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

Human iPSC-based neurodevelopmental models of globoid cell leukodystrophy uncover patient- and cell type-specific disease phenotypes

“…The LV-mediated supply of physiological or slightly supraphysiological GALC activity (3-4 fold the ND levels) in GLD iPSCs normalizes psychosine levels, confirming the potential of gene transfer to correct the primary storage in GALC-deficient human cells. In line with our previous study (Meneghini et al, 2017), we did not experience LV-mediated toxicity in hiPSCs, which were viable and functional for several passages after transduction, maintaining pluripotency and stable GALC activity.…”

“…All the selected GLD iPSC lines displayed normal phenotypic and functional properties despite having undetectable GALC enzymatic activity and significant psychosine storage. The dispensability of lysosomal enzymatic activity for somatic cell reprogramming and iPSC maintenance as well as the tolerability to lipid storage has been described in human iPSCs in other LSD models (Canals et al, 2015;Lojewski et al, 2014;Meneghini et al, 2017;Trilck et al, 2013). The LV-mediated supply of physiological or slightly supraphysiological GALC activity (3-4 fold the ND levels) in GLD iPSCs normalizes psychosine levels, confirming the potential of gene transfer to correct the primary storage in GALC-deficient human cells.…”

“…Human iPSCs have been differentiated into neurons and/or glial cells to model the neural defects associated to several lysosomal storage diseases (LSDs) including mucopolysaccharidosis (Bayó-Puxan et al, 2018;Canals et al, 2015;Kobolák et al, 2019;Lemonnier et al, 2011;Vallejo-Diez et al, 2018), Niemann−Pick type C (Maetzel et al, 2014;Ordoñez and Steele, 2016;Trilck et al, 2013;Yu et al, 2014), Pompe disease (Higuchi et al, 2014), Gaucher disease (Panicker et al, 2012;Sun et al, 2015;Tiscornia et al, 2013), GM1 and GM2 gangliosidosis (Son et al, 2015) (Allende et al, 2018), NCL (Lojewski et al, 2014), and MLD (Frati et al, 2018;Meneghini et al, 2017). To our knowledge this is the first report describing a human iPSC-based neural model of GLD.…”

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

“…In this regard, genome editing tools enable creating isogenic controls iPSCs that differ from the original counterpart only on the gene/s responsible for the disease [43,44]. Despite these limitations, iPSCs have shown to be very promising for recapitulating complex genetically disorders especially when combined with gene editing techniques [33,45,46]. Timothy syndrome Patient-derived fibroblasts [77,78] The selective modification of cells' genetic information was the groundbreaking discovery that revolutionized the concept of cell models.…”

Generation of UCiPSCs-Derived Neurospheres for Cell Therapy and its Application in Cell Shipment at Ambient Temperature

“…Compared to these, Zhou et.al discovered that urinary cells provide us a non-invasive, practical and unlimited source for reprogramming [11]. Since the discovery of induced pluripotent stem cells (iPSCs) [6], induced neural stem cells (iNSCs) have progressed rapidly toward applications in neurodegenerative disease [12]. The availability of patient-speci c iPSCs-derived NSCs may alleviate di culties of immunological issues and ethical concerns.…”