Lens Regeneration: The Application of iSyTE and In Silico Approaches to Evaluate Gene Expression in Lens Organoids.

Several protocols have been established for the generation of lens organoids from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and other cells with regenerative potential in humans or various animal models. It is important to examine how well the regenerated lens organoids reflect lens biology, in terms of its development, homeostasis, and aging. Toward this goal, the iSyTE database (integrated Systems Tool for Eye gene discovery; https://research.bioinformatics.udel.edu/iSyTE/ ), a bioinformatics resource tool that contains meta-analyzed gene expression data in wild-type lens across different embryonic, postnatal, and adult stages, can serve as a resource for comparative analysis. This article outlines the approaches toward effective use of iSyTE to gain insights into normal gene expression in the mouse lens, enriched expression in the lens, and differential gene expression in select mouse gene-perturbation cataract/lens defects models, which in turn can be used to evaluate expression of key lens-relevant genes in lens organoids by transcriptomics (e.g., RNA-sequencing (RNA-seq), microarrays, etc.) or other downstream methods (e.g., RT-qPCR, etc.).

Generation of Functional Lentoid Bodies from Human-Induced Pluripotent Stem Cells.

The pathological mechanisms of cataract remain largely unknown due to the lack of appropriate in vitro cellular models. We developed a stable in vitro system, namely, a "fried egg" differentiation method to generate functional lentoid bodies (LBs) from induced pluripotent stem cells (iPSCs). The iPSCs-derived LBs exhibited crystalline lens-like morphology and a transparent structure, and expressed lens-specific markers. TEM examination and optical analysis further demonstrated that it has the same cell arrangement structure and magnifying ability as lens.

Discovery of 5-phenyl-3-ureidothiophene-2-carboxamides as protective agents for ALS patient iPSC-derived motor neurons.

We discovered novel neuroprotective compounds by phenotypic screening using SOD1-mutant amyotrophic lateral sclerosis (ALS) patient induced pluripotent stem cell (iPSC)-derived motor neurons. Mechanistic analysis showed that the protective effect of initial hit compound 1 was likely due to the inhibition of MAP4Ks, including MAP4K4, a member of the MAP4K kinase family. Structural transformation led to compound 15f, which showed improved MAP4K4 inhibitory activity and superior neuroprotective effects compared to 1 in motor neurons. The results suggest that structural optimization based on MAP4K4 inhibitory activity might improve the neuroprotective effect of this series of compounds.

Functional outcome of the anterior vaginal wall in a pelvic surgery injury rat model after treatment with stem cell-derived progenitors of smooth muscle cells.

BACKGROUND: Stem-cell-derived therapy is a promising option for tissue regeneration. Human iPSC-derived progenitors of smooth muscle cells (pSMCs) exhibit limited proliferation and differentiation, which minimizes the risk of tumor formation while restoring smooth muscle cells (SMCs). Up to 29% of women suffer from recurrence of vaginal prolapse after prolapse surgery. Therefore, there is a need for therapies that can restore vaginal function. SMCs contribute to vaginal tone and contractility. We sought to examine whether human pSMCs can restore vaginal function in a rat model. METHODS: Female immunocompromised RNU rats were divided into 5 groups: intact controls (n = 12), VSHAM (surgery + saline injection, n = 35), and three cell-injection groups (surgery + cell injection using pSMCs from three patients, n = 14/cell line). The surgery to induce vaginal injury was analogous to prolapse surgery. Menopause was induced by surgical ovariectomy. The vagina, urethra, bladder were harvested 10 weeks after surgery (5 weeks after cell injection). Organ bath myography was performed to evaluate the contractile function of the vagina, and smooth muscle thickness was examined by tissue immunohistochemistry. Collagen I, collagen III, and elastin mRNA and protein expressions in tissues were assessed. RESULTS: Vaginal smooth muscle contractions induced by carbachol and KCl in the cell-injection groups were significantly greater than those in the VSHAM group. Collagen I protein expression in the vagina of the cell-injections groups was significantly higher than in the VSHAM group. Vaginal elastin protein expression was similar between the cell-injection and VSHAM groups. In the urethra, gene expression levels of collagen I, III, and elastin were all significantly greater in the cell-injection groups than in the VSHAM group. Collagen I, III, and elastin protein expression of the urethra did not show a consistent trend between cell-injection groups and the VSHAM group. CONCLUSIONS: Human iPSC-derived pSMCs transplantation appears to be associated with improved contractile function of the surgically injured vagina in a rat model. This is accompanied by changes in extracellular protein expression the vagina and urethra. These observations support further efforts in the translation of pSMCs into a treatment for regenerating the surgically injured vagina in women who suffer recurrent prolapse after surgery.

Experimental Cell Models for Investigating Neurodegenerative Diseases.

Experimental models play a pivotal role in biomedical research, facilitating the understanding of disease mechanisms and the development of novel therapeutics. This is particularly true for neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and motor neuron disease, which present complex challenges for research and therapy development. In this work, we review the recent literature about experimental models and motor neuron disease. We identified three main categories of models that are highly studied by scientists. In fact, experimental models for investigating these diseases encompass a variety of approaches, including modeling the patient's cell culture, patient-derived induced pluripotent stem cells, and organoids. Each model offers unique advantages and limitations, providing researchers with a range of tools to address complex biological questions. Here, we discuss the characteristics, applications, and recent advancements in terms of each model system, highlighting their contributions to advancing biomedical knowledge and translational research.

Expanded ATXN1 alters transcription and calcium signaling in SCA1 human motor neurons differentiated from induced pluripotent stem cells.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited and lethal neurodegenerative disease caused by the abnormal expansion of CAG repeats in the ATAXIN-1 (ATXN1) gene. Pathological studies identified dysfunction and loss of motor neurons (MNs) in the brain stem and spinal cord, which are thought to contribute to premature lethality by affecting the swallowing and breathing of SCA1 patients. However, the molecular and cellular mechanisms of MN pathogenesis remain unknown. To study SCA1 pathogenesis in human MNs, we differentiated induced pluripotent stem cells (iPSCs) derived from SCA1 patients and their unaffected siblings into MNs. We examined proliferation of progenitor cells, neurite outgrowth, spontaneous and glutamate-induced calcium activity of SCA1 MNs to investigate cellular mechanisms of pathogenesis. RNA sequencing was then used to identify transcriptional alterations in iPSC-derived MN progenitors (pMNs) and MNs which could underlie functional changes in SCA1 MNs. We found significantly decreased spontaneous and evoked calcium activity and identified dysregulation of genes regulating calcium signaling in SCA1 MNs. These results indicate that expanded ATXN1 causes dysfunctional calcium signaling in human MNs.

An improved approach to generate IL-15+/+/TGFßR2-/- iPSC-derived natural killer cells using TALEN.

We present a TALEN-based workflow to generate and maintain dual-edited (IL-15+/+/TGFßR2-/-) iPSCs that produce enhanced iPSC-derived natural killer (iNK) cells for cancer immunotherapy. It involves using a cell lineage promoter for knocking in (KI) gene(s) to minimize the potential effects of expression of any exogenous genes on iPSCs. As a proof-of-principle, we KI IL-15 under the endogenous B2M promoter and show that it results in high expression of the sIL-15 in iNK cells but minimal expression in iPSCs. Furthermore, given that it is known that knockout (KO) of TGFßR2 in immune cells can enhance resistance to the suppressive TGF-ß signaling in the tumor microenvironment, we develop a customized medium containing Nodal that can maintain the pluripotency of iPSCs with TGFßR2 KO, enabling banking of these iPSC clones. Ultimately, we show that the dual-edited IL-15+/+/TGFßR2-/- iPSCs can be efficiently differentiated into NK cells that show enhanced autonomous growth and are resistant to the suppressive TGF-ß signaling.

Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs.

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends, capturing the target region for sequencing on PacBio's Sequel, Sequel II, or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands. Key features • This protocol is adapted from PacBio's previous "no-amp targeted sequencing utilizing the CRISPR-Cas9 system." • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type, blood, or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio.

Induction of Pluripotent Stem Cells from Muscle Cells of Large Yellow Croaker (Larimichthys Crocea) Via Electrotransfection.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future.

ARID1A-BAF coordinates ZIC2 genomic occupancy for epithelial-to-mesenchymal transition in cranial neural crest specification.

The BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS), a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/- induced pluripotent stem cells to model CNCC specification, we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination.

Remyelinating Drugs at a Crossroad: How to Improve Clinical Efficacy and Drug Screenings.

Axons wrapped around the myelin sheath enable fast transmission of neuronal signals in the Central Nervous System (CNS). Unfortunately, myelin can be damaged by injury, viral infection, and inflammatory and neurodegenerative diseases. Remyelination is a spontaneous process that can restore nerve conductivity and thus movement and cognition after a demyelination event. Cumulative evidence indicates that remyelination can be pharmacologically stimulated, either by targeting natural inhibitors of Oligodendrocyte Precursor Cells (OPCs) differentiation or by reactivating quiescent Neural Stem Cells (qNSCs) proliferation and differentiation in myelinating Oligodendrocytes (OLs). Although promising results were obtained in animal models for demyelination diseases, none of the compounds identified have passed all the clinical stages. The significant number of patients who could benefit from remyelination therapies reinforces the urgent need to reassess drug selection approaches and develop strategies that effectively promote remyelination. Integrating Artificial Intelligence (AI)-driven technologies with patient-derived cell-based assays and organoid models is expected to lead to novel strategies and drug screening pipelines to achieve this goal. In this review, we explore the current literature on these technologies and their potential to enhance the identification of more effective drugs for clinical use in CNS remyelination therapies.

KCNJ16-depleted kidney organoids recapitulate tubulopathy and lipid recovery upon statins treatment.

BACKGROUND: The KCNJ16 gene has been associated with a novel kidney tubulopathy phenotype, viz. disturbed acid-base homeostasis, hypokalemia and altered renal salt transport. KCNJ16 encodes for Kir5.1, which together with Kir4.1 constitutes a potassium channel located at kidney tubular cell basolateral membranes. Preclinical studies provided mechanistic links between Kir5.1 and tubulopathy, however, the disease pathology remains poorly understood. Here, we aimed at generating and characterizing a novel advanced in vitro human kidney model that recapitulates the disease phenotype to investigate further the pathophysiological mechanisms underlying the tubulopathy and potential therapeutic interventions. METHODS: We used CRISPR/Cas9 to generate KCNJ16 mutant (KCNJ16+/- and KCNJ16-/-) cell lines from healthy human induced pluripotent stem cells (iPSC) KCNJ16 control (KCNJ16WT). The iPSCs were differentiated following an optimized protocol into kidney organoids in an air-liquid interface. RESULTS: KCNJ16-depleted kidney organoids showed transcriptomic and potential functional impairment of key voltage-dependent electrolyte and water-balance transporters. We observed cysts formation, lipid droplet accumulation and fibrosis upon Kir5.1 function loss. Furthermore, a large scale, glutamine tracer flux metabolomics analysis demonstrated that KCNJ16-/- organoids display TCA cycle and lipid metabolism impairments. Drug screening revealed that treatment with statins, particularly the combination of simvastatin and C75, prevented lipid droplet accumulation and collagen-I deposition in KCNJ16-/- kidney organoids. CONCLUSIONS: Mature kidney organoids represent a relevant in vitro model for investigating the function of Kir5.1. We discovered novel molecular targets for this genetic tubulopathy and identified statins as a potential therapeutic strategy for KCNJ16 defects in the kidney.

An induced pluripotent stem cell-based approach for hair follicle development and regeneration.

Because hair loss is a common concern for many individuals, potential regenerative therapies of hair follicles have been extensively researched. Induced pluripotent stem cells (iPSCs) are a promising avenue for hair follicle regeneration. This review explores current iPSC-based approaches and highlights their potential applications and challenges in hair restoration. The principles of iPSC technology, iPSC differentiation into hair follicle precursor cells, and potential clinical implications for hair follicle regeneration are also discussed. This overview of iPSCs and their applications aims to contribute to our understanding of their role in hair restoration and potential future therapeutic applications.

Progesterone receptor is constitutively expressed in induced Pluripotent Stem Cells (iPSCs).

Induced Pluripotent Stem Cells (iPSCs) are nowadays a common starting point for wide-ranging applications including 3D disease modeling (i.e. organoids) and in future regenerative medicine. Physiological processes like homeostasis, cell differentiation, development and reproduction are tightly regulated by hormones through binding to their transmembrane or nuclear receptors of target cells. Considering their pleiotropic effect, take into account also their expression in an iPSCs-based disease modeling would better recapitulate the molecular events leading to 3D organoid development and disease study. Here we reported the expression pattern of estrogen receptor (ERα) and progesterone receptor (PR) in four different iPSCs, obtained from CD34 + progenitor cells and skin fibroblasts with four different methods. Expression of ERα and PR mRNA were significantly downregulated in iPSCs as well as fibroblasts compared to MCF7 positive control. Immunofluorescence (IF) staining detected only the expression of PR protein in all the different iPSCs cell lines, while ERα was not detectable. By flow cytometry analysis we observed that the ~ 65% of the total population of iPSCs cells expressed only PR, with 100% fold increase compared to HSPCs and fibroblasts, while ERα was not expressed. Our results collectively demonstrated for the first time that the reprogramming of somatic cells into iPSCs leads to the expression of PR receptor.

A Novel Stem Cell Model to Study Preeclampsia Endothelial Dysfunction.

Preeclampsia is a common pregnancy complication affecting 5% to 7% of all pregnancies worldwide annually. While the pathogenesis is not fully understood, maternal endothelium dysfunction is thought to be a central component to preeclampsia development. Studies to dissect maternal endothelial dysfunction, particularly on a patient-specific basis, are hampered by limited access to systemic primary endothelial cells (ECs). The objective of this study was to establish a replenishable, patient-specific in vitro EC model to allow robust mechanistic studies to dissect endothelial dysfunction in preeclampsia. Induced pluripotent stem cells (iPSCs) from three women with a history of normotensive pregnancies were differentiated into ECs. The established ECs were exposed to pooled sera from normotensive pregnancies, preeclamptic pregnancies, normotensive postpartum for non-pregnant comparison and controls. Endothelial functions including nitric oxide (NO) release, cell migration, tube formation and viability were evaluated. Levels of NO release were significantly lower after incubation with preeclamptic sera compared to the fetal bovine serum (FBS) control, and normotensive and non-pregnant (postpartum) sera treatments were also lower than FBS but higher than preeclamptic sera treatments. Tube formation and cell migration were also impaired with preeclamptic sera compared to FBS controls. Cell viabilities remained unaffected by any sera treatment. Consistent outcomes were obtained across all three patient-specific lines treated with the same pooled sera. Establishment of patient-derived iPSC-ECs treated with pregnancy sera serves as a novel model to explore the interplay between individual maternal endothelial health and circulating factors that lead to endothelial dysfunction in preeclampsia.

Immunomodulatory effects of the induced pluripotent stem cells through expressing IGF-related factors and IL-10 in vitro.

BACKGROUND: Induced Pluripotent Stem Cells (IPSCs) represent an innovative strategy for addressing challenging diseases, including various rheumatologic conditions. Aside from their regenerative capacities, some studies have shown the potential of these cells in the modulation of inflammatory responses. The underlying mechanisms by which they exert their effects have yet to be fully comprehended. Therefore, we aimed to explore the gene expression linked to the IGF pathway as well as IL-10 and TGF-ß, which are known to exert immunomodulatory effects. METHODS: A C57/Bl6 pregnant mouse was used for obtaining mouse embryonic fibroblasts (MEFs), then the IPSCs were induced using lentiviral vectors expressing the pluripotency genes (OCT4, SOX2, KLF1, and c-MYC). Cells were cultured for 72 h in DMEM high glucose plus leukemia inhibitory factor; Evaluating the gene expression was conducted using specific primers for Igf1, Igf2, Igfbp3, Igfbp4, Irs1, Il-10, and Tgf-ß genes, as well as SYBR green qPCR master mix. The data were analyzed using the 2-ΔΔCT method and were compared by employing the t test; the results were plotted using GraphPad PRISM software. MEFs were utilized as controls. RESULTS: Gene expression analyses revealed that Igf-1, Igf-bp3, Igf-bp4, and Il-10 were significantly overexpressed (p ≤ .01), while Igf-2 and Tgf-b genes were significantly downregulated in the lysates from IPSCs in comparison with the control MEFs. The Irs1 gene expression was not altered significantly. CONCLUSION: IPSCs are potentially capable of modulating inflammatory responses through the expression of various anti-inflammatory mediators from the IGF signaling, as well as IL-10. This discovery uncovers a previously unknown dimension of IPSCs' therapeutic effects, potentially leading to more advanced in vivo research and subsequent clinical trials.

Generation of Marfan syndrome-specific induced pluripotent stem cells harboring FBN1 mutations.

Marfan syndrome (MFS) is a hereditary condition caused by mutations in the FBN1 gene. Genetic mutations in the FBN1 locus impact the function of the encoded protein, Fibrillin 1, a structural molecule forming microfibrils found in the connective tissue. MFS patients develop severe cardiovascular complications including thoracic aortic aneurysm and aortic dissection, which predispose them to an enhanced risk of premature death. Here, we generated two induced pluripotent stem cell (iPSC) lines harboring mutations in the FBN1 gene (p.C1942C>A and c.1954 T>C), directly derived from MFS patients. We have shown that both iPSC lines displayed expression of pluripotency markers, normal karyotype and ability of trilineage differentiation, representing a valuable tool for the identification of new therapeutic strategies for intervening in this disease.

Atelocollagen supports three-dimensional culture of human induced pluripotent stem cells.

As autologous induced pluripotent stem cell (iPSC) therapy requires a custom-made small-lot cell production line, and the cell production method differs significantly from the existing processes for producing allogeneic iPSC stocks for clinical use. Specifically, mass culture to produce stock is no longer necessary; instead, a series of operations from iPSC production to induction of differentiation of therapeutic cells must be performed continuously. A three-dimensional (3D) culture method using small, closed-cell manufacturing devices is suitable for autologous iPSC therapy. The use of such devices avoids the need to handle many patient-derived specimens in a single clean room; handling of cell cultures in an open system in a cell processing facility increases the risk of infection. In this study, atelocollagen beads were evaluated as a 3D biomaterial to assist 3D culture in the establishment, expansion culture, and induction of differentiation of iPSCs. It was found that iPSCs can be handled in a closed-cell device with the same ease as use of a two-dimensional (2D) culture when laminin-511 is added to the medium. In conclusion, atelocollagen beads enable 3D culture of iPSCs, and the quality of the obtained cells is at the same level as those derived from 2D culture.

Altered transcriptomes, cell type proportions, and dendritic spine morphology in hippocampus of suicide decedents.

BACKGROUND: Suicide is a manner of death resulting from complex environmental and genetic risks that affect millions of people globally. Both structural and functional studies identified the hippocampus as one of the vulnerable brain regions contributing to suicide risk. METHODS: We have identified the hippocampal tissue transcriptomes, gene ontology, cell type proportions, and dendritic spine morphology in controls (n = 28) and suicide decedents (n = 22). In addition, the transcriptomic signature in iPSC-derived neuronal precursor cells (NPCs) and neurons were also investigated in controls (n = 2) and suicide decedents (n = 2). RESULTS: The hippocampal tissue transcriptomic data revealed that NPAS4 gene expression was downregulated while ALDH1A2, NAAA, and MLXIPL gene expressions were upregulated in hippocampal tissue of suicide decedents. The gene ontology identified 29 significant pathways including NPAS4-associated gene ontology terms "excitatory post-synaptic potential", "regulation of postsynaptic membrane potential" and "long-term memory" indicating alteration of glutamatergic synapses in the hippocampus of suicide decedents. The cell type deconvolution identified decreased excitatory neuron proportion and an increased inhibitory neuron proportion providing evidence of excitation/inhibition imbalance in the hippocampus of suicide decedents. In addition, suicide decedents had increased dendric spine density in the hippocampus, due to an increase of thin (relatively unstable) dendritic spines, compared to controls. The transcriptomes of iPSC-derived hippocampal-like NPCs and neurons revealed 31 and 33 differentially expressed genes in NPC and neurons, respectively, of suicide decedents. CONCLUSIONS: Our findings will provide new insights into the hippocampal neuropathology of suicide.

miR-92a-3p and miR-320a are Upregulated in Plasma Neuron-Derived Extracellular Vesicles of Patients with Frontotemporal Dementia.

Despite the efforts to identify fluid biomarkers to improve diagnosis of Frontotemporal dementia (FTD), only a few candidates have been described in recent years. In a previous study, we identified three circulating miRNAs (miR-92a-3p, miR-320a and miR-320b) differentially expressed in FTD patients with respect to healthy controls and/or Alzheimer's disease (AD) patients. Now, we investigated whether those changes could be due to miRNAs contained in neuron-derived extracellular vesicles (NDEVs). We also evaluated miRNAs content in total plasma EVs and in CSF samples. The analysis of plasma NDEVs carried out on 40 subjects including controls (n = 13), FTD (n = 13) and AD (n = 14) patients, showed that both miR-92a-3p and miR-320a levels were triplicated in the FTD group if compared with CT and AD patients. Increased levels of the same miRNAs were found also in CSF derived from FTD group compared to CTs. No differences were observed in expression levels of miR-320b among the three groups. Worthy of note, all miRNAs analysed were increased in an FTD cell model, MAPT IVS10 + 16 neurons. Our results suggest that miR-92a and miR-320a in NDEVs could be proposed as FTD biomarkers.