopmental disorders,result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele,respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless,gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences,reprogramming methods,and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here,we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally,these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS. View Publication

BackgroundHuman pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI),but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models,representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore,we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation.MethodsWe used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs,and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next,we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry.ResultsIn vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (If) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs,all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However,in contrast to our hypothesis,both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT).ConclusionsWhile genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro,this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model,implying more complex mechanism(s) are operational in vivo. View Publication

Cardiac hypertrophy is a cellular process characterized by the increased size of cardiomyocytes in response to a high workload or stress. 17-beta estradiol (E2) has cardioprotective and anti-hypertrophic effects by maintaining mitochondrial network and function. MUL1 is a mitochondrial ubiquitin ligase directly involved in the control of mitochondrial fission and mitophagy. Studies from our group and others have previously shown that cardiomyocyte hypertrophy is associated with mitochondrial fission and dysfunction. These findings led us to study in vitro whether E2 regulates MUL1 to prevent cardiac hypertrophy,mitochondrial fission,and dysfunction induced by the catecholamine norepinephrine (NE). Our results showed that NE induces hypertrophy in cultured rat cardiomyocytes. Pre-treatment with E2 (10-100?nM) prevented the NE-dependent increases in cell perimeter and the hypertrophic stress markers ANP and BNP at both the protein and mRNA levels. NE induced the fragmentation of the mitochondrial network and reduced ATP levels,effects that were both prevented by E2. In silico analysis suggested a putative binding site for estrogen receptors on the MUL1 gene promoter. In accordance with this finding,E2 prevented increases in MUL1 mRNA and protein levels induced by NE. Our data also showed that a siRNA MUL1 knockdown counteracted NE-induced cardiomyocyte hypertrophy and mitochondrial dysfunction,mirroring the protective effect triggered by E2. In contrast,a MUL1 adenovirus did not prevent the E2 protection from cardiomyocyte hypertrophy. Further,in vivo analysis in a transgenic mouse model overexpressing MUL1 revealed that only young male mice overexpressed the protein. Consequently,they exhibited increased levels of the hypertrophic marker ANP,an elevated heart weight,and larger cardiomyocyte size. Therefore,our data demonstrate that 17-beta estradiol prevents cardiac myocyte hypertrophy by regulating MUL1. View Publication

BackgroundSpinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study,we compared different isoforms of basic fibroblast growth factor 2 (FGF2),overexpressed in stably transfected Human embryonic stem cells (hESCs),following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB).MethodsIn the present work,hESCs bioengineered to overexpress 18,23,and 31 kD isoforms of FGF2,were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA,followed by Tukey’s or Dunnett’s multiple comparison tests. Significance was set at *p < 0.05,**p < 0.01,***p < 0.001,and ****p < 0.0001.ResultsFor the first set of experiments,rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival,glial reaction,and synaptic coverage,two weeks after the lesion,indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently,the second set of experiments was performed with that isoform,so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival,glial reaction,synaptic coverage,and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection,coupled with immunomodulation,attenuation of astrogliosis,and preservation of inputs to the rescued motoneurons. Behaviorally,the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery.ConclusionTransgenic hESCs were an effective delivery platform for neurotrophic factors,rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury,while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03676-6. View Publication

Cell replacement therapy is a promising therapeutic option for dry age-related macular degeneration (AMD). In this study,we outline our design for scalable manufacture with appropriate quality gates and present in vivo data for establishing preclinical safety and efficacy of an induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) product,thus laying the foundation for Phase 1/2a trial approval in India (ClinicalTrials.gov ID: NCT06394232; date of registration: 23rd September 2024). Escalating doses of RPE cell suspension in immunocompromised animals demonstrated absence of tumor formation up to 9?months post-injection. Good Laboratory Practices (GLP) toxicology and tolerability studies in rabbits and non-human primates (NHP) respectively showed no major adverse events. RPE transplanted into immune suppressed RCS rats showed integration,neuroprotection and rescue of visual function. In addition,we provide a detailed description of the modifications in GMP manufacturing protocol to create a final product with a unique composition and Chemistry,Manufacturing and Controls (CMC) studies performed during product development. View Publication

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However,they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX),Daunorubicin (DNR),Epirubicin (EPI),and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class,or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this,we studied the effects of treatment of DOX,DNR,EPI,MTX,and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum,while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling,a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time,giving rise to four distinct gene expression response signatures,denoted as TOP2i early-acute,early-sustained,and late response genes,and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators,which mediate AC sensitivity across breast cancer patients. However,there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability,we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed,DOX response eQTLs are enriched in genes that respond to all TOP2i. Next,we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i,respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes,including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity. Author summaryAnthracycline drugs such as Doxorubicin are effective treatments for breast cancer; however,they can cause cardiotoxicity in some women. It is unclear whether women would experience the same toxicity for all drugs in this class,or whether specific drugs would be better tolerated in specific individuals. We used an in vitro system of induced pluripotent stem cell-derived cardiomyocytes from six healthy females to test the effects of five breast cancer drugs on cell heath and global gene expression. We identified a strong shared cellular and gene expression response to drugs from the same class. However,there is more variation in gene expression levels between individuals following treatment with each anthracycline compared to untreated cells. We found that many genes in regions previously associated with Doxorubicin-induced cardiotoxicity in cancer patients,respond to at least two drugs in the class. This suggests that drugs in the same class induce similar effects on an individual’s heart. This work contributes to our understanding of how drug response,in the context of off-target effects,varies across individuals. View Publication

Induced pluripotent stem cells (iPSCs) can be generated from various adult cells,genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however,their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells,here,we aimed to generate human iPSCs overexpressing interferon-beta (IFNB,IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-?. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation,IFNB-iPSCs formed embryoid bodies and upregulated endoderm,mesoderm,and some ectoderm markers. However,an upregulation of key neuroectoderm markers,PAX6 and LHX2,was compromised. A negative effect of IFN-? on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-?. The study describes new IFN-?-producing iPSC lines suitable for the generation of various types of IFN-?-producing cells for future experimental and clinical applications,and it unravels an inhibitory effect of IFN-? on stem cell neuroectoderm differentiation. View Publication

Parkinson’s disease (PD) is a neurodegenerative illness characterized by motor and non-motor features. Hallmarks of the disease include an extensive loss of dopaminergic neurons in the substantia nigra pars compacta,evidence of neuroinflammation,and the accumulation of misfolded proteins leading to the formation of Lewy bodies. While PD etiology is complex and identifying a single disease trigger has been a challenge,accumulating evidence indicates that non-neuronal and peripheral factors may likely contribute to disease onset and progression. The brain is shielded from peripheral factors by the blood-brain barrier (BBB),which tightly controls the entry of systemic molecules and cells from the blood to the brain. The BBB integrates molecular signals originating from the luminal (blood) and abluminal (brain) sides of the endothelial wall,regulating these exchanges. Of particular interest are erythrocytes,which are not only the most abundant cell type in the blood,but they also secrete extracellular vesicles (EVs) that display disease-specific signatures over the course of PD. Erythrocyte-derived EVs (EEVs) could provide a route by which pathological molecular signals travel from the periphery to the central nervous system. The primary objective of this study was to evaluate,in a human-based platform,mechanisms of EEV transport from the blood to the brain under physiological conditions. The secondary objective was to determine the ability of EEVs,generated by erythrocytes of healthy donors or patients,to induce PD-like features. We leveraged two in vitro models of the BBB,the transwell chambers and a microfluidic BBB chip generated using human induced pluripotent stem cells. Our findings suggest that EEVs transcytose from the vascular to the brain compartment of the human BBB model via a caveolin-dependant mechanism. Furthermore,EEVs derived from individuals with PD altered BBB integrity compared to healthy EEV controls,and clinical severity aggravated the loss of barrier integrity and increased EEV extravasation into the brain compartment. PD-derived EEVs reduced ZO-1 and Claudin 5 tight junction levels in BMEC-like cells and induced the selective atrophy of dopaminergic neurons. In contrast,non-dopaminergic neurons were not affected by treatment with PD EEVs. In summary,our data suggest that EEV interactions at the human BBB can be studied using a highly translational human-based brain chip model,and EEV toxicity at the neurovascular unit is exacerbated by disease severity.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00646-9. HighlightsErythrocytes secrete extracellular vesicles that can transcytose into the brain via a caveolin-dependant mechanism.A microfluidic brain chip can be used to evaluate mechanisms of transcytosis across the blood-brain barrier.The clinical severity of Parkinson’s disease affects how erythrocyte-derived extracellular vesicles interact with cerebral endothelial cells.Erythrocyte-derived extracellular vesicles generated from donors with Parkinson’s disease alter the blood-brain barrier and induce atrophy of dopaminergic neurons.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00646-9. View Publication