Generation of iPSC lines from two patients affected by febrile seizure due to inherited missense mutation in SCN1A gene.

Abstract Here, we described the generation of human induced pluripotent stem cell lines (hiPSCs) from fibroblasts isolated by punch biopsies of two siblings carrying inherited mutation (c.434 T > C) in the SCN1A gene, encoding for the neuronal voltage gated sodium channel NaV1.1. The mutation leads to the substitution of a highly conserved methionine with a threonine (M145T) in the protein sequence, leading to infant febrile seizures (FS). The older brother, affected by complex FS, also developed temporal lobe epilepsy (TLE) during adolescence

Stem Cells: The Game Changers of Human Cardiac Disease Modelling and Regenerative Medicine

A comprehensive understanding of the molecular basis and mechanisms underlying cardiac diseases is mandatory for the development of new and effective therapeutic strategies. The lack of appropriate in vitro cell models that faithfully mirror the human disease phenotypes has hampered the understanding of molecular insights responsible of heart injury and disease development. Over the past decade, important scientific advances have revolutionized the field of stem cell biology through the remarkable discovery of reprogramming somatic cells into induced pluripotent stem cells (iPSCs). These advances allowed to achieve the long-standing ambition of modelling human disease in a dish and, more interestingly, paved the way for unprecedented opportunities to translate bench discoveries into new therapies and to come closer to a real and effective stem cell-based medicine. The possibility to generate patient-specific iPSCs, together with the new advances in stem cell differentiation procedures and the availability of novel gene editing approaches and tissue engineering, has proven to be a powerful combination for the generation of phenotypically complex, pluripotent stem cell-based cellular disease models with potential use for early diagnosis, drug screening, and personalized therapy. This review will focus on recent progress and future outcome of iPSCs technology toward a customized medicine and new therapeutic options

Modeling Cardiac Disease Mechanisms Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Progress, Promises and Challenges

Cardiovascular diseases (CVDs) are a class of disorders affecting the heart or blood vessels. Despite progress in clinical research and therapy, CVDs still represent the leading cause of mortality and morbidity worldwide. The hallmarks of cardiac diseases include heart dysfunction and cardiomyocyte death, inflammation, fibrosis, scar tissue, hyperplasia, hypertrophy, and abnormal ventricular remodeling. The loss of cardiomyocytes is an irreversible process that leads to fibrosis and scar formation, which, in turn, induce heart failure with progressive and dramatic consequences. Both genetic and environmental factors pathologically contribute to the development of CVDs, but the precise causes that trigger cardiac diseases and their progression are still largely unknown. The lack of reliable human model systems for such diseases has hampered the unraveling of the underlying molecular mechanisms and cellular processes involved in heart diseases at their initial stage and during their progression. Over the past decade, significant scientific advances in the field of stem cell biology have literally revolutionized the study of human disease in vitro. Remarkably, the possibility to generate disease-relevant cell types from induced pluripotent stem cells (iPSCs) has developed into an unprecedented and powerful opportunity to achieve the long-standing ambition to investigate human diseases at a cellular level, uncovering their molecular mechanisms, and finally to translate bench discoveries into potential new therapeutic strategies. This review provides an update on previous and current research in the field of iPSC-driven cardiovascular disease modeling, with the aim of underlining the potential of stem-cell biology-based approaches in the elucidation of the pathophysiology of these life-threatening diseases

Waveguiding and SERS Simplified Raman Spectroscopy on Biological Samples

Biomarkers detection at an ultra-low concentration in biofluids (blood, serum, saliva, etc.) is a key point for the early diagnosis success and the development of personalized therapies. However, it remains a challenge due to limiting factors like (i) the complexity of analyzed media, and (ii) the aspecificity detection and the poor sensitivity of the conventional methods. In addition, several applications require the integration of the primary sensors with other devices (microfluidic devices, capillaries, flasks, vials, etc.) where transducing the signal might be difficult, reducing performances and applicability. In the present work, we demonstrate a new class of optical biosensor we have developed integrating an optical waveguide (OWG) with specific plasmonic surfaces. Exploiting the plasmonic resonance, the devices give consistent results in surface enhanced Raman spectroscopy (SERS) for continuous and label-free detection of biological compounds. The OWG allows driving optical signals in the proximity of SERS surfaces (detection area) overcoming spatial constraints, in order to reach places previously optically inaccessible. A rutile prism couples the remote laser source to the OWG, while a Raman spectrometer collects the SERS far field scattering. The present biosensors were implemented by a simple fabrication process, which includes photolithography and nanofabrication. By using such devices, it was possible to detect cell metabolites like Phenylalanine (Phe), Adenosine 5-triphosphate sodium hydrate (ATP), Sodium Lactate, Human Interleukin 6 (IL6), and relate them to possible metabolic pathway variation

Insights into the Genetic Profile of Two Siblings Affected by Unverricht-Lundborg Disease Using Patient-Derived hiPSCs

Unverricht-Lundborg disease (ULD), also known as progressive myoclonic epilepsy 1 (EPM1), is a rare autosomal recessive neurodegenerative disorder characterized by a complex symptomatology that includes action- and stimulus-sensitive myoclonus and tonic-clonic seizures. The main cause of the onset and development of ULD is a repeat expansion of a dodecamer sequence localized in the promoter region of the gene encoding cystatin B (CSTB), an inhibitor of lysosomal proteases. Although this is the predominant mutation found in most patients, the physio-pathological mechanisms underlying the disease complexity remain largely unknown. In this work, we used patient-specific iPSCs and their neuronal derivatives to gain insight into the molecular and genetic machinery responsible for the disease in two Italian siblings affected by different phenotypes of ULD. Specifically, fragment length analysis on amplified CSTB promoters found homozygous status for dodecamer expansion in both patients and showed that the number of dodecamer repeats is the same in both. Furthermore, the luciferase reporter assay showed that the CSTB promoter activity was similarly reduced in both lines compared to the control. This information allowed us to draw important conclusions: (1) the phenotypic differences of the patients do not seem to be strictly dependent on the genetic mutation around the CSTB gene, and (2) that some other molecular mechanisms, not yet clearly identified, might be taken into account. In line with the inhibitory role of cystatin B on cathepsins, molecular investigations performed on iPSCs-derived neurons showed an increased expression of lysosomal cathepsins (B, D, and L) and a reduced expression of CSTB protein. Intriguingly, the increase in cathepsin expression does not appear to be correlated with the residual amount of CSTB, suggesting that other mechanisms, in addition to the regulation of cathepsins, could be involved in the pathological complexity of the disease

Generation of human induced pluripotent stem cell lines (UNIMGi003-A and UNIMGi004-A) from two Italian siblings affected by Unverricht-Lundborg disease

Unverricht-Lundborg disease (ULD) is an inherited form of progressive myoclonus epilepsy caused by mutations in the gene encoding Cystatin B (CSTB), an inhibitor of lysosomal proteases. The most common mutation described in ULD patients is an unstable expansion of a dodecamer sequence located in the CSTB gene promoter. This expansion is causative of the downregulation of CSTB gene expression and, consequently, of its inhibitory activity. Here we report the generation of induced pluripotent stem cell (iPSC) lines from two Italian siblings having a family history of ULD and affected by different clinical and pathological phenotypes of the disease

Establishment and characterization of induced pluripotent stem cells (iPSCs) from central nervous system lupus erythematosus

Involvement of the central nervous system (CNS) is an uncommon feature in systemic lupus erythematosus (SLE), making diagnosis rather difficult and challenging due to the poor specificity of neuropathic symptoms and neurological symptoms. In this work, we used human-induced pluripotent stem cells (hiPSCs) derived from CNS-SLE patient, with the aim to dissect the molecular insights underlying the disease by gene expression analysis and modulation of implicated pathways. CNS-SLE-derived hiPSCs allowed us to provide evidence of Erk and Akt pathways involvement and to identify a novel cohort of potential biomarkers, namely CHCHD2, IDO1, S100A10, EPHA4 and LEFTY1, never reported so far. We further extended the study analysing a panel of oxidative stress-related miRNAs and demonstrated, under normal or stress conditions, a strong dysregulation of several miRNAs in CNS-SLE-derived compared to control hiPSCs. In conclusion, we provide evidence that iPSCs reprogrammed from CNS-SLE patient are a powerful useful tool to investigate the molecular mechanisms underlying the disease and to eventually develop innovative therapeutic approaches

Human iPSC Modeling of Genetic Febrile Seizure Reveals Aberrant Molecular and Physiological Features Underlying an Impaired Neuronal Activity

Mutations in SCN1A gene, encoding the voltage-gated sodium channel (VGSC) NaV1.1, are widely recognized as a leading cause of genetic febrile seizures (FS), due to the decrease in the Na+ current density, mainly affecting the inhibitory neuronal transmission. Here, we generated induced pluripotent stem cells (iPSCs)-derived neurons (idNs) from a patient belonging to a genetically well-characterized Italian family, carrying the c.434T > C mutation in SCN1A gene (hereafter SCN1AM145T). A side-by-side comparison of diseased and healthy idNs revealed an overall maturation delay of SCN1AM145T cells. Membranes isolated from both diseased and control idNs were injected into Xenopus oocytes and both GABA and AMPA currents were successfully recorded. Patch-clamp measurements on idNs revealed depolarized action potential for SCN1AM145T, suggesting a reduced excitability. Expression analyses of VGSCs and chloride co-transporters NKCC1 and KCC2 showed a cellular “dysmaturity” of mutated idNs, strengthened by the high expression of SCN3A, a more fetal-like VGSC isoform, and a high NKCC1/KCC2 ratio, in mutated cells. Overall, we provide strong evidence for an intrinsic cellular immaturity, underscoring the role of mutant NaV1.1 in the development of FS. Furthermore, our data are strengthening previous findings obtained using transfected cells and recordings on human slices, demonstrating that diseased idNs represent a powerful tool for personalized therapy and ex vivo drug screening for human epileptic disorders