Production of an induced pluripotent stem cell line CSSi018-A (14192) from a patient with hypomyelinating leukodystrophy 7 (HLD7) carrying biallelic variants of POLR3A (c.1802 T > A; c.4072G > A).

Hypomyelinating leukodystrophies (HLD) are a group of heterogeneous genetic disorders characterized by a deficit in myelin deposition during brain development. Specifically, 4H-Leukodystrophy is a recessive disease due to biallelic mutations in the POLR3A gene, which encodes one of the subunits forming the catalytic core of RNA polymerase III (PolIII). The disease also presents non-neurological signs such as hypodontia and hypogonadotropic hypogonadism. Here, we report the generation of a human induced pluripotent stem cell (hiPSC) line from fibroblasts of the first identified carrier of the biallelic POLR3A variants c.1802 T > A and c.4072G > A.

Phase I clinical trial of intracerebroventricular transplantation of allogeneic neural stem cells in people with progressive multiple sclerosis.

We report the analysis of 1 year of data from the first cohort of 15 patients enrolled in an open-label, first-in-human, dose-escalation phase I study (ClinicalTrials.gov: NCT03282760, EudraCT2015-004855-37) to determine the feasibility, safety, and tolerability of the transplantation of allogeneic human neural stem/progenitor cells (hNSCs) for the treatment of secondary progressive multiple sclerosis. Participants were treated with hNSCs delivered via intracerebroventricular injection in combination with an immunosuppressive regimen. No treatment-related deaths nor serious adverse events (AEs) were observed. All participants displayed stability of clinical and laboratory outcomes, as well as lesion load and brain activity (MRI), compared with the study entry. Longitudinal metabolomics and lipidomics of biological fluids identified time- and dose-dependent responses with increased levels of acyl-carnitines and fatty acids in the cerebrospinal fluid (CSF). The absence of AEs and the stability of functional and structural outcomes are reassuring and represent a milestone for the safe translation of stem cells into regenerative medicines.

Different states of stemness of glioblastoma stem cells sustain glioblastoma subtypes indicating novel clinical biomarkers and high-efficacy customized therapies.

BACKGROUND: Glioblastoma (GBM) is the most malignant among gliomas with an inevitable lethal outcome. The elucidation of the physiology and regulation of this tumor is mandatory to unravel novel target and effective therapeutics. Emerging concepts show that the minor subset of glioblastoma stem cells (GSCs) accounts for tumorigenicity, representing the true target for innovative therapies in GBM. METHODS: Here, we isolated and established functionally stable and steadily expanding GSCs lines from a large cohort of GBM patients. The molecular, functional and antigenic landscape of GBM tissues and their derivative GSCs was highlited in a side-by-side comprehensive genomic and transcriptomic characterization by ANOVA and Fisher's exact tests. GSCs' physio-pathological hallmarks were delineated by comparing over time in vitro and in vivo their expansion, self-renewal and tumorigenic ability with hierarchical linear models for repeated measurements and Kaplan-Meier method. Candidate biomarkers performance in discriminating GBM patients' classification emerged by classification tree and patients' survival analysis. RESULTS: Here, distinct biomarker signatures together with aberrant functional programs were shown to stratify GBM patients as well as their sibling GSCs population into TCGA clusters. Of importance, GSCs cells were demonstrated to fully resemble over time the molecular features of their patient of origin. Furthermore, we pointed out the existence of distinct GSCs subsets within GBM classification, inherently endowed with different self-renewal and tumorigenic potential. Particularly, classical GSCs were identified by more undifferentiated biological hallmarks, enhanced expansion and clonal capacity as compared to the more mature, relatively slow-propagating mesenchymal and proneural cells, likely endowed with a higher potential for infiltration either ex vivo or in vivo. Importantly, the combination of DCX and EGFR markers, selectively enriched among GSCs pools, almost exactly predicted GBM patients' clusters together with their survival and drug response. CONCLUSIONS: In this study we report that an inherent enrichment of distinct GSCs pools underpin the functional inter-cluster variances displayed by GBM patients. We uncover two selectively represented novel functional biomarkers capable of discriminating GBM patients' stratification, survival and drug response, setting the stage for the determination of patient-tailored diagnostic and prognostic strategies and, mostly, for the design of appropriate, patient-selective treatment protocols.

APOGEE 2: multi-layer machine-learning model for the interpretable prediction of mitochondrial missense variants.

Mitochondrial dysfunction has pleiotropic effects and is frequently caused by mitochondrial DNA mutations. However, factors such as significant variability in clinical manifestations make interpreting the pathogenicity of variants in the mitochondrial genome challenging. Here, we present APOGEE 2, a mitochondrially-centered ensemble method designed to improve the accuracy of pathogenicity predictions for interpreting missense mitochondrial variants. Built on the joint consensus recommendations by the American College of Medical Genetics and Genomics/Association for Molecular Pathology, APOGEE 2 features an improved machine learning method and a curated training set for enhanced performance metrics. It offers region-wise assessments of genome fragility and mechanistic analyses of specific amino acids that cause perceptible long-range effects on protein structure. With clinical and research use in mind, APOGEE 2 scores and pathogenicity probabilities are precompiled and available in MitImpact. APOGEE 2's ability to address challenges in interpreting mitochondrial missense variants makes it an essential tool in the field of mitochondrial genetics.

Local delivery of hrBMP4 as an anticancer therapy in patients with recurrent glioblastoma: a first-in-human phase 1 dose escalation trial.

BACKGROUND: This Phase 1 study evaluates the intra- and peritumoral administration by convection enhanced delivery (CED) of human recombinant Bone Morphogenetic Protein 4 (hrBMP4) - an inhibitory regulator of cancer stem cells (CSCs) - in recurrent glioblastoma. METHODS: In a 3 + 3 dose escalation design, over four to six days, fifteen recurrent glioblastoma patients received, by CED, one of five doses of hrBMP4 ranging from 0·5 to 18 mg. Patients were followed by periodic physical, neurological, blood testing, magnetic resonance imaging (MRI) and quality of life evaluations. The primary objective of this first-in-human study was to determine the safety, dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of hrBMP4. Secondary objectives were to assess potential efficacy and systemic exposure to hrBMP4 upon intracerebral infusion. RESULTS: Intra- and peritumoral infusion of hrBMP4 was safe and well-tolerated. We observed no serious adverse events related to this drug. Neither MTD nor DLT were reached. Three patients had increased hrBMP4 serum levels at the end of infusion, which normalized within 4 weeks, without sign of toxicity. One patient showed partial response and two patients a complete (local) tumor response, which was maintained until the most recent follow-up, 57 and 30 months post-hrBMP4. Tumor growth was inhibited in areas permeated by hrBMP4. CONCLUSION: Local delivery of hrBMP4 in and around recurring glioblastoma is safe and well-tolerated. Three patients responded to the treatment. A complete response and long-term survival occurred in two of them. This warrants further clinical studies on this novel treatment targeting glioblastoma CSCs. TRIAL REGISTRATION: ClinicaTrials.gov identifier: NCT02869243.

Circadian profile, daytime activity, and the Parkinson's phenotype: A motion sensor pilot study with neurobiological underpinnings.

Circadian rhythm impairment may play a role in Parkinson's disease (PD) pathophysiology. Recent literature associated circadian rhythm features to the risk of developing Parkinson and to its progression through stages. The association between the chronotype and the phenotype should be verified on a clinical and biological point of view. Herein we investigate the chronotype of a sample of 50 PD patients with the Morningness Eveningness Questionnaire and monitor their daily activity with a motion sensor embedded in a smartphone. Fibroblasts were collected from PD patients (n = 5) and from sex/age matched controls (n = 3) and tested for the circadian expression of clock genes (CLOCK, BMAL1, PER1, CRY1), and for cell morphology, proliferation, and death. Our results show an association between the chronotype and the PD phenotype. The most representative clinical chronotypes were "moderate morning" (56%), the "intermediate" (24%) and, in a minor part, the "definite morning" (16%). They differed for axial motor impairment, presence of motor fluctuations and quality of life (p < 0.05). Patients with visuospatial dysfunction and patients with a higher PIGD score had a blunted motor daily activity (p = 0.006 and p = 0.001, respectively), independently by the influence of age and other motor scores. Fibroblasts obtained by PD patients (n = 5) had an impaired BMAL1 cycle compared to controls (n = 3, p = 0.01). Moreover, a PD flat BMAL1 profile was associated with the lowest cell proliferation and the largest cell morphology. This study contributes to the growing literature on CR abnormalities in the pathophysiology of Parkinson's disease providing a link between the clinical and biological patient chronotype and the disease phenomenology.

Corrigendum: Deepening the understanding of CNVs on chromosome 15q11-13 by using hiPSCs: An overview.

[This corrects the article DOI: 10.3389/fcell.2022.1107881.].

Generation of an induced pluripotent stem cell line CSSi015-A (9553), carrying a point mutation c.2915C > T in the human calcium sensing receptor (CasR) gene.

Familial Hypocalciuric Hypercalcemia (FHH1) is a rare autosomal dominant disease with low penetrance, caused by inactivating mutations of the calcium-sensing receptor (CaSR) gene, characterized by significant hypercalcemia, inappropriately normal serum PTH levels and a low urinary calcium level. Human induced pluripotent stem cells (hiPSCs) from a patient carrying a previously identified heterozygous mutation, a p.T972M amino acid substitution in cytoplasmic tail of CasR, were produced using a virus, xeno-free and non-integrative protocol.

Human Neural Stem Cell-Based Drug Product: Clinical and Nonclinical Characterization.

Translation of cell therapies into clinical practice requires the adoption of robust production protocols in order to optimize and standardize the manufacture and cryopreservation of cells, in compliance with good manufacturing practice regulations. Between 2012 and 2020, we conducted two phase I clinical trials (EudraCT 2009-014484-39, EudraCT 2015-004855-37) on amyotrophic lateral sclerosis secondary progressive multiple sclerosis patients, respectively, treating them with human neural stem cells. Our production process of a hNSC-based medicinal product is the first to use brain tissue samples extracted from fetuses that died in spontaneous abortion or miscarriage. It consists of selection, isolation and expansion of hNSCs and ends with the final pharmaceutical formulation tailored to a specific patient, in compliance with the approved clinical protocol. The cells used in these clinical trials were analyzed in order to confirm their microbiological safety; each batch was also tested to assess identity, potency and safety through morphological and functional assays. Preclinical, clinical and in vitro nonclinical data have proved that our cells are safe and stable, and that the production process can provide a high level of reproducibility of the cultures. Here, we describe the quality control strategy for the characterization of the hNSCs used in the above-mentioned clinical trials.

Retinoic acid-induced 1 gene haploinsufficiency alters lipid metabolism and causes autophagy defects in Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment.

Generation and characterization of CSSi016-A (9938) human pluripotent stem cell line carrying two biallelic variants in MTMR5/SBF1 gene resulting in a case of severe CMT4B3.

Charcot-Marie-Tooth type 4B3 (CMT4B3) is a rare subtype of hereditary neuropathy associated with variants in the MTMR5/SBF1 gene. Herein, we report the generation and characterization of a hiPSC line from a 12-year-old Italian girl with early onset severe polyneuropathy with motor and axonal involvement, harboring biallelic variants in the MTMR5/SBF1 gene. Fibroblasts were reprogrammed using non-integrating episomal plasmids, and iPSCs successfully passed the stemness and pluripotency tests. Patient-specific hiPSCs were produced to obtain a disease model for the study of this rare condition.

AQP4-dependent glioma cell features affect the phenotype of surrounding cells via extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) are membrane-enclosed particles released systemically by all cells, including tumours. Tumour EVs have been shown to manipulate their local environments as well as distal targets to sustain the tumour in a variety of tumours, including glioblastoma (GBM). We have previously demonstrated the dual role of the glial water channel aquaporin-4 (AQP4) protein in glioma progression or suppression depending on its aggregation state. However, its possible role in communication mechanisms in the microenvironment of malignant gliomas remains to be unveiled. RESULTS: Here we show that in GBM cells AQP4 is released via EVs that are able to affect the GBM microenvironment. To explore this role, EVs derived from invasive GBM cells expressing AQP4-tetramers or apoptotic GBM cells expressing orthogonal arrays of particles (AQP4-OAPs) were isolated, using a differential ultracentrifugation method, and were added to pre-seeded GBM cells. Confocal microscopy analysis was used to visualize the interaction and uptake of AQP4-containing EVs by recipient cells. Chemoinvasion and Caspase3/7 activation assay, performed on recipient cells after EVs uptake, revealed that EVs produced by AQP4-tetramers expressing cells were able to drive surrounding tumour cells toward the migratory phenotype, whereas EVs produced by AQP4-OAPs expressing cells drive them toward the apoptosis pathway. CONCLUSION: This study demonstrates that the different GBM cell phenotypes can be transferred by AQP4-containing EVs able to influence tumour cell fate toward invasiveness or apoptosis. This study opens a new perspective on the role of AQP4 in the brain tumour microenvironment associated with the EV-dependent communication mechanism.

Human neural stem cells drug product: Microsatellite instability analysis.

INTRODUCTION: In central nervous system neurodegenerative disorders, stem cell-based therapies should be considered as a promising therapeutic approach. The safe use of human Neural Stem Cells (hNSCs) for the treatment of several neurological diseases is currently under evaluation of phase I/II clinical trials. Clinical application of hNSCs require the development of GMP standardized protocols capable of generating high quantities of reproducible and well characterized stem cells bearing stable functional and genetic properties. AIM: The aim of this study was to evaluate possible instabilities or modifications of the microsatellite loci in different culture passages because high culture passages represent an in vitro replicative stress leading to senescence. Experimental method: The hNSCs were characterized at different culture time points, from passage 2 to passage 25, by genetic typing at ten microsatellite loci. CONCLUSION: We showed that genetic stability at microsatellite loci is maintained by the cells even at high passages adding a further demonstration of the safety of our hNSCs GMP culture method.

KDM6A missense variants hamper H3 histone demethylation in lung squamous cell carcinoma.

KDM6A is the disease causative gene of type 2 Kabuki Syndrome, a rare multisystem disease; it is also a known cancer driver gene, with multiple somatic mutations found in a few cancer types. In this study, we looked at eleven missense variants in lung squamous cell carcinoma, one of the most common lung cancer subtypes, to see how they affect the KDM6A catalytic mechanisms. We found that they influence the interaction with histone H3 and the exposure of the trimethylated Lys27, which is critical for wild-type physiological function to varying degrees, by altering the conformational transition.

Generation of an induced pluripotent stem cells line, CSSi014-A 9407, carrying the variant c.479C>T in the human iduronate 2-sulfatase (hIDS) gene.

Mucopolysaccharidosis type II (Hunter Syndrome) is a rare X-linked inherited lysosomal storage disorder presenting a wide genetic heterogeneity. It is due to pathogenic variants in the IDS gene, causing the deficit of the lysosomal hydrolase iduronate 2-sulfatase, degrading the glycosaminoglycans (GAGs) heparan- and dermatan-sulfate. Based on the presence/absence of neurocognitive signs, commonly two forms are recognized, the severe and the attenuate ones. Here we describe a line of induced pluripotent stem cells, generated from dermal fibroblasts, carrying the mutation c.479C>T, and obtained from a patient showing an attenuated phenotype. The line will be useful to study the disease neuropathogenesis.

Production of CSSi013-A (9360) iPSC line from an asymptomatic subject carrying an heterozygous mutation in TDP-43 protein.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease affecting both upper and lower motoneurons. The transactive response DNA binding protein (TARDBP) gene, encoding for TDP-43, is one of the most commonly mutated gene associated with familial cases of ALS (10%). We generated a human induced pluripotent stem cell (hiPSC) line from the fibroblasts of an asymptomatic subject carrying the TARDBP p.G376D mutation. This mutation is very rare and was described in a large Apulian family, in which all ALS affected members are carriers of the mutation. The subject here described is the first identified asymptomatic carrier of the mutation.

Characterization of the p.L145F and p.S135N Mutations in SOD1: Impact on the Metabolism of Fibroblasts Derived from Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of the upper and lower motor neurons (MNs). About 10% of patients have a family history (familial, fALS); however, most patients seem to develop the sporadic form of the disease (sALS). SOD1 (Cu/Zn superoxide dismutase-1) is the first studied gene among the ones related to ALS. Mutant SOD1 can adopt multiple misfolded conformation, lose the correct coordination of metal binding, decrease structural stability, and form aggregates. For all these reasons, it is complicated to characterize the conformational alterations of the ALS-associated mutant SOD1, and how they relate to toxicity. In this work, we performed a multilayered study on fibroblasts derived from two ALS patients, namely SOD1L145F and SOD1S135N, carrying the p.L145F and the p.S135N missense variants, respectively. The patients showed diverse symptoms and disease progression in accordance with our bioinformatic analysis, which predicted the different effects of the two mutations in terms of protein structure. Interestingly, both mutations had an effect on the fibroblast energy metabolisms. However, while the SOD1L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1S135N fibroblasts showed a metabolic shift toward glycolysis. Our study suggests that SOD1 mutations might lead to alterations in the energy metabolism.

Growth factor independence underpins a paroxysmal, aggressive Wnt5aHigh/EphA2Low phenotype in glioblastoma stem cells, conducive to experimental combinatorial therapy.

BACKGROUND: Glioblastoma multiforme (GBM) is an incurable tumor, with a median survival rate of only 14-15 months. Along with heterogeneity and unregulated growth, a central matter in dealing with GBMs is cell invasiveness. Thus, improving prognosis requires finding new agents to inhibit key multiple pathways, even simultaneously. A subset of GBM stem-like cells (GSCs) may account for tumorigenicity, representing, through their pathways, the proper cellular target in the therapeutics of glioblastomas. GSCs cells are routinely enriched and expanded due to continuous exposure to specific growth factors, which might alter some of their intrinsic characteristic and hide therapeutically relevant traits. METHODS: By removing exogenous growth factors stimulation, here we isolated and characterized a subset of GSCs with a "mitogen-independent" phenotype (I-GSCs) from patient's tumor specimens. Differential side-by-side comparative functional and molecular analyses were performed either in vitro or in vivo on these cells versus their classical growth factor (GF)-dependent counterpart (D-GSCs) as well as their tissue of origin. This was performed to pinpoint the inherent GSCs' critical regulators, with particular emphasis on those involved in spreading and tumorigenic potential. Transcriptomic fingerprints were pointed out by ANOVA with Benjamini-Hochberg False Discovery Rate (FDR) and association of copy number alterations or somatic mutations was determined by comparing each subgroup with a two-tailed Fisher's exact test. The combined effects of interacting in vitro and in vivo with two emerging GSCs' key regulators, such as Wnt5a and EphA2, were then predicted under in vivo experimental settings that are conducive to clinical applications. In vivo comparisons were carried out in mouse-human xenografts GBM model by a hierarchical linear model for repeated measurements and Dunnett's multiple comparison test with the distribution of survival compared by Kaplan-Meier method. RESULTS: Here, we assessed that a subset of GSCs from high-grade gliomas is self-sufficient in the activation of regulatory growth signaling. Furthermore, while constitutively present within the same GBM tissue, these GF-independent GSCs cells were endowed with a distinctive functional and molecular repertoire, defined by highly aggressive Wnt5aHigh/EphA2Low profile, as opposed to Wnt5aLow/EphA2High expression in sibling D-GSCs. Regardless of their GBM subtype of origin, I-GSCs, are endowed with a raised in vivo tumorigenic potential than matched D-GSCs, which were fast-growing ex-vivo but less lethal and invasive in vivo. Also, the malignant I-GSCs' transcriptomic fingerprint faithfully mirrored the original tumor, bringing into evidence key regulators of invasiveness, angiogenesis and immuno-modulators, which became candidates for glioma diagnostic/prognostic markers and therapeutic targets. Particularly, simultaneously counteracting the activity of the tissue invasive mediator Wnt5a and EphA2 tyrosine kinase receptor addictively hindered GSCs' tumorigenic and invasive ability, thus increasing survival. CONCLUSION: We show how the preservation of a mitogen-independent phenotype in GSCs plays a central role in determining the exacerbated tumorigenic and high mobility features distinctive of GBM. The exploitation of the I-GSCs' peculiar features shown here offers new ways to identify novel, GSCs-specific effectors, whose modulation can be used in order to identify novel, potential molecular therapeutic targets. Furthermore, we show how the combined use of PepA, the anti-Wnt5a drug, and of ephrinA1-Fc to can hinder GSCs' lethality in a clinically relevant xenogeneic in vivo model thus being conducive to perspective, novel combinatorial clinical application.

Culturing and Expansion of "Clinical Grade" Neural Stem Cells from the Fetal Human Central Nervous System.

NSCs have been demonstrated to be very useful in grafts into the mammalian central nervous system to investigate the exploitation of NSC for the therapy of neurodegenerative disorders in animal models of neurodegenerative diseases. To push cell therapy in CNS on stage of clinical application, it is necessary to establish a continuous and standardized, clinical grade (i.e., produced following the good manufacturing practice guidelines) human neural stem cell lines.In this chapter we will illustrate some of the protocols for the production and characterization routinely used into our GMP "cell factory" for the production of "clinical grade" human neural stem cell lines already in use in clinical trials on neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS- Clinicaltrials.gov number NCT01640067) and secondary progressive multiple sclerosis (SPMS- Clinicaltrials.gov number NCT03282760).

Deepening the understanding of CNVs on chromosome 15q11-13 by using hiPSCs: An overview.

The human α7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is widely expressed in the central and peripheral nervous systems. This receptor is implicated in both brain development and adult neurogenesis thanks to its ability to mediate acetylcholine stimulus (Ach). Copy number variations (CNVs) of CHRNA7 gene have been identified in humans and are genetically linked to cognitive impairments associated with multiple disorders, including schizophrenia, bipolar disorder, epilepsy, Alzheimer's disease, and others. Currently, α7 receptor analysis has been commonly performed in animal models due to the impossibility of direct investigation of the living human brain. But the use of model systems has shown that there are very large differences between humans and mice when researchers must study the CNVs and, in particular, the CNV of chromosome 15q13.3 where the CHRNA7 gene is present. In fact, human beings present genomic alterations as well as the presence of genes of recent origin that are not present in other model systems as well as they show a very heterogeneous symptomatology that is associated with both their genetic background and the environment where they live. To date, the induced pluripotent stem cells, obtained from patients carrying CNV in CHRNA7 gene, are a good in vitro model for studying the association of the α7 receptor to human diseases. In this review, we will outline the current state of hiPSCs technology applications in neurological diseases caused by CNVs in CHRNA7 gene. Furthermore, we will discuss some weaknesses that emerge from the overall analysis of the published articles.