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BackgroundRadiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass,irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications,neurodegeneration,and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity,homeostasis,and repair. Here,we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines.MethodsWe generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome,their modulation,and the molecular pathways they elicit.ResultsIncreasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ? 5 Gy) were treated with iMSC CM,endothelial cell viability,adherence,spheroid compactness,and proangiogenic sprout formation were significantly ameliorated,and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently,iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore,iMSC CM suppressed IR-induced NF?B activation,TNF-? release,and ROS production in THP1 cells. Additionally,iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3,iMSCs,or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2,IL6,IL8/CXCL8,ANG (Angiogenin),GRO?/CXCL1,and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors,the PI3K/AKT/mTOR modulator PRAS40 and ?-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism,translation,mitochondrial respiration,DNA damage repair,and neurodevelopment.ConclusionsThe iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03847-5. View Publication

Type 1 interferons (IFN-Is) exhibit significant antiviral,antitumor,and immunoregulatory properties,demonstrating substantial therapeutic potential. However,IFN-Is are pleiotropic cytokines,and the available data on their effect under specific pathological conditions are inconclusive. Furthermore,the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types,including human immune cells. In this study,we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4,SOX2,TRA 1-60,and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline,all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line,126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types,as well as for generating different types of cells with controllable IFN-β expression. View Publication

Neural crest stem cells (NCSCs) compose a highly migratory,multipotent,stem cell population arising from the neural plate border of the embryonic ectoderm. Investigating the development of NCSCs is critical in understanding both embryonic development and abnormal events that underlie neurocristopathies. Suggested seeding densities in in vitro human induced pluripotent stem cells (hiPSCs) differentiation protocols,varying between 10,000 cells/cm 2 and 200,000 cells/cm 2,demonstrate a lack of consensus on the optimal conditions to obtain NCSCs. Aiming to maximize the differentiation efficiency of hiPSCs towards the NCSCs lineage,we investigated the effect of the initial seeding density on NCSCs lineage commitment,both in fibroblast- and human peripheral blood mononuclear cell (PBMC)-derived hiPSCs. Cultures were characterized with gene and protein expression analysis assessing stemness ( OCT3/4 and NANOG ),neural crest identity ( SNAI2 and SOX10 ) and neuroectoderm identity ( PAX6 and SOX1 ). We demonstrate that reaching a confluent monolayer of cells by the end of the differentiating protocol is crucial to obtaining NCSCs from hiPSCs. To achieve this,our results indicated 17,000 cells/cm 2 is the optimal initial seeding density. Under this protocol,a confluent monolayer was reached after 8 days of differentiation and an average of 89% SOX10 positive cells were obtained. The fold change of SNAI2 and SOX10 expression was 11-fold and 17-fold higher,respectively,in cultures seeded with 17,000 cells/cm 2,compared to the highest tested density of 200,000 cells/cm 2 . In contrast,seeding 200,000 cells/cm 2 induced neuroectoderm-like cells,confirmed by an average of 45% of cells marking positive for PAX6. With this work,we demonstrate the importance of achieving cellular confluency during NCSCs differentiation. View Publication

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality,inter-batch consistency,cryopreservation and scale remain,reducing experimental reproducibility and clinical translation. Here,we report a robust stirred suspension cardiac differentiation protocol,and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines,the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs,bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks,which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible,scalable,and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications,and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols. Subject terms: Cardiovascular biology,Induced pluripotent stem cells,Cardiovascular models View Publication

Immunodeficiency,Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2,accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family,which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here,we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34 + -blood cells of a patient homozygous for the p.Cys408Gly mutation,the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain,perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency,including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation,ICF2-iPSCs showed decreased vitality and a lower percentage of CD34 + /CD43 + /CD45 + progenitors. Overall,the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype. View Publication

Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives,particularly extracellular vesicles (EVs),for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs,alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction,indicating a potential mechanism for their immunomodulatory effects. Furthermore,iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally,iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably,priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics,given their potent immunomodulatory and regenerative properties. The online version contains supplementary material available at 10.1038/s41598-024-75956-3. View Publication

Effective therapies for Alzheimer’s disease (AD) remain to be developed. APOE4 is the strongest genetic risk factor for late-onset AD. Pericyte degeneration and blood–brain barrier (BBB) disruption are thought to be early biomarkers of AD and contribute to cognitive decline in APOE4 carriers,representing potential therapeutic targets. Our previous studies have shown that pericyte transplantation is one of the most effective strategies for BBB restoration,exhibiting great therapeutic potential for APOE4-related BBB damage and AD phenotypes. Methods: APOE4/4 mice were treated with pericytes derived from APOE3/3 human induced pluripotent stem cells (hiPSCs). Behavioral tests,AD pathologies,and BBB integrity were assessed. Subsequently,temporal and spatial distribution of the transplanted pericytes was analyzed using tdTomato+ lentivirus labeling. Next,therapeutic effects of apoptotic vesicles (ApoVs) generated from APOE3/3 pericytes were evaluated in APOE4/4 pericytes in vitro. Additionally,transcriptomic and proteomic profiling were performed to identify key effector molecules in pericyte-derived ApoVs. Finally,the therapeutic effects of ApoVs derived from pericytes were evaluated in APOE4/4 mice. Results: Early,multiple transplantations of pericytes derived from APOE3/3 hiPSCs robustly rescued cognitive decline and AD pathologies,restored BBB integrity,and prevented in situ pericyte degeneration in aged APOE4/4 mice. Intriguingly,ApoVs released from the infused cells,rather than the transplanted pericytes,were predominantly distributed in the brain,which were ingested by in situ APOE4/4 pericytes and then promoted functional recovery. We further characterized insulin growth factor-2 (IGF-2) as a key factor in APOE3/3 pericyte-derived ApoVs. Infusion of the in vitro generated ApoVs from APOE3/3 pericytes demonstrated distinct therapeutic effects in APOE4/4 mice,which were reversed by IGF2 knockout. Conclusions: APOE3/3 pericytes or APOE3/3 pericyte-derived IGF2-rich ApoVs may offer promising therapeutic strategies for APOE4-associated AD. View Publication

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However,access to such cells is limited,and studies systematically mapping the spectrum of their inflammatory states are scarce. Here,we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust,accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling,revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli,including pro-inflammatory cytokines (TNFα,interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1),TLR3 (Poly(I:C)),TLR4 (lipopolysaccharide,LPS),and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g.,oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally,the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1,LPS,or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism,highlighting the role of soluble mediators in the signal propagation. Altogether,this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation. View Publication

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in COL7A1. Patient-derived induced pluripotent stem cells (iPSCs) enable the personalized study of RDEB pathogenesis and potential therapies. However,current skin cell differentiation protocols via 2D culture perform suboptimally when applied to engineered 3D skin constructs (ESC). Here,we present an approach to source fibroblasts (iFBs) and keratinocytes (iKCs) from iPSC-derived skin organoids using an optimized differentiation protocol,and utilize them to engineer ESCs modeling wild-type and RDEB phenotypes. The resulting iPSC-derived skin cells display marker expression consistent with primary counterparts and produce ESCs exhibiting significant extracellular matrix remodeling,protein deposition,and epidermal differentiation. RDEB constructs recapitulated hallmark disease features,including absence of collagen VII and reduced iFB proliferation. This work establishes a robust and scalable strategy for generating physiologically-relevant,iPSC-derived skin constructs,offering a powerful model for studying RDEB mechanisms and advancing personalized regenerative medicine. View Publication

Summary Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290,which causes missplicing and premature termination,but the basis of this sensitivity is unclear. Here,we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups,despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups,explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290,restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention. View Publication