title: Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy.
abstract: Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Vasco
authors.initials: V
authors.lastname: Meneghini
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Frati
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Sala
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: De Cicco
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Luciani
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Cavazzin
authors.affiliation: National Research Council, Milan, Italy.|Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Marianna
authors.initials: M
authors.lastname: Paulis
authors.affiliation: BioFlag Ltd., Pula, Cagliari, Italy.
authors.forename: Wieslawa
authors.initials: W
authors.lastname: Mentzen
authors.affiliation: Biochemistry and Molecular Biology Unit, Department of Chemistry, Biology and Biotechnologies, University of Perugia, Perugia, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Morena
authors.affiliation: Division of Neuroscience, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Giannelli
authors.affiliation: Anatomy and Histopathology Department, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Sanvito
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.|National Research Council, Milan, Italy.|Humanitas Clinical and Research Center, Rozzano, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Villa
authors.affiliation: BioFlag Ltd., Pula, Cagliari, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bulfone
authors.affiliation: Division of Neuroscience, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Vania
authors.initials: V
authors.lastname: Broccoli
authors.affiliation: Biochemistry and Molecular Biology Unit, Department of Chemistry, Biology and Biotechnologies, University of Perugia, Perugia, Italy.
authors.forename: Sabata
authors.initials: S
authors.lastname: Martino
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Milan, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Gritti
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002465:Cell Movement; D065150:Cellular Reprogramming; D000067470:Cellular Reprogramming Techniques; D002553:Cerebroside-Sulfatase / Q000096:biosynthesis* / Q000235:genetics; D018920:Coculture Techniques; D004195:Disease Models, Animal; D004790:Enzyme Induction; D018507:Gene Expression Regulation, Developmental; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000201:enzymology / Q000637:transplantation*; D007966:Leukodystrophy, Metachromatic / Q000201:enzymology / Q000235:genetics / Q000503:physiopathology / Q000601:surgery*; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009416:Nerve Regeneration; D058953:Neural Stem Cells / Q000201:enzymology / Q000637:transplantation*; D010641:Phenotype; D033581:Stem Cell Transplantation / Q000379:methods*; D013433:Sulfoglycosphingolipids / Q000378:metabolism; D059467:Transcriptome
chemical_list: D013433:Sulfoglycosphingolipids; D002553:Cerebroside-Sulfatase
keywords: Gene therapy; Intracerebral transplantation; Metachromatic leukodystrophy; Neural stem cell; Oligodendrocytes; Pluripotent stem cells