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    • Potential sources of NSCs include cell lines pluripotent

    2018-10-24

    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., 1995), but they may have unstable genomes or pose a risk of tumorigenesis (Koso et al., 2012; Mi et al., 2005; Snyder et al., 1995). Protocols for the differentiation of human embryonic stem uric acid (ESCs) allow for potentially unlimited expansion of transplantable NSCs, but ESC-derived NSCs are generally incompatible with the host immune system (Koch et al., 2009). 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, 2013; Walton et al., 2008; Wright et al., 2006). 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., 2013). 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., 1999). 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., 2012). Intravenous enzyme replacement therapy (ERT) is ineffectual in the CNS due to the impermeability of the blood-brain barrier (Augustine and Mink, 2013), and while intrathecal ERT may be efficacious, it requires regular infusions (Kakkis et al., 2004). 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., 2013; Snyder and Wolfe, 1996). 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, 1996). Here, we utilize the NOD/SCID/MPS VII model (Hofling et al., 2003) 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.
    Results
    Discussion 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., 2012; Koch et al., 2009). 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., 2009). 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., 2010). 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., 2013; Lemonnier et al., 2011), and in primary canine MPS VII NSCs (Walton and Wolfe, 2007). 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.