br Experimental Procedures Murine ESC lines E Tg A

Experimental Procedures Murine ESC lines E14Tg2A (passages 25–38) and K/l EB5 (transgenic Rax-GFP ESCs, used with the kind permission of Y. Sasai, RIKEN Center) and H9 hESCs (WiCell) were cultured as described (Bertacchi et al., 2013; Lupo et al., 2013). All animal protocols were reviewed and approved by the Animal Protocol Review Committee at ITB-CNR of Pisa. Detailed experimental procedures are available in Supplemental Information.
Acknowledgments
Introduction Sanfilippo syndrome, also known as mucopolysaccharidosis type III (MPS III), is a lysosomal storage disorder (LSD) with an autosomal recessive inheritance pattern. Four different subtypes have been described (type A, OMIM 252900; type B, OMIM 252920; type C, OMIM 252930; and type D, OMIM 252940), which share clinical characteristics, including severe early onset CNS degeneration that typically results in death within the second or third decade of life (Valstar et al., 2008). Each subtype is caused by mutations in a different gene encoding for enzymes involved in the degradation pathway of the glycosaminoglycan (GAG) heparan sulfate (Neufeld and Muenzer, 2001). The lack of activity of any of these enzymes leads to the accumulation of partially degraded heparan sulfate chains within the lysosomes. Subtype C (MPS IIIC) is caused by mutations in the HGSNAT gene, encoding acetyl-CoA α-glucosaminide N-acetyltransferase (EC 2.3.1.78), a lysosomal membrane enzyme. The prevalence of MPS IIIC ranges between 0.07 and 0.42 per 100,000 births, depending on the population (Poupetová et al., 2010). The HGSNAT gene was identified by two independent groups in 2006 (Fan et al., 2006; Hřebíček et al., 2006), and 64 different mutations have been identified since then (Human Gene Mutation Database Professional 2014.3). A mouse model has been very recently developed (Martins et al., 2015), but a cellular model for Sanfilippo type C has yet to be developed. The ability to reprogram somatic cyclobenzaprine hcl back to a pluripotent state (Takahashi and Yamanaka, 2006; Takahashi et al., 2007) has created new opportunities for generating in vitro models of disease-relevant cells differentiated from patient-specific induced pluripotent stem cell (iPSC) lines (recently reviewed by Cherry and Daley, 2013; Inoue et al., 2014; Trounson et al., 2012). This approach has been shown to be particularly useful in the case of congenital or early-onset monogenic diseases. In particular, iPSC-based models of various LSD have been established, including Gaucher disease (Mazzulli et al., 2011; Panicker et al., 2012; Park et al., 2008; Schöndorf et al., 2014; Tiscornia et al., 2013), Hurler syndrome (Tolar et al., 2011), Pompe disease (Higuchi et al., 2014; Huang et al., 2011), Sanfilippo B syndrome (Lemonnier et al., 2011), and Niemann-Pick type C1 (Maetzel et al., 2014; Trilck et al., 2013). In all these cases, disease-relevant cell types derived from patient-specific iPSCs not only displayed morphologic, biochemical, and/or functional hallmarks of the disease but also have the capacity of being used as a drug-screening platform to find therapies that are capable of reverting LSD-related phenotypes.
Results
Discussion iPSC technology has been widely used to model different types of diseases, including those affecting the CNS (Durnaoglu et al., 2011; Okano and Yamanaka, 2014). Some other LSDs have been modeled using iPSC technology, which were later differentiated to the human cellular type of interest for each case. For Pompe’s disease, cardiomyocytes exhibit the highest accumulation of glycogen, impaired autophagy, vacuolation, mitochondrial aberrances, and shorter survival times, features that were reverted after the overexpression of the normal gene (Huang et al., 2011). In the case of Hurler disease, hematopoietic and non-hematopoietic cells showed GAG accumulation and could be rescued by introducing the normal copy of the gene (Tolar et al., 2011). For Sanfilippo B syndrome, patient-derived neurons presented storage vesicles and Golgi disorganization (Lemonnier et al., 2011). In the case of Gaucher disease, iPSC-derived macrophages showed impaired lysosomal function and red blood cell clearance, recapitulating the hallmarks of the disease in this cell type, which could be reverted after administration of the recombinant enzyme (Panicker et al., 2012). Moreover, Gaucher disease-specific macrophages and neurons displayed low enzyme activity that could be partially rescued using small compounds with chaperone activity (Tiscornia et al., 2013); and dopaminergic neurons accumulated glucosylceramide and α-synuclein and showed autophagy and lysosomal defects and dysregulation of calcium homeostasis, all of which could be reverted after gene correction (Schöndorf et al., 2014). Finally, for Niemann-Pick type C1, iPSC-derived neurons exhibited spontaneous action potentials, confirming their maturation and accumulated cholesterol (Trilck et al., 2013). In another work regarding this disease, hepatic and neuronal cells presented lower cell viability, cholesterol storage, and impaired autophagy, features that could be reverted after gene correction (Maetzel et al., 2014). In many of these studies, the phenotypes observed could not be analyzed in fibroblasts, highlighting the importance of developing iPSC-derived models. Gene complementation provides an important experimental control that allows the assurance that the phenotypes detected are due to the genetic defect in the patient, rather than reprogramming artifacts.