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• W2051234805 abstract "•Sly disease patient fibroblasts converted to iPSCs yield transplantable NSCs•A PiggyBac transposon-based approach corrects the lysosomal enzyme deficiency•Widespread migration of transplanted NSCs occurs in neonates, but not in adults•Reversal of microglial pathology in a zone surrounding corrected grafts Neural stem cell (NSC) transplantation is a promising strategy for delivering therapeutic proteins in the brain. We evaluated a complete process of ex vivo gene therapy using human induced pluripotent stem cell (iPSC)-derived NSC transplants in a well-characterized mouse model of a human lysosomal storage disease, Sly disease. Human Sly disease fibroblasts were reprogrammed into iPSCs, differentiated into a stable and expandable population of NSCs, genetically corrected with a transposon vector, and assessed for engraftment in NOD/SCID mice. Following neonatal intraventricular transplantation, the NSCs engraft along the rostrocaudal axis of the CNS primarily within white matter tracts and survive for at least 4 months. Genetically corrected iPSC-NSCs transplanted post-symptomatically into the striatum of adult Sly disease mice reversed neuropathology in a zone surrounding the grafts, while control mock-corrected grafts did not. The results demonstrate the potential for ex vivo gene therapy in the brain using human NSCs from autologous, non-neural tissues. Neural stem cell (NSC) transplantation is a promising strategy for delivering therapeutic proteins in the brain. We evaluated a complete process of ex vivo gene therapy using human induced pluripotent stem cell (iPSC)-derived NSC transplants in a well-characterized mouse model of a human lysosomal storage disease, Sly disease. Human Sly disease fibroblasts were reprogrammed into iPSCs, differentiated into a stable and expandable population of NSCs, genetically corrected with a transposon vector, and assessed for engraftment in NOD/SCID mice. Following neonatal intraventricular transplantation, the NSCs engraft along the rostrocaudal axis of the CNS primarily within white matter tracts and survive for at least 4 months. Genetically corrected iPSC-NSCs transplanted post-symptomatically into the striatum of adult Sly disease mice reversed neuropathology in a zone surrounding the grafts, while control mock-corrected grafts did not. The results demonstrate the potential for ex vivo gene therapy in the brain using human NSCs from autologous, non-neural tissues. Neural stem cells (NSCs), the self-renewing precursors of neurons and glia within the developing and adult brain, have the potential to serve as delivery vehicles for therapeutics in the CNS. Transplanted NSCs have been shown to migrate, engraft long term, and secrete beneficial levels of exogenous protein (Aboody et al., 2011Aboody K. Capela A. Niazi N. Stern J.H. Temple S. Translating stem cell studies to the clinic for CNS repair: current state of the art and the need for a Rosetta stone.Neuron. 2011; 70: 597-613Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, Snyder et al., 1995Snyder E.Y. Taylor R.M. Wolfe J.H. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain.Nature. 1995; 374: 367-370Crossref PubMed Scopus (429) Google Scholar). There are numerous potential sources of transplantable NSCs, and the development of efficacious therapies will depend on improved methods for their derivation and propagation (Conti and Cattaneo, 2010Conti L. Cattaneo E. Neural stem cell systems: physiological players or in vitro entities?.Nat. Rev. Neurosci. 2010; 11: 176-187Crossref PubMed Scopus (4) Google Scholar). Potential sources of NSCs include cell lines, pluripotent stem cell-derived NSCs, and primary NSCs harvested from fetal or adult animals. Some immortalized NSC lines can engraft and migrate extensively (Snyder et al., 1995Snyder E.Y. Taylor R.M. Wolfe J.H. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain.Nature. 1995; 374: 367-370Crossref PubMed Scopus (429) Google Scholar), but they may have unstable genomes or pose a risk of tumorigenesis (Koso et al., 2012Koso H. Takeda H. Yew C.C. Ward J.M. Nariai N. Ueno K. Nagasaki M. Watanabe S. Rust A.G. Adams D.J. et al.Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.Proc. Natl. Acad. Sci. USA. 2012; 109: E2998-E3007Crossref PubMed Scopus (52) Google Scholar, Mi et al., 2005Mi R. Luo Y. Cai J. Limke T.L. Rao M.S. Höke A. Immortalized neural stem cells differ from nonimmortalized cortical neurospheres and cerebellar granule cell progenitors.Exp. Neurol. 2005; 194: 301-319Crossref PubMed Scopus (46) Google Scholar, Snyder et al., 1995Snyder E.Y. Taylor R.M. Wolfe J.H. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain.Nature. 1995; 374: 367-370Crossref PubMed Scopus (429) Google Scholar). Protocols for the differentiation of human embryonic stem cells (ESCs) allow for potentially unlimited expansion of transplantable NSCs, but ESC-derived NSCs are generally incompatible with the host immune system (Koch et al., 2009Koch P. Opitz T. Steinbeck J.A. Ladewig J. Brüstle O. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration.Proc. Natl. Acad. Sci. USA. 2009; 106: 3225-3230Crossref PubMed Scopus (377) Google Scholar). Primary NSCs derived from the patient would circumvent immune rejection, but are difficult to obtain and have a relatively limited capacity for expansion and engraftment (Chaubey and Wolfe, 2013Chaubey S. Wolfe J.H. Transplantation of CD15-enriched murine neural stem cells increases total engraftment and shifts differentiation toward the oligodendrocyte lineage.Stem Cells Transl. Med. 2013; 2: 444-454Crossref PubMed Scopus (13) Google Scholar, Walton et al., 2008Walton R.M. Magnitsky S.G. Seiler G.S. Poptani H. Wolfe J.H. Transplantation and magnetic resonance imaging of canine neural progenitor cell grafts in the postnatal dog brain.J. Neuropathol. Exp. Neurol. 2008; 67: 954-962Crossref PubMed Scopus (9) Google Scholar, Wright et al., 2006Wright L.S. Prowse K.R. Wallace K. Linskens M.H. Svendsen C.N. Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro.Exp. Cell Res. 2006; 312: 2107-2120Crossref PubMed Scopus (117) Google Scholar). A compelling potential solution to this problem involves the use of patient-specific induced pluripotent stem cells (iPSCs), which offer a readily obtainable source of immunologically compatible cells that possess the broad expansion and engraftment potential of ESCs (Guha et al., 2013Guha P. Morgan J.W. Mostoslavsky G. Rodrigues N.P. Boyd A.S. Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells.Cell Stem Cell. 2013; 12: 407-412Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). The combination of iPSC technology, ESC to NSC differentiation methods, and ex vivo gene therapy offers a promising template for treating a wide range of CNS disorders. Lysosomal storage diseases (LSDs), which are among the most prevalent monogenetic disorders affecting the brain, may be particularly amenable to this strategy (Meikle et al., 1999Meikle P.J. Hopwood J.J. Clague A.E. Carey W.F. Prevalence of lysosomal storage disorders.JAMA. 1999; 281: 249-254Crossref PubMed Scopus (1662) Google Scholar). Most often the result of a nonfunctional lysosomal hydrolase, LSDs result in the pathological accumulation of various proteins, lipids, and sugars within the cell. Neuropathology is a significant component of most LSDs, and neurons are particularly susceptible to the accumulation of waste products and associated inflammatory processes (Platt et al., 2012Platt F.M. Boland B. van der Spoel A.C. The cell biology of disease: lysosomal storage disorders: the cellular impact of lysosomal dysfunction.J. Cell Biol. 2012; 199: 723-734Crossref PubMed Scopus (452) Google Scholar). Intravenous enzyme replacement therapy (ERT) is ineffectual in the CNS due to the impermeability of the blood-brain barrier (Augustine and Mink, 2013Augustine E.F. Mink J.W. Enzyme replacement in neuronal storage disorders in the pediatric population.Curr. Treat. Options Neurol. 2013; 15: 634-651Crossref PubMed Scopus (5) Google Scholar), and while intrathecal ERT may be efficacious, it requires regular infusions (Kakkis et al., 2004Kakkis E. McEntee M. Vogler C. Le S. Levy B. Belichenko P. Mobley W. Dickson P. Hanson S. Passage M. Intrathecal enzyme replacement therapy reduces lysosomal storage in the brain and meninges of the canine model of MPS I.Mol. Genet. Metab. 2004; 83: 163-174Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). NSC-based gene therapy is a potential approach to overcome these obstacles. Donor NSCs can secrete therapeutic levels of lysosomal enzymes, which traffic to host cell lysosomes via the mannose-6-phosphate pathway in a process known as cross-correction (Simonato et al., 2013Simonato M. Bennett J. Boulis N.M. Castro M.G. Fink D.J. Goins W.F. Gray S.J. Lowenstein P.R. Vandenberghe L.H. Wilson T.J. et al.Progress in gene therapy for neurological disorders.Nat. Rev. Neurol. 2013; 9: 277-291Crossref PubMed Scopus (160) Google Scholar, Snyder and Wolfe, 1996Snyder E.Y. Wolfe J.H. Central nervous system cell transplantation: a novel therapy for storage diseases?.Curr. Opin. Neurol. 1996; 9: 126-136Crossref PubMed Scopus (46) Google Scholar). Sly disease (MPS VII) is a prototypical LSD with which to test the efficacy of NSC transplantation due to the availability of cognate animal models and sensitive enzyme assays (Wolfe and Sands, 1996Wolfe J.H. Sands M.S. Murine mucopolysaccharidosis type VII: a model system for somatic gene therapy of the central nervous system.in: Lowenstein P. Enquist L. Gene Transfer into Neurones, towards Gene Therapy of Neurological Disorders. J. Wiley and Sons, Essex1996: 263-274Google Scholar). Here, we utilize the NOD/SCID/MPS VII model (Hofling et al., 2003Hofling A.A. Vogler C. Creer M.H. Sands M.S. Engraftment of human CD34+ cells leads to widespread distribution of donor-derived cells and correction of tissue pathology in a novel murine xenotransplantation model of lysosomal storage disease.Blood. 2003; 101: 2054-2063Crossref PubMed Scopus (34) Google Scholar) to demonstrate an integrated process by which patient somatic cells can be reprogrammed, differentiated into a relevant cell type (NSCs), genetically corrected, and transplanted to yield a therapeutic effect in a mouse homolog of the human disease. Frozen dermal fibroblasts from a patient with MPS VII (GM02784; Coriell Institute) were thawed, expanded, and transduced with vesicular stomatitis virus G protein (VSV-G) pseudotyped retroviral vectors expressing OCT4, SOX2, KLF-4, and c-MYC to initiate reprogramming. Despite the fact that these fibroblasts had been frozen for ∼30 years, colonies of ESC-like cells emerged alongside aggregates of partially reprogrammed cells, consistent with previous reports (Chan et al., 2009Chan E.M. Ratanasirintrawoot S. Park I.H. Manos P.D. Loh Y.H. Huo H. Miller J.D. Hartung O. Rho J. Ince T.A. et al.Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.Nat. Biotechnol. 2009; 27: 1033-1037Crossref PubMed Scopus (396) Google Scholar). One line was selected for further characterization. To confirm the pluripotency of MPS VII iPSCs, we performed standard in vitro and in vivo assays. The putative MPS VII-iPSC line displayed typical ESC/iPSC-like colony morphology (Figure 1A) and expressed multiple markers of pluripotency, including SSEA4, Tra-1-60, and Tra-1-81 (Figures 1B–1D). Cells injected into immunodeficient mice formed teratomas containing the three primary germ layers. H&E-stained teratoma sections revealed structures typical of endoderm, ectoderm, and mesoderm (Figures 1E–1H), and immunostained teratoma sections were positive for markers of each lineage (Figures 1I–1K). iPSCs were passaged >40 times with no change in morphology or pluripotency marker expression. MPS VII and control iPSCs were passaged at least 20 times before being subjected to an adapted NSC differentiation protocol (Koch et al., 2009Koch P. Opitz T. Steinbeck J.A. Ladewig J. Brüstle O. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration.Proc. Natl. Acad. Sci. USA. 2009; 106: 3225-3230Crossref PubMed Scopus (377) Google Scholar) (Figure 2). After iPSCs were removed from the mouse embryonic fibroblast (MEF) feeder layer and grown in suspension culture containing fetal bovine serum (FBS), they formed large spherical aggregates. These embryoid bodies were plated and grown in a minimal neural induction medium. A variety of cell types grew outward from the plated aggregates, including many with neurite-like extensions. After approximately 2 weeks, neural tube-like structures began to form on some cell aggregates, consisting of a raised ring surrounding a central lumen (Elkabetz et al., 2008Elkabetz Y. Panagiotakos G. Al Shamy G. Socci N.D. Tabar V. Studer L. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage.Genes Dev. 2008; 22: 152-165Crossref PubMed Scopus (506) Google Scholar). These rosette structures were isolated and grown as neurospheres for 2 days, after which they were dissociated and plated, yielding an adherent monolayer. No obvious differences were observed between normal and MPS VII iPSCs during the differentiation procedure at any point, and both yielded a relatively homogenous population of putative NSCs (Figure 2). The majority of the iPSC-NSCs (89.9% ± 2.9%) retained expression of the NSC marker nestin (Figures 3A and 3B). The rate of spontaneous differentiation into MAP2-positive neurons (9.5% ± 0.8%) and glial fibrillary acidic protein (GFAP)-positive astrocytes (0.4% ± 0.6%) was low (Figures 3A and 3B). Importantly, no cells expressed the pluripotency marker Tra-1-60 or the reprogramming factor OCT4 (Figures 3A and 3B). The generation and culture of iPSCs from frozen MPS VII fibroblasts and the subsequent differentiation and propagation of iPSC-NSCs did not introduce any gross chromosomal abnormalities, as shown by a normal 46,XX karyotype (Figure 3C). To test the differentiation capacity of these cells, we grew them in terminal differentiation medium without growth factors for 1 month. Differentiating conditions yielded neurons and astrocytes (Figure 3D), as measured by MAP2 (86.4% ± 1.6%) and GFAP (13.9% ± 8.2%) expression, respectively (Figure 3E). The majority of cells (74.8% ± 5.2%) were positive for the inhibitory neurotransmitter GABA, while there was no evidence of tyrosine hydroxylase-positive dopaminergic neurons (Figures 3D and 3E). In order to monitor the in vivo fate of iPSC-NSCs, we first attempted to label cells with GFP using lentiviral vectors with a tropism for multiple neural cell types in vitro and in vivo (Watson et al., 2002Watson D.J. Kobinger G.P. Passini M.A. Wilson J.M. Wolfe J.H. Targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV envelope proteins.Mol. Ther. 2002; 5: 528-537Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Survival of MPS VII iPSC-NSCs was very low following application of these vectors despite attempts using multiple MOIs, pseudotypes, and transduction conditions. To avoid this apparent toxicity, we used a PiggyBac transposon-based approach instead. The PiggyBac vector expressed a GFP gene and a puromycin resistance gene. The PiggyBac plasmid was electroporated along with a nonintegrating transposase expression plasmid into MPS VII iPSC-NSCs. Two days after electroporation, GFP expression was visible in many transfected cells (Figure 4A). Following a week of puromycin treatment, nearly all cells were GFP positive (Figure 4B) and retained nestin expression (Figure 4C). To assess the engraftment potential of MPS VII iPSC-NSCs, we intraventricularly injected GFP-labeled cells into neonatal mice, which provide a more hospitable environment for engraftment relative to the adult brain (Snyder et al., 1995Snyder E.Y. Taylor R.M. Wolfe J.H. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain.Nature. 1995; 374: 367-370Crossref PubMed Scopus (429) Google Scholar). Over 100 NOD-SCID neonates were injected with iPSC-NSCs between passages 15 and 25 with no evidence of deleterious effects. By 1-month post-transplant, cells had engrafted along the rostrocaudal axis of the brain and were primarily found in periventricular regions and white matter tracts (Figure 4E). GFP-labeled MPS VII iPSC-NSCs were also transplanted into NOD/SCID/MPS VII neonates. At 4-weeks post-transplant, the distribution of engrafted cells was similar to the distribution in non-MPS VII NOD/SCID littermates, with cells found predominantly in and around ventricles and white matter (Figure S2). Thus, the disease did not alter the engraftment properties of the donor cells. Regardless of location, the engrafted cells expressed nestin and had an immature morphology (Figure 4E, lower). The iPSC-NSCs survived for at least 4 months (Figure 5), with the donor cells predominantly located in the white matter. Engrafted iPSC-NSCs remained in an immature stage even after 4 months in vivo, as indicated by human-specific nestin immunostaining (Figure 5B). Despite their immature phenotype, transplanted iPSC-NSCs quickly exited the cell cycle. At 1-week post-transplant, only a few cells expressed the cell proliferation marker Ki67, and no Ki67-positive cells were seen at 4- or 16-weeks post-transplant (Figure S1). The MPS VII iPSC-NSCs were genetically corrected and introduced into an adult MPS VII host in order to evaluate their therapeutic potential. The iPSC-NSCs were electroporated with a PiggyBac vector expressing the GUSB cDNA driven by the CAG promoter. A separate culture of the same passage of NSCs was prepared as a negative control by electroporating them with a mock-correction vector containing the GUSB cDNA in the reverse orientation. After puromycin selection, the mock-corrected MPS VII iPSC-NSCs had negligible GUSB activity (1.3 ± 1.3 nmol 4-MU/μg protein/hr) while the corrected cells showed strong GUSB activity (116.8 ± 2.7 nmol 4-MU/μg protein/hr, p < 0.01), comparable to an iPSC-NSC line derived from a healthy control (112.3 ± 3.1 nmol 4-MU/μg protein/hr, p > 0.05) (Figure 6A). MPS VII is a progressive disease with extensive pathology present by 2 months of age (Levy et al., 1996Levy B. Galvin N. Vogler C. Birkenmeier E.H. Sly W.S. Neuropathology of murine mucopolysaccharidosis type VII.Acta Neuropathol. 1996; 92: 562-568Crossref PubMed Scopus (45) Google Scholar, Snyder et al., 1995Snyder E.Y. Taylor R.M. Wolfe J.H. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain.Nature. 1995; 374: 367-370Crossref PubMed Scopus (429) Google Scholar). Therefore, 2-month-old NOD/SCID/MPS VII mice were injected bilaterally into the striatum with 50,000 corrected MPS VII iPSC-NSCs in one hemisphere and 50,000 mock-corrected cells in the contralateral hemisphere. Animals were sacrificed 1 month later. Engrafted cells survived, remained nestin positive, and did not migrate away from the injection site. GUSB enzymatic activity, detected by a histochemical reaction (Snyder et al., 1995Snyder E.Y. Taylor R.M. Wolfe J.H. Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain.Nature. 1995; 374: 367-370Crossref PubMed Scopus (429) Google Scholar), was limited to the injection site of the hemisphere receiving corrected cells (Figure 6B). To test whether the lack of differentiation and migration was unique to our MPS VII line or to the diseased environment, we transplanted iPSC-NSCs derived from a healthy human control, as well as MPS VII iPSC-NSCs, into NOD/SCID (non-MPS VII) adult mice. At 1-month post-transplant, engrafted cells from both lines remained at the injection site and stained positive for human-specific nestin (Figure S3). This demonstrates that iPSC-NSCs from a normal or a diseased source had the same properties when transplanted into adult brain parenchyma. To determine whether corrected or mock-corrected MPS VII iPSC-NSCs affected neuropathology in MPS VII mice, we used changes in activated microglia as a marker of neuropathology. Neuroinflammation is a common finding in neurodegenerative diseases, and microarray analysis of the MPS VII brain has shown that CD68, the major marker of microglial activation, is transcribed at very high levels (Parente et al., 2012Parente M.K. Rozen R. Cearley C.N. Wolfe J.H. Dysregulation of gene expression in a lysosomal storage disease varies between brain regions implicating unexpected mechanisms of neuropathology.PLoS ONE. 2012; 7: e32419Crossref PubMed Scopus (36) Google Scholar). We confirmed via immunostaining that CD68 was highly upregulated in NOD/SCID/MPS VII animals early in the disease process, preceding the appearance of other commonly used markers of neuropathology (Figure S4). CD68-positive microglia were uniformly distributed throughout the MPS VII brain by 3 months of age (Figure 7). One month following adult transplantation, there was a striking reduction in CD68 immunoreactivity surrounding the corrected MPS VII iPSC-NSC grafts, but not the mock-corrected grafts (Figures 7A and 7B). The density of CD68-positive cells was quantified in a 0.5-mm2 region of interest (ROI) surrounding each injection tract (Figure 7C). There was a significant difference between the region surrounding corrected grafts (49.2 ± 9.9 cells/mm2) versus the region surrounding mock-corrected grafts (145.2 ± 13.2 cells/mm2, p < 0.0001) (Figure 7C). There was not a significant difference between the region surrounding mock-corrected grafts and a comparable region of untreated MPS VII striatum (166.1 ± 13.4 cells/mm2, p > 0.05). Immunostaining for CD68 and the pan-microglial marker showed that the microglia in untreated MPS VII mice as well as the microglia surrounding mock-corrected iPSC-NSCs had a distended, amoeboid-type morphology (Figure 7D). In contrast, the region surrounding corrected iPSC-NSCs contained smaller, ramified-type microglia, which closely resembled the microglia in normal control brains (Figure 7D). As a control for the effect of localized GUSB overexpression on CD68-positive microglia in the absence of transplanted iPSC-NSCs, we injected adult MPS VII mice with an AAV-GUSB vector (Passini et al., 2003Passini M.A. Watson D.J. Vite C.H. Landsburg D.J. Feigenbaum A.L. Wolfe J.H. Intraventricular brain injection of adeno-associated virus type 1 (AAV1) in neonatal mice results in complementary patterns of neuronal transduction to AAV2 and total long-term correction of storage lesions in the brains of beta-glucuronidase-deficient mice.J. Virol. 2003; 77: 7034-7040Crossref PubMed Scopus (169) Google Scholar). At 1-month post-injection, there was localized clearing of CD68-positive microglia around the injection site (Figure S5), similar to that seen after corrected MPS VII iPSC-NSC transplantation. Here we demonstrate the feasibility of ex vivo gene therapy for the treatment of neuropathology accompanying metabolic disease using patient-derived somatic cells from a readily accessible source (e.g., skin biopsy). By reprogramming patient fibroblasts into pluripotent stem cells and subsequently generating genetically corrected tissue-specific stem cells, we evaluated a therapeutic strategy that can be applied to many genetic diseases affecting the brain. We show, via xenotransplantation into a mouse homolog of the human disease, that such a strategy can reverse pathologic lesions surrounding the engrafted cells. The success of NSC-based therapy will depend on protocols that yield well-characterized and expandable lines suitable for transplantation. We chose an NSC differentiation protocol for its ability to generate a self-renewing population of relatively homogenous NSCs from ESC and iPSC lines (Falk et al., 2012Falk A. Koch P. Kesavan J. Takashima Y. Ladewig J. Alexander M. Wiskow O. Tailor J. Trotter M. Pollard S. et al.Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons.PLoS ONE. 2012; 7: e29597Crossref PubMed Scopus (195) Google Scholar, Koch et al., 2009Koch P. Opitz T. Steinbeck J.A. Ladewig J. Brüstle O. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration.Proc. Natl. Acad. Sci. USA. 2009; 106: 3225-3230Crossref PubMed Scopus (377) Google Scholar). The in vitro characteristics of MPS VII iPSC-NSCs generated here were consistent with reports utilizing similar ESC-based protocols in regards to the immunophenotype and ability to generate predominately GABAergic neurons upon withdrawal of growth factors (Koch et al., 2009Koch P. Opitz T. Steinbeck J.A. Ladewig J. Brüstle O. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration.Proc. Natl. Acad. Sci. USA. 2009; 106: 3225-3230Crossref PubMed Scopus (377) Google Scholar). We found that GUSB deficiency did not compromise the ability of human MPS VII iPSCs to generate embryoid bodies or differentiate toward neural lineages, in contrast to a previous report on a mouse MPS VII iPSC line (Meng et al., 2010Meng X.L. Shen J.S. Kawagoe S. Ohashi T. Brady R.O. Eto Y. Induced pluripotent stem cells derived from mouse models of lysosomal storage disorders.Proc. Natl. Acad. Sci. USA. 2010; 107: 7886-7891Crossref PubMed Scopus (37) Google Scholar). Disease-related phenotypes have been reported, in vitro, in iPSCs derived from patients with other LSDs, such as Niemann-Pick type C or MPS IIIB (Bergamin et al., 2013Bergamin N. Dardis A. Beltrami A. Cesselli D. Rigo S. Zampieri S. Domenis R. Bembi B. Beltrami C.A. A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient’s skin.Orphanet J. Rare Dis. 2013; 8: 34Crossref PubMed Scopus (25) Google Scholar, Lemonnier et al., 2011Lemonnier T. Blanchard S. Toli D. Roy E. Bigou S. Froissart R. Rouvet I. Vitry S. Heard J.M. Bohl D. Modeling neuronal defects associated with a lysosomal disorder using patient-derived induced pluripotent stem cells.Hum. Mol. Genet. 2011; 20: 3653-3666Crossref PubMed Scopus (68) Google Scholar), and in primary canine MPS VII NSCs (Walton and Wolfe, 2007Walton R.M. Wolfe J.H. Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease.J. Neuropathol. Exp. Neurol. 2007; 66: 760-769Crossref PubMed Scopus (16) Google Scholar). However, there was no evidence in our study that the MPS VII iPSC-NSCs or their progeny had a disease-related phenotypic difference in vitro. The GUSB deficiency also did not impair engraftment as we observed no apparent differences in numbers or distribution of donor NSCs between genetically corrected and mock-corrected MPS VII iPSC-NSCs after transplantation into MPS VII mice. Transplantation of iPSC-NSCs within the neonatal brain yielded stable engraftment across the neuroaxis for at least 4 months, but only within and adjacent to white matter tracts. Although the precise mechanisms are unclear, NSCs from various sources display a tropism for white matter in both normal and pathological contexts (Carney and Shah, 2011Carney B.J. Shah K. Migration and fate of therapeutic stem cells in different brain disease models.Neuroscience. 2011; 197: 37-47Crossref PubMed Scopus (27) Google Scholar, Gupta et al., 2012Gupta N. Henry R.G. Strober J. Kang S.M. Lim D.A. Bucci M. Caverzasi E. Gaetano L. Mandelli M.L. Ryan T. et al.Neural stem cell engraftment and myelination in the human brain.Sci. Transl. Med. 2012; 4: 155ra137Crossref PubMed Scopus (215) Google Scholar, Maciaczyk et al., 2009Maciaczyk J. Singec I. Maciaczyk D. Klein A. Nikkhah G. Restricted spontaneous in vitro differentiation and region-specific migration of long-term expanded fetal human neural precursor cells after transplantation into the adult rat brain.Stem Cells Dev. 2009; 18: 1043-1058Crossref PubMed Scopus (30) Google Scholar, Tabar et al., 2005Tabar V. Panagiotakos G. Greenberg E.D. Chan B.K. Sadelain M. Gutin P.H. Studer L. Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain.Nat. Biotechnol. 2005; 23: 601-606Crossref PubMed Scopus (149) Google Scholar). The affinity for white-matter tracts may be useful for treating leukodystrophies or as a pathway for NSC dissemination. The limited migration of transplanted NSCs within gray matter may be a barrier to widespread delivery for some diseases with global CNS pathology; however, axonal transport can facilitate wider distribution of lysosomal proteins within this group of diseases (Passini et al., 2002Passini M.A. Lee E.B. Heuer G.G. Wolfe J.H. Distribution of a lysosomal enzyme in the adult brain by axonal transport and by cells of the rostral migratory stream.J. Neurosci. 2002; 22: 6437-6446PubMed Google Scholar). We transplanted iPSC-NSCs into neonatal mice in order to evaluate their behavior in a more appropriate developmental context. Many cues required for the survival and migration of NSCs are present in neonates, but not in healthy adults (Guzman et al., 2007Guzman R. Uchida N. Bliss T.M. He D. Christopherson K.K. Stellwagen D. Capela A. Greve J. Malenka R.C. Moseley M.E. et al.Long-term monitoring of transplanted human neural stem cells in developmental and patholo" @default.
• W2051234805 created "2016-06-24" @default.
• W2051234805 creator A5013856846 @default.
• W2051234805 creator A5034329487 @default.
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• W2051234805 date "2015-05-01" @default.
• W2051234805 modified "2023-10-16" @default.
• W2051234805 title "Ex Vivo Gene Therapy Using Patient iPSC-Derived NSCs Reverses Pathology in the Brain of a Homologous Mouse Model" @default.
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