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Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of cancer chemotherapy that can often limit treatment options for cancer patients or have life-long neurodegenerative consequences that reduce the patient’s quality of life. CIPN is caused by the detrimental actions of various chemotherapeutic agents on peripheral axons. Currently,there are no approved preventative measures or treatment options for CIPN,highlighting the need for the discovery of novel therapeutics and improving our understanding of disease mechanisms. In this study,we utilized human-induced pluripotent stem cell (hiPSC)-derived motor neurons as a platform to mimic axonal damage after treatment with vincristine,a chemotherapeutic used for the treatment of breast cancers,osteosarcomas,and leukemia. We screened a total of 1902 small molecules for neuroprotective properties in rescuing vincristine-induced axon growth deficits. From our primary screen,we identified 38 hit compounds that were subjected to secondary dose response screens. Six compounds showed favorable pharmacological profiles – AZD7762,A-674563,Blebbistatin,Glesatinib,KW-2449,and Pelitinib,all novel neuroprotectants against vincristine toxicity to neurons. In addition,four of these six compounds also showed efficacy against vincristine-induced growth arrest in human iPSC-derived sensory neurons. In this study,we utilized high-throughput screening of a large library of compounds in a therapeutically relevant assay. We identified several novel compounds that are efficacious in protecting different neuronal subtypes from the toxicity induced by a common chemotherapeutic agent,vincristine which could have therapeutic potential in the clinic. The online version contains supplementary material available at 10.1007/s00018-024-05340-x. 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

Pericytes play a crucial role in controlling inflammation and vascular functions in the central nervous system,which are disrupted in Parkinson’s disease (PD). Still,there is a lack of studies on the impact of pericytes on neurodegenerative diseases,and their involvement in the pathology of PD is unclear. Our objective was to investigate the molecular and functional differences between healthy pericytes and pericytes with the LRRK2 G2019S mutation,which is one of the most common mutations associated with PD. Our study employed pericyte-like cells obtained from induced pluripotent stem cells produced from PD patients with the LRRK2 G2019S mutation as well as from healthy individuals. We examined the gene expression profiles of the cells and analyzed how the alterations reflect on their functionality. We have shown differences in the expression of genes related to inflammation and angiogenesis. Furthermore,we observe modified migration speed in PD pericyte-like cells as well as enhanced secretion of inflammatory mediators,such as soluble VCAM-1 and MCP-1,in these pericyte-like cells following exposure to proinflammatory stimuli. In summary,our findings support the notion that pericytes play a role in the inflammatory and vascular changes observed in PD. Further investigation of pericytes could provide valuable insight into understanding the pathogenesis of PD. The online version contains supplementary material available at 10.1186/s12987-024-00592-y. View Publication

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless,dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS),taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated,the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition,transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together,our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity. Subject terms: Neuroimmunology,Innate immunity,Neural stem cells View Publication

Nanoplastics (NPs) are emerging environmental pollutants that pose growing concerns due to their potential health risks. However,the effects of inhaled NP exposure during pregnancy on fetal brain development remain poorly understood. In this study,we investigated the impact of maternal exposure to polypropylene nanoplastics (PP-NPs) on fetal brain development and neurobehavioral outcomes in a mouse model and further explored its mechanism in human cerebral organoids. Maternal exposure to PP-NPs significantly impaired neuronal differentiation and proliferation in the fetal cortex. Neurobehavioral assessments revealed significant deficits in offspring following maternal exposure,including impaired spatial memory,reduced motor coordination,and heightened anxiety-like behavior. Furthermore,human brain organoids exposed to PP-NPs exhibited reduced growth and neuronal differentiation,with significant downregulation of key neuronal markers such as TUJ1,MAP2,and PAX6. Transcriptomic analysis identified alterations in gene expression,particularly in neuroactive ligand-receptor interaction pathway. Molecular docking and fluorescence co-localization analysis further suggested CYSLTR1 and PTH1R as key molecular targets of PP-NPs. These findings provide novel insights into the toxicological effects of NPs on the developing brain and emphasize the need for preventive measures to protect fetal neurodevelopment during pregnancy. The online version contains supplementary material available at 10.1186/s12951-025-03561-1. View Publication

Spinal cord injury (SCI) remains a significant clinical challenge and poses a dramatic threat to the life quality of patients due to limited neural regeneration and detrimental post-injury alternations in tissue microenvironment. We developed a therapeutic approach by transplanting spinal neural progenitor cells (spNPGs),derived from human induced pluripotent stem cell (iPSC)-generated neuromesodermal progenitors,into a contusive SCI model in NOD-SCID mice. Single-cell RNA sequencing mapped the in vitro differentiation of iPSC-spNPGs,confirming their specification into spinal neuronal lineages. Single-nucleus transcriptomics at 1 week post-transplantation showed that the grafted cells differentiated in vivo into motor neurons and two interneuron subtypes (V2 and dI4). Additionally,spNPGs integrated into host neural circuits,enhancing synaptic connectivity,while simultaneously modulating the injury microenvironment by shifting microglia and astrocyte polarization toward anti-inflammatory and neuroprotective phenotypes. This dual mechanism promoted axonal regrowth,remyelination,and significant sensorimotor recovery,as evidenced by improved locomotor scores. Our findings highlight the therapeutic potential of human iPSC-spNPGs in reconstructing neural networks and mitigating secondary damage,providing compelling preclinical evidence for advancing stem cell-based SCI therapies. Subject terms: Stem-cell differentiation,Spinal cord injury View Publication

Renal failure due to drug nephrotoxicity or disease is frequently observed in patients. The development of in vitro models able to recapitulate kidney biology offers new possibilities to study drug toxicity or model diseases. Induced pluripotent stem cell–derived kidney organoids already show promise,but several drawbacks must be overcome to maintain them in culture,among which is the presence of non-renal cell populations such as cartilage. We modified the culture protocol and maintained kidney organoids in medium containing FGF9 for 1 additional week compared to the control protocol (Takasato). In comparison to the control,the FGF9-treated kidney organoids had reduced cartilage at day 7 + 25 and diminished chondrocyte marker expression. Importantly,the renal structures assessed by immunofluorescence were unaffected by the FGF9 treatment. This reduction of cartilage produces a higher quality kidney organoid that can be maintained longer in culture to improve their maturation for further in vivo work. Subject terms: Pluripotent stem cells,Stem-cell differentiation,Kidney View Publication

The generation of retinal models from human induced pluripotent stem cells holds significant potential for advancing our understanding of retinal development,neurodegeneration,and the in vitro modeling of neurodegenerative disorders. The retina,as an accessible part of the central nervous system,offers a unique window into these processes,making it invaluable for both study and early diagnosis. This study investigates the impact of the Frontotemporal Dementia-linked IVS 10?+?16 MAPT mutation on retinal development and function using 2D and 3D retinal models derived from human induced pluripotent stem cells. Our findings reveal that the MAPT mutation leads to delayed retinal cell differentiation and maturation,with tau-mutant disease models exhibiting sustained higher expression of retinal progenitor cell markers and a reduced presence of post-mitotic neurons. Both 2D and 3D tau-mutant retinal models demonstrated an imbalance in tau isoforms,favoring 4R tau,along with increased tau phosphorylation,altered neurite morphology,and impaired cytoskeletal maturation. These changes are associated with impaired synaptic development,reduced neuronal connectivity,and enhanced cellular stress responses,including the increased formation of stress granules,markers of apoptosis and autophagy,and the presence of intracellular toxic tau aggregates. This study highlights the value of retinal models derived from human induced pluripotent stem cells in exploring the mechanisms underlying retinal pathology associated with tau mutations. These models offer essential insights into the development of therapeutic strategies for neurodegenerative diseases characterized by tau aggregation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-024-01920-x. View Publication

Propionic acidemia (PA) is a metabolic disorder caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC) due to mutations in the PCCA or PCCB genes,which encode the two PCC subunits. PA may lead to several types of cardiomyopathy and has been linked to cardiac electrical abnormalities such as QT interval prolongation,life-threatening arrhythmias,and sudden cardiac death. To gain insights into the mechanisms underlying PA-induced proarrhythmia,we recorded action potentials (APs) and ion currents using whole-cell patch-clamp in ventricular-like induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) from a PA patient carrying two pathogenic mutations in the PCCA gene (p.Cys616_Val633del and p.Gly477Glufs*9) (PCCA cells) and from a healthy subject (healthy cells). In cells driven at 1 Hz,PCC deficiency increased the latency and prolonged the AP duration (APD) measured at 20% of repolarization,without modifying resting membrane potential or AP amplitude. Moreover,delayed afterdepolarizations appeared at the end of the repolarization phase in unstimulated and paced PCCA cells. PCC deficiency significantly reduced peak sodium current (INa) but increased the late INa (INaL) component. In addition,L-type Ca2+ current (ICaL) density was reduced,while the inward and outward density of the Na+/Ca2+ exchanger current (INCX) was increased in PCCA cells compared to healthy ones. In conclusion,our results demonstrate that at the cellular level,PCC deficiency can modify the ion currents controlling cardiac excitability,APD,and intracellular Ca2+ handling,increasing the risk of arrhythmias independently of the progressive late-onset cardiomyopathy induced by PA disease. View Publication

BackgroundChronic ischemic limb disease often leads to amputation,which remains a significant clinical problem. Smooth-muscle cells (SMCs) are crucially involved in the development and progression of many cardiovascular diseases,but studies with primary human SMCs have been limited by a lack of availability. Here,we evaluated the efficiency of two novel protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into SMCs and assessed their potency for the treatment of ischemic limb disease.MethodshiPSCs were differentiated into SMCs via a conventional two-dimensional (2D) protocol that was conducted entirely with cell monolayers,or via two protocols that consisted of an initial five-day three-dimensional (3D) spheroid phase followed by a six-day 2D monolayer phase (3D?+?2D differentiation). The 3D phases were conducted in shaker flasks on an orbital shaker (the 3D?+?2D shaker protocol) or in a PBS bioreactor (the 3D?+?2D bioreactor protocol). Differentiation efficiency was evaluated via the expression of SMC markers (smooth-muscle actin [SMA],smooth muscle protein 22 [SM22],and Calponin-1),and the biological activity of the differentiated hiPSC-SMCs was evaluated via in-vitro assessments of migration (scratch assay),contraction in response to the treatment with a prostaglandin H2 analog (U46619),and tube formation on Geltrex,as well as in-vivo measurements of perfusion (fluorescence angiography) and vessel density in the limbs of mice that were treated with hiPSC-SMCs after experimentally induced hind-limb ischemia (HLI).ResultsBoth 3D?+?2D protocols yielded?>?5.6?×?107 hiPSC-SMCs/differentiation,which was?~?nine-fold more than that produced via 2D differentiation,and flow cytometry analyses confirmed that?>?98% of the 3D?+?2D-differentiated hiPSC-SMCs expressed SMA,?>?81% expressed SM22,and?>?89% expressed Calponin-1. hiPSC-SMCs obtained via the 3D?+?2D shaker protocol also displayed typical SMC-like migratory,contraction,and tube-formation activity in-vitro and significantly improved measurements of perfusion,vessel density,and SMA-positive arterial density in the ischemic limb of mouse HLI model.ConclusionsOur dynamic 3D?+?2D protocols produced an exceptionally high yield of hiPSC-SMCs. Transplantation of these hiPSC-SMCs results in significantly improved recovery of ischemic limb after ischemic injury in mice. View Publication

SummaryRibosomopathies arise from the disruptions in ribosome biogenesis within the nucleolus,which is organized via liquid-liquid phase separation (LLPS). The roles of LLPS in ribosomopathies remain poorly understood. Here,we generated human induced pluripotent stem cell (hiPSC) models of ribosomopathy caused by mutations in small nucleolar RNA (snoRNA) gene SNORD118. Mutant hiPSC-derived neural progenitor cells (NPCs) or neural crest cells (NCCs) exhibited ribosomopathy hallmark cellular defects resulting in reduced organoid growth,recapitulating developmental delay in patients. SNORD118 mutations in NPCs disrupted nucleolar morphology and LLPS properties coupled with impaired ribosome biogenesis and a translational downregulation of fibrillarin (FBL),the key LLPS effector acting via the intrinsically disordered region (IDR) motif. IDR-depleted FBL failed to rescue NPC defects,whereas a chimeric FBL with swapped IDR motif from an unrelated protein mitigated ribosomopathy and organoid growth defects. Thus,SNORD118 human iPSC models revealed aberrant phase separation and nucleolar functions as potential pathogenic mechanisms in ribosomopathies. Graphical abstract Highlights•SNORD118 mutant hiPSC-derived cells and organoids recapitulate the ribosomopathy defects•Mutations impair ribosome biogenesis and translation of phase separation effector FBL•Phase separation and nucleolar organization are defective in SNORD118 mutant cells•Impaired phase separation causes ribosomopathy and growth defects in hiPSC models Natural sciences; Biological sciences; Cell biology; Stem cell research View Publication

A growing body of clinical literature has described neurodevelopmental delays in infants with chronic prenatal opioid exposure and withdrawal. Despite this,the mechanism of how opioids impact the developing brain remains unknown. Here,we developed an in vitro model of prenatal morphine exposure and withdrawal using healthy human induced pluripotent stem cell (iPSC)-derived midbrain neural progenitors in monolayer. To optimize our model,we identified that a longer neural induction and regional patterning period increases expression of canonical opioid receptors mu and kappa in midbrain neural progenitors compared to a shorter protocol (OPRM1,two-tailed t-test,p =? 0.004; OPRK1,p =? 0.0003). Next,we showed that the midbrain neural progenitors derived from a longer iPSC neural induction also have scant toll-like receptor 4 (TLR4) expression,a key player in neonatal opioid withdrawal syndrome pathophysiology. During morphine withdrawal,differentiating neural progenitors experience cyclic adenosine monophosphate overshoot compared to cell exposed to vehicle (p =? 0.0496) and morphine exposure conditions (p,=? 0.0136,1-way ANOVA). Finally,we showed that morphine exposure and withdrawal alters proportions of differentiated progenitor cell fates (2-way ANOVA,F =? 16.05,p View Publication