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

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.

The pathological cascade leading from primary storage to neural cell dysfunction and death in metachromatic leukodystrophy (MLD) has been poorly elucidated in human-derived neural cell systems. In the present study, we have modeled the progression of pathological events during the differentiation of patient-specific iPSCs to neuroepithelial progenitor cells (iPSC-NPCs) and mature neurons, astrocytes, and oligodendrocytes at the morphological, molecular, and biochemical level. We showed significant sulfatide accumulation and altered sulfatide composition during the differentiation of MLD iPSC-NPCs into neuronal and glial cells. Changes in sulfatide levels and composition were accompanied by the expansion of the lysosomal compartment, oxidative stress, and apoptosis. The neuronal and glial differentiation capacity of MLD iPSC-NPCs was significantly impaired. We showed delayed appearance and/or reduced levels of oligodendroglial and astroglial markers as well as reduced number of neurons and disorganized neuronal network. Restoration of a functional Arylsulfatase A (ARSA) enzyme in MLD cells using lentiviral-mediated gene transfer normalized sulfatide levels and composition, globally rescuing the pathological phenotype. Our study points to MLD iPSC-derived neural progeny as a useful in vitro model to assess the impact of ARSA deficiency along NPC differentiation into neurons and glial cells. In addition, iPSC-derived neural cultures allowed testing the impact of ARSA reconstitution/overexpression on disease correction and, importantly, on the biology and functional features of human NPCs, with important therapeutic implications.