A new patient‐derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α‐dystroglycan
2019; Springer Nature; Volume: 20; Issue: 11 Linguagem: Inglês
10.15252/embr.201947967
ISSN1469-3178
AutoresJihee Kim, Beatrice Lana, Silvia Torelli, David Ryan, Francesco Catapano, Pierpaolo Ala, Christin Luft, Elizabeth Stevens, Evangelos Konstantinidis, Sandra Louzada, Beiyuan Fu, Amaia Paredes‐Redondo, AW Edith Chan, Fengtang Yang, Derek L. Stemple, Pentao Liu, Robin Ketteler, David L. Selwood, Francesco Muntoni, Yung‐Yao Lin,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle30 September 2019Open Access Transparent process A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan Jihee Kim Jihee Kim Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author Beatrice Lana Beatrice Lana Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author Silvia Torelli Silvia Torelli UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author David Ryan David Ryan Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Francesco Catapano Francesco Catapano UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Pierpaolo Ala Pierpaolo Ala UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Christin Luft Christin Luft MRC Laboratory for Molecular Cell Biology, University College London, London, UK Search for more papers by this author Elizabeth Stevens Elizabeth Stevens UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Evangelos Konstantinidis Evangelos Konstantinidis Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author Sandra Louzada Sandra Louzada Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Beiyuan Fu Beiyuan Fu Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Amaia Paredes-Redondo Amaia Paredes-Redondo Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author AW Edith Chan AW Edith Chan The Wolfson Institute for Biomedical Research, University College London, London, UK Search for more papers by this author Fengtang Yang Fengtang Yang Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Derek L Stemple Derek L Stemple orcid.org/0000-0002-8296-9928 Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Pentao Liu Pentao Liu Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Robin Ketteler Robin Ketteler orcid.org/0000-0002-2786-7291 MRC Laboratory for Molecular Cell Biology, University College London, London, UK Search for more papers by this author David L Selwood David L Selwood orcid.org/0000-0002-6817-5064 The Wolfson Institute for Biomedical Research, University College London, London, UK Search for more papers by this author Francesco Muntoni Francesco Muntoni UCL Great Ormond Street Institute of Child Health, London, UK NIHR Biomedical Research Centre at Great Ormond Street Hospital, London, UK Search for more papers by this author Yung-Yao Lin Corresponding Author Yung-Yao Lin [email protected] orcid.org/0000-0002-0435-7694 Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author Jihee Kim Jihee Kim Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author Beatrice Lana Beatrice Lana Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author Silvia Torelli Silvia Torelli UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author David Ryan David Ryan Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Francesco Catapano Francesco Catapano UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Pierpaolo Ala Pierpaolo Ala UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Christin Luft Christin Luft MRC Laboratory for Molecular Cell Biology, University College London, London, UK Search for more papers by this author Elizabeth Stevens Elizabeth Stevens UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Evangelos Konstantinidis Evangelos Konstantinidis Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author Sandra Louzada Sandra Louzada Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Beiyuan Fu Beiyuan Fu Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Amaia Paredes-Redondo Amaia Paredes-Redondo Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author AW Edith Chan AW Edith Chan The Wolfson Institute for Biomedical Research, University College London, London, UK Search for more papers by this author Fengtang Yang Fengtang Yang Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Derek L Stemple Derek L Stemple orcid.org/0000-0002-8296-9928 Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Pentao Liu Pentao Liu Wellcome Sanger Institute, Hinxton, Cambridge, UK Search for more papers by this author Robin Ketteler Robin Ketteler orcid.org/0000-0002-2786-7291 MRC Laboratory for Molecular Cell Biology, University College London, London, UK Search for more papers by this author David L Selwood David L Selwood orcid.org/0000-0002-6817-5064 The Wolfson Institute for Biomedical Research, University College London, London, UK Search for more papers by this author Francesco Muntoni Francesco Muntoni UCL Great Ormond Street Institute of Child Health, London, UK NIHR Biomedical Research Centre at Great Ormond Street Hospital, London, UK Search for more papers by this author Yung-Yao Lin Corresponding Author Yung-Yao Lin [email protected] orcid.org/0000-0002-0435-7694 Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK Search for more papers by this author Author Information Jihee Kim1,2,‡, Beatrice Lana1,2,‡, Silvia Torelli3, David Ryan4, Francesco Catapano3, Pierpaolo Ala3, Christin Luft5, Elizabeth Stevens3, Evangelos Konstantinidis1,2, Sandra Louzada4, Beiyuan Fu4, Amaia Paredes-Redondo1,2, AW Edith Chan6, Fengtang Yang4, Derek L Stemple4, Pentao Liu4, Robin Ketteler5, David L Selwood6, Francesco Muntoni3,7,‡ and Yung-Yao Lin *,1,2,‡ 1Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK 2Stem Cell Laboratory, National Bowel Research Centre, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK 3UCL Great Ormond Street Institute of Child Health, London, UK 4Wellcome Sanger Institute, Hinxton, Cambridge, UK 5MRC Laboratory for Molecular Cell Biology, University College London, London, UK 6The Wolfson Institute for Biomedical Research, University College London, London, UK 7NIHR Biomedical Research Centre at Great Ormond Street Hospital, London, UK ‡These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +44 2078 822339; E-mail: [email protected] EMBO Reports (2019)20:e47967https://doi.org/10.15252/embr.201947967 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development. Synopsis Defective glycosylation of α-dystroglycan is a pathological hallmark of secondary dystroglycanopathies that often affect the central nervous system. An unbiased screen identifies 4BPPNit that augments glycosylation of α-dystroglycan as validated in a patient-derived iPSC model. First human FKRP-iPSC line was generated from a dystroglycanopathy patient with severe CNS abnormalities. Targeted gene correction of FKRP by CRISPR/Cas9 restores α-dystroglycan glycosylation in iPSC-derived neural cells. An unbiased high-throughput chemical screen for increased glycosylation of α-dystroglycan identifies 4BPPNit. 4BPPNit significantly augments glycosylation of α-dystroglycan in human FKRP-iPSC derived neural cells. Introduction Post-translational processing of dystroglycan is crucial for its function as an extracellular matrix (ECM) receptor in a variety of fetal and adult tissues. The dystroglycan precursor is cleaved into non-covalently associated α- and β-subunits, forming an integral component of a multiprotein complex. The mature α-dystroglycan is a heavily glycosylated peripheral membrane protein with molecular weight range from 100 to 156 kDa, dependent on tissue-specific glycosylation. The β-dystroglycan is a 43-kDa transmembrane protein that links to the actin cytoskeleton via interaction with dystrophin 1. Defective O-linked glycosylation of α-dystroglycan is a common pathological hallmark associated with number of genetic syndromes, encompassing symptoms from muscular dystrophies to ocular defects, cognitive deficits, and structural cortical malformations (cobblestone lissencephaly) in the central nervous system (CNS). This group of autosomal recessive disorders are commonly referred to as secondary dystroglycanopathies 2, 3. Currently, there is no cure for dystroglycanopathies. The effort of identifying causative gene mutations in dystroglycanopathies has shed light on a novel mammalian glycosylation pathway 4, 5. To date, at least 18 genes have been implicated in dystroglycanopathies and their products elaborate sequentially the functional glycosylation of α-dystroglycan (core M3 glycan) required for binding with (ECM) ligands, e.g., laminins, perlecan, and neurexin (Fig 1A) 4, 5. The genes involved include those for dolichol-phosphate-mannose synthesis: GMPPB, DPM1, DPM2, DPM3 and DOLK 6-13; genes required for O-mannosylation and subsequent sugar addition: POMT1, POMT2, POMGNT1, POMGNT2/GTDC2, and B3GALNT2 14-21 and an O-mannose-specific kinase gene: POMK/SGK196 21, 22. Recent studies have demonstrated that the ISPD gene encodes a CDP-ribitol pyrophosphorylase that generates the reduced nucleotide sugar for the addition of tandem ribitol-5-phosphate to α-dystroglycan by ribitol-5-phosphate transferases, encoded by the FKTN and FKRP genes (Fig 1A) 23-30. Furthermore, TMEM5 and B4GAT1/B3GNT1 genes encode enzymes to prime the phospho-ribitol with xylose and then glucuronic acid 27, 31-34. LARGE1 encodes a bifunctional enzyme to synthesize the subsequent extension of xylose-glucuronic acid disaccharide repeats that function as the binding sites for laminins (IIH6 epitope) (Fig 1A) 35, 36. Overexpression of LARGE1 in human cell lines and transgenic mice results in enhanced functional glycosylation of α-dystroglycan and similar genetic strategies have been proposed as a potential therapeutic approach in dystroglycanopathies 37, 38. Figure 1. Functional glycosylation of α-dystroglycan and characterization of dystroglycanopathy patient-specific iPSCs Current model of the core M3 functional glycan structure on α-dystroglycan and enzymes involved in its synthesis. ECM ligands, such as laminins, bind to the Xyl-GlucA disaccharide repeats (IIH6 epitope). Man, mannose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Rbo5P, ribitol-5-phosphate; Xyl, xylose; GlcA, glucuronic acid. Representative images of immunostaining demonstrate that FKRP-iPSCs express specific pluripotency-associated markers, including NANOG, OCT4, Tra-1-60, and SSEA4. FKRP-iPSCs have a normal karyotype. In vitro differentiation of FKRP-iPSCs to cells representing ectoderm (β-III Tubulin, Tuj1), mesoderm (SMA, smooth muscle actin), and endoderm (AFP, α-fetoprotein). Data information: Scale bars, 50 μm. Download figure Download PowerPoint Among dystroglycanopathies, allelic FKRP mutations are the more prevalent and cause a wide spectrum of clinical severities that range from the mild late-onset limb-girdle muscular dystrophy without neurological deficits (e.g. LGMD2I) to congenital muscular dystrophy with severe CNS abnormalities (e.g. Walker–Warburg syndrome) 39. Malformations of cortical development are pathological features commonly encountered in patients at the severe end of the dystroglycanopathy spectrum 40, 41. Although murine models have been widely used to study mammalian cortical malformations, successful translation from animal models to patients has been limited. To facilitate drug discovery and development for dystroglycanopathy, there is an unmet need for isogenic, physiology-relevant human cell models to test potential drug candidates in a human- and neural-specific context. Recent studies have demonstrated success in using patient-specific induced pluripotent stem cells (iPSCs) to model a variety of neurodevelopment and neurodegenerative disease and their use in high-throughput screening 42, 43. We hypothesized that patient-specific iPSC-derived neural cells can recapitulate pathological hallmarks in dystroglycanopathies and can be used for testing potential drug candidates. Here, we generated the first human FKRP-iPSCs from a dystroglycanopathy patient with severe CNS abnormalities. We carried out CRISPR/Cas9-mediated genome editing 44, 45 to correct the FKRP mutation in FKRP-iPSCs or knock-in the FKRP mutation in unrelated wild-type iPSCs. We showed that, for the first time, targeted gene correction of FKRP restores tissue-specific α-dystroglycan functional glycosylation in iPSC-derived neural cells, whereas targeted gene mutation of FKRP in wild-type cells leads to the expected disruption of functional glycosylation of α-dystroglycan. In parallel, we screened 31,954 compounds for increased glycosylation of α-dystroglycan in the H2K 2B4 mouse myoblast cell line 46. We identified and validated a compound, 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit; PubChem CID 2837349), that significantly augmented tissue-specific functional glycosylation of α-dystroglycan in human FKRP-iPSC-derived neural cells. This effect appears to be secondary to the upregulated LARGE1 gene expression, suggesting a mechanism by which 4BPPNit leads to increased functional glycosylation of α-dystroglycan. This proof-of-concept study demonstrates that isogenic, physiology-relevant human iPSC-based platform can contribute to facilitating drug development for dystroglycanopathies. Results Generation and characterization of FKRP-iPSCs derived from a dystroglycanopathy patient with CNS abnormalities We obtained dermal fibroblasts from a female individual, previously diagnosed with congenital muscular dystrophy with CNS abnormalities, including marked cognitive delay, microcephaly, cerebellar cysts, and cerebellar dysplasia. Clinical features summarized in Table 1 are consistent with previously reported descriptions 40, 47, 48. Genetic analysis revealed homozygous FKRP c.1364C>A (p.A455D) mutations in this affected individual; both parents are heterozygous carriers of this mutation. To generate dystroglycanopathy iPSCs, we reprogrammed the patient's fibroblasts carrying the FKRP c.1364C>A mutations using a six-factor reprogramming technology based on a doxycycline-inducible system 49. The initial characterization of five independent FKRP-iPSC clonal lines by qPCR revealed the gene expression of pluripotency markers, such as NANOG, OCT4 and REX1 (Appendix Fig S1). Among these clonal iPSC lines, we focused on the FKRP-iPSC line (clone 1-6). Immunocytochemistry confirmed the expression of pluripotency markers, such as NANOG, OCT4, Tra-1-60 and SSEA4 in this FKRP-iPSC line (Fig 1B), which exhibited the normal karyotype (Fig 1C). In addition, in vitro differentiation of the FKRP-iPSCs formed embryoid bodies with cell types representing all three embryonic germ layers, confirmed by immunocytochemistry in specific cell lineage markers, such as β-III tubulin (Tuj1) for ectoderm, smooth muscle actin (SMA) for mesoderm, and α-fetoprotein (AFP) for endoderm (Fig 1D). Table 1. Clinical features of the affected individual with FKRP mutations Subject FKRP variants c.1364C>A (p.A455D) Zygosity Homozygous Sex Female Age at assessment 11 months Symptoms at presentation Hypotonia; floppiness; paucity of spontaneous movements Feeding difficulties From first few months of life Respiratory difficulties Frequent chest infection from 6 months Orthopedic complications Hip subluxation Max functional achievement Partial antigravity in arms and legs; unable to sit Serum CK (NR < 200 U/l) 5000-7090 CNS function Partially alert Head circumference Microcephaly MRI Cerebellar cysts and cerebellar dysplasia Muscle biopsy Dystrophic; severely reduced IIH6 staining Targeted gene correction of FKRP-iPSCs using CRISPR/Cas9-mediated genome editing A common issue of patient-specific iPSCs in disease modeling is the lack of appropriate isogenic control cells, causing concerns about the effect of genetic backgrounds on phenotypic variability 50. To overcome this issue, we applied a precise genome-editing strategy to correct the FKRP c.1364C>A (p.A455D) mutation using site-specific endonuclease CRISPR/Cas9 stimulated homologous recombination 44, 45, which consists of a single-guide RNA (sgRNA), the Cas9 nuclease, and a donor-targeting vector. To minimize potential off-target effects, we used an optimized computational algorithm (http://crispr.mit.edu) to identify appropriate sgRNAs for utilizing the CRISPR/Cas9 to generate DNA double-strand breaks (DSB) near the FKRP mutation (Fig 2A). The sgRNA used in this study has high on-target and low off-target scores (Appendix Table S1). A transposon-based selection cassette piggyBac (PGK-puro∆tk) was used in the targeting donor vector to enable both positive and negative selections to facilitate the screening process for edited events 51. Figure 2. Targeted gene correction of FKRPA455D-iPSCs by CRISPR/Cas9-mediated genome editing Cas9 protein and the specific sgRNA targeting the human FKRP locus. The FKRP c.1364C>A (p.A455D) mutation is 43 bases upstream of the sgRNA target sequences. Red arrowheads indicate putative cleavage site. PAM, protospacer-adjacent motif. A schematic diagram shows the genome-editing strategy based on CRISPR/Cas9-stimulated homologous recombination, followed by positive selection with puromycin and negative selection with FIAU. Homology left and right arms on the targeting donor vector are indicated as black boxes, flanking the piggyBac (PGK-puro∆tk) selection cassette, which is under the control of PGK promoter. PCR genotyping primers are shown as blue arrows. Note that the TTAA sequences are designed to accommodate the selection cassette excision sites, yet code the same amino acids. Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected-iPSC clones (5D17, 5D23, and 3B17), compared with their parental FKRPA455D-iPSCs. Selection cassette excision sites are identified in the corrected-iPSC lines. CRISPR/Cas9 corrected-iPSC lines show a normal karyotype. Download figure Download PowerPoint We PCR-amplified two 1-kb fragments flanking the CRISPR/Cas9 target site close to the FKRP c.1364C>A allele, which was simultaneously corrected (FKRP c.1364C). The two fragments (homology left and right arms) flanking a piggyBac (PGK-puro∆tk) selection cassette were assembled together into a targeting donor vector (Fig 2B). DNA sequences at the junctions between piggyBac (PGK-puro∆tk) selection cassette and homology arms were modified to accommodate TTAA sequences, yet coding the same amino acids after excision of the selection cassette (Fig 2B). We electroporated the site-specific CRISPR/Cas9 plasmids with the targeting donor vector (FKRP c.1364C) into the iPSCs. By positive selection with puromycin, iPSCs with an integrated donor vector formed puromycin-resistant clones, which were picked for rapid PCR genotyping (Appendix Fig S2A and B). We identified 3 homozygously and 27 heterozygously targeted independent clones (Appendix Table S2), which were confirmed by sequencing (data not shown). To excise the piggyBac (PGK-puro∆tk) selection cassette, 2 homozygously targeted FKRP (c.1364C)-iPSC clones (5.19 and 3.16) were electroporated with piggyBac transposase expression plasmid, followed by negative selection in culture media containing 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodo-2,4(1H,3H)-pyrimidinedione (FIAU), a thymidine analogue that is processed to toxic metabolites in the presence of piggyBac (PGK-puro∆tk). PCR genotyping identified 11 biallelicly corrected-iPSC clones that had the selection cassette completely excised without re-integration (Appendix Fig S2C and Table S2). The biallelicly corrected-iPSC clones (5D17, 5D23, and 3B17) were sequenced to confirm a precise correction of the FKRP mutation and the engineered selection cassette excision site (Fig 2C). The biallelicly corrected-iPSC lines retained normal karyotypes (Fig 2D). In addition, sequencing of the top 5 potential off-target sites (Appendix Table S1) confirmed that no mutations were introduced during genome editing (data not shown). Generation of neural stem cells and cortical neurons from FKRP- and CRISPR/Cas9 corrected-iPSCs To investigate the potential of iPSCs for modeling neural pathogenesis in dystroglycanopathies, we used a serum-free neural induction medium to derive primitive neural stem cells (NSCs) from iPSCs 52. Immunocytochemistry confirmed that NSCs derived from FKRP- and CRISPR/Cas9 corrected-iPSC lines expressed the classic NSC markers, including SOX1, SOX2, and nestin (Fig 3A and B). In terms of the efficiency of neural induction, no discernible difference was observed between NSCs derived from FKRP- and the three corrected-iPSC lines, 5D17, 5D23, and 3B17 (Fig 3C and D). Quantification of SOX1- and SOX2-positive NSCs showed >99% efficiency from both FKRP- and three corrected-iPSC lines (Fig 3C and D). Figure 3. Characterization of NSCs and cortical neurons derived from FKRP- and CRISPR/Cas9 corrected-iPSCs A, B. Representative images of NSCs derived from FKRP- and corrected-iPSC lines expressing SOX1, SOX2, and nestin. C, D. Quantification of percentage of SOX1+ (C) and SOX2+ (D) cells in culture. The efficiency of neural induction is more than 99% in FKRP- and corrected-iPSC (5D17, 5D23, and 3B17) lines. Data are mean ± s.d. n = 4 technical replicates. E, F. FKRP- and corrected-NSC lines can be further differentiated to cortical neural progenitor cells, expressing PAX6, OTX2, and vimentin. G–I. Quantification of percentage of PAX6+ (G) and OTX2+ (H) cells in culture. About 91-98% of cells derived from FKRP, 5D17, 5D23, and 3B17 NSC lines express PAX6 (G). About 93-96% of cells derived from FKRP, 5D17, 5D23, and 3B17 NSC lines express OTX2 (H). Of the OTX2+ population, about 60-67% cells are also Ki67+ cycling progenitors (I). Data are mean ± s.d. n = 4 technical replicates. J, K. Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23, and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labeled by Tuj1). Right panels are enlarged images from the insets of left panels. Data information: Scale bars, 50 μm. Download figure Download PowerPoint Subsequently, we directed the differentiation of FKRP- and corrected-NSC lines toward cortical projection neurons using a well-established protocol, which recapitulates important stages in human cortical development 53. After switching to the Neural Maintenance Medium for one week, we confirmed the identity of FKRP- and corrected-iPSC-derived cortical stem and progenitor cells, which expressed the classic cortical stem cell markers, PAX6, OTX2, and vimentin (Fig 3E and F); the proliferating cells were Ki67-positive (Fig 3E and F). The efficiency of cortical induction between FKRP and three lines of corrected cortical progenitors were very similar. The PAX6+ cells in culture were about 91-98%, and OTX2+ cells are about 93-96% (Fig 3G and H); 60-67% of OTX2+ cells were also Ki67+ cycling progenitors (Fig 3I). After three weeks in Neural Maintenance Medium, FKRP and corrected progenitor-derived cells showed punctate staining of vesicular glutamate transporter 1 (vGlut1) in their neurites labeled by neuron-specific tubulin, Tuj1 (Fig 3J and K). This confirmed the generation of glutamatergic projection neurons during cortical neurogenesis in culture. Although the FKRP(A455D) variant does not appear to be required during early neurogenesis, we cannot exclude that this mutation might be responsible for pathological phenotypes at a later stage. Tissue-specific functional glycosylation of α-dystroglycan is restored in cortical neurons derived from CRISPR/Cas9-corrected-iPSCs Next, we investigated the presence of functional glycosylation of α-dystroglycan in cortical neurons derived from our corrected-iPSC lines using immunoblotting with the IIH6 antibody, which recognizes the laminin-binding glyco-epitope on α-dystroglycan 54. Wild-type mouse muscle and brain lysates were used as positive controls to show differential glycosylation of α-dystroglycan in a tissue-specific manner. In addition, lysate of cortical neurons derived from a non-isogenic, human wild-type iPSC line (WT-iPSCs) was included 49. We showed that the molecular weight of glycosylated α-dystroglycan in WT-iPSC-derived cortical neurons is similar to that in mouse brain lysate (~120 kDa) and less than that in muscle lysate (~156 kDa) (Fig 4A), consistent with previo
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