Centronuclear myopathies (CNMs) are a group of inherited rare muscle disorders characterised by the abnormal position of the nucleus in the center of the muscle fiber. One of CNM is the X-Linked Myotubular Myopathy, caused by mutations in the myotubularin (MTM1) gene (XLMTM), characterised by profound muscle hypotonia and weakness, severe bulbar and respiratory involvement. Here, we generated an induced pluripotent stem cell (iPSC) line from a patient with a severe form of XLMTM. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. This new MTM1-mutant iPSC line could facilitate disease-modelling and therapy development studies for XLMTM

Devito, LG

Healy, L

Lionello, VM

Muntoni, F

Tedesco, FS

English

UCL Discovery

Stem Cell Research 69 (2023) 103079Available online 21 March 20231873-5061/© 2023 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Lab Resource: Single Cell Line Generation of an MTM1-mutant iPSC line (CRICKi008-A) from an individual with X-linked myotubular myopathy (XLMTM) Liani G. Devito a,1, Valentina M. Lionello b,c,1, Francesco Muntoni d, Francesco Saverio Tedesco b,c,d,*, Lyn Healy a,* a Human Embryo and Stem Cell Unit, The Francis Crick Institute, London, UK b Stem Cells and Neuromuscular Regeneration Laboratory, The Francis Crick Institute, London, UK c Department of Cell and Developmental Biology, University College London, London, UK d Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health and Great Ormond Street Hospital for Children, London, UK  A B S T R A C T   Centronuclear myopathies (CNMs) are a group of inherited rare muscle disorders characterised by the abnormal position of the nucleus in the center of the muscle fiber. One of CNM is the X-Linked Myotubular Myopathy, caused by mutations in the myotubularin (MTM1) gene (XLMTM), characterised by profound muscle hypotonia and weakness, severe bulbar and respiratory involvement. Here, we generated an induced pluripotent stem cell (iPSC) line from a patient with a severe form of XLMTM. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. This new MTM1-mutant iPSC line could facilitate disease-modelling and therapy development studies for XLMTM.   1. Resource table  Unique stem cell line identifier CRICKi008-A Alternative name(s) of stem cell line iFCI006 Institution THE FRANCIS CRICK INSTITUTE Contact information of distributor lyn.healy@crick.ac.uk liani.devito@crick.ac.uk saver io.tedesco@crick.ac.uk Type of cell line iPSC Origin Human Additional origin info Age: 6 months Cell Source Dermal Fibroblast Clonality Clonal Method of reprogramming mRNA Genetic Modification NO Associated disease XLMTM caused by MTM1 mutation Gene/locus c.594C>G (p.Tyr198X) in MTM1 exon 8 Date archived/stock date AUGUST 2022 Cell line repository/bank https://hpscreg.eu/cell-line/CRICKi008-A Ethical approval This study was approved by The London – West London & GTAC Research Ethics Committee (formerly known as the Hammersmith, Queen Charlotte’s and Chelsea Research Ethics Committee). Fibroblasts were supplied by the MRC CNMD Biobank London (REC reference number 06/Q0406/33). Human cell work was conducted under the approval of the National (continued on next column)  (continued ) Health Service (NHS) Health Research Authority Research Ethics Committee reference no. 13/LO/1826; Integrated Research Application System (IRAS) project no. 141,100.  2. Resource utility We report a patient-derived iPSC line for use as a disease-specific cellular model to further understand the pathogenesis of rare muscle disorder XLMTM caused by MTM1 mutation. This new MTM1-mutant iPSC line could facilitate disease-modelling and therapy development studies for XLMTM (see Table 1). 3. Resource details Centronuclear myopathies (CNMs) are a group of incurable muscle disorders characterized by the abnormal position of the nucleus in the center of the muscle fiber. The most severe CNM is the X-Linked Myo-tubular Myopathy, caused by mutations in the myotubularin (MTM1) gene (XLMTM, MIM #310400), characterised by profound muscle hy-potonia and weakness (often with neonatal onset) severe bulbar and respiratory dysfunction and premature death. XLMTM remains * Corresponding authors at: The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK (F.S. Tedesco). E-mail addresses: saverio.tedesco@crick.ac.uk (F.S. Tedesco), lyn.healy@crick.ac.uk (L. Healy).   1 Equal contribution. Contents lists available at ScienceDirect Stem Cell Research journal homepage: www.elsevier.com/locate/scr https://doi.org/10.1016/j.scr.2023.103079 Received 7 February 2023; Accepted 18 March 2023   Stem Cell Research 69 (2023) 1030792incurable, and several affected males present symptoms at birth and invariably develop respiratory failure in infancy or childhood. Here we report the generation and characterisation of a novel iPSC line derived from a male patient with a severe infantile form of XLMTM with a pathogenic MTM1 mutation (Bachmann et al., 2017). Patient dermal fibroblasts were obtained from UCL MRC Neuro-muscular Disease Biobank and reprogrammed using the non-integrating mRNA-based protocol (StemRNA™ 3rd Gen Reprogramming Kit, REPROCELL) that combines non-modified RNA (NM-RNA) and micro-RNA technology. It contains six reprogramming factors, Oct4, Sox2, Klf4, cMyc, Nanog, Lin 28, and three immune evasion factors, E3, K3 and B18. Cells were reprogrammed in a feeder-free system according to the manufacturer’s instructions. Colonies with a typical pluripotent stem cell morphology were individually and manually selected to establish clonal feeder-free iPSC lines. Cells showed typical iPSC morphology after a few passages (Fig. 1A). Dideoxynucleotide sequencing (Sanger Sequencing) confirmed the presence of the pathogenic c.594C>G nonsense mutation in exon 8 of the MTM1 gene (Fig. 1B). Copy number variation analysis by chromo-somal microarray confirmed the male sex of the individual with a benign partial chromosomal gain on chromosome Y (Fig. 1B). Stem cell identity of the CRICKi008-A was confirmed by the expression of pluripotency markers OCT4 and SSEA4, on Flow Cytometry analyses (Fig. 1C). In vitro differentiation (direct and spontaneous) confirmed the cell line’s ability to different onto all three germ layers (Fig. 1D and 1E). Identical genetic identity to the donor of the iPSC was confirmed by short tandem repeat (STR) profiling. Taken together, these results prove that we have successfully produced an iPSC line from a patient with a severe form of XLMTM. 4. Materials and methods 4.1. iPSC cell generation and expansion Dermal fibroblasts were thawed at passage 4 and seeded them at a density of 5x104/well in 2 wells of a 6-well plate coated with iMatrix (Stemgent). Cells were plated in Fibroblast Expansion Medium (DMEM (Gibco)/ Glutamax (Gibco)/ 10% Hyclone FBS (Thermo Scientific)) and cultured for 24 h in 37 ◦C, 5% CO2 and 21% O2. Then, on the first day of reprogramming, Day 0, the medium was switched to NutriStem medium (Stemgent), and cells were transferred to a hypoxic incubator at 37 ◦C, 5% CO2 and 5% O2. Fibroblasts were reprogrammed 1-day post-seeding using the StemRNA™ 3rd Gen Reprogramming Kit (Stemgent) according to the manufacturer’s instructions. Briefly, the NM-RNA cocktail was added to RNAiMAX transfection reagent (Gibco) and transfected into the cells for four consecutive days with medium change 6 h post-transfection. iPSC-like colonies started to appear 9 days following the first trans-fections; they were manually picked and transferred to Matrigel-coated 6-well plates with mTeSR1 medium (StemCell Technologies) containing 10 µM Y-27362. The medium was changed after 24 h. Colonies were expanded by splitting at a 1:3 to 1:6 ratio every 4–6 days and maintained in a hypoxic incubator at 37 ◦C, 5% CO2 and 5% O2. 4.2. Pluripotency markers We evaluated the pluripotency status of the CRICKi008-A line by Flow Cytometry using the BD StemflowTM Human and Mouse Pluripo-tent Stem Cell Analysis Kit (BD) as per the manufacturer’s instruction. Briefly, we detached the cells using Accutase (Sigma-Aldrich) and passed through a 70 μM cell strainer to eliminate cell clumps. Cells were then washed with DPBS (without Ca2 Mg2) (Thermo Fisher Scientific) and resuspended at 1 × 106 cells/ ml before adding the Live/ Dead staining (Thermo Fisher) for 30 min at room temperature. After another cell wash step with DPBS, cells were fixed in 4% paraformaldehyde (BD Stemflow Analyses kit component) for 20 min. For the permeabilization step, we used the 1X Perm/ Wash buffer (BD Stemflow Analyses kit component) for 10 min, followed by a wash in the same buffer. We incubated the cells with the antibodies (Table 2) for 30 min. We used DIVA software to analyse the cells and FlowJo to analyse the data. 4.3. Spontaneous differentiation into three germ layers We tested the differentiation capacity of the CRICKi008-A line by spontaneous differentiation and embryoid body (EB) formation. Using the AggreWell™ 800 Microwell Plates, we seeded 5x106/well in APEL 2 medium (STEMCELL Technologies) and cultured the EBs for 14 days in a hypoxic incubator at 37 ◦C, 5% CO2 and 5% O2. The expression of the lineage-specific markers was assessed by TaqMan hPSC Scorecard Assay (Thermo Scientific). Briefly, we isolated RNA using the QIAgen RNeasy mini kit and measured concentration by a Nanodrop. We used the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher) for cDNA preparation. We ana-lysed the data using the hPSC Scorecard™ – Analysis group (Thermo Fisher). 4.4. Direct differentiation into three germ layers For direct differentiation, we used the STEMdiff™ Trilineage Dif-ferentiation Kit (STEMCELL Technologies) as per manufacturer’s in-structions. We evaluated the differentiation potential of the CRICKi008- A line by immunostaining for lineage-specific markers on Day 5 (Mesoderm and Endoderm) and Day 7 (Ectoderm) as previously described (Devito et al., 2021). Differentiated cells were washed twice in DPBS (Ca2 Mg2) (Thermo Fisher Scientific) before fixation with 3.7% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Table 1 Characterization and validation.  Classification Test Result Data Morphology Microscopic photography Normal morphology at passage 6–8 Fig. 1 panel A Phenotype Quantitative analysis Flow Cytometry Staining of pluripotency markers: OCT4+, SSEA4+ and SSEA1- Fig. 1 panel C Genotype CNV analysis: Karyostat assay (Thermo Scientific) with resolution >2 Mb for chromosomal gains and >1 Mb for chromosomal losses  Male/ female individual; no chromosome aberrations compared to reference dataset Fig. 1 panel B  STR analysis All 16 sites matched submitted in archive with journal Mutation analysis Sanger Sequencing State if heterozygous/ homozygous, type of mutation Fig. 1 panel B Microbiology and virology Mycoplasma Mycoplasma testing by RT-PCR Negative not shown but available with author Differentiation potential In vitro differentiation Direct differentiation to three germ layers confirmed by immunostaining Fig. 1 panel E List of recommended germ layer markers Spontaneous In vitro differentiation: EB formation followed by TaqMan™ hPSC Scorecard™ Panel, Fast 96-well (Cat. N. A15876) Expression of markers (qPCR) of Endoderm, Mesoderm and Ectoderm germ layer confirmed by Scorecard Fig. 1 Panel D  L.G. Devito et al.                                                                                                                                                                                                                                Stem Cell Research 69 (2023) 1030793Cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature, then incubated with primary antibodies (Table 2) overnight at 4 ◦C. The following day, cells were washed twice with DPBS and incubated with secondary antibodies (Table 2) for 30 min at room temperature. 4.5. Dideoxy nucleotide sequencing We performed PCR amplification (primers listed in Table 2) using the Q5 High-Fidelity 2X Master-Mix (BioLabs) on cell line genomic DNA extracted using the QIAamp DNA micro Kit (Qiagen). Then we purified the PCR product using the Monarch PCR and DNA cleanup Kit (BioLabs). Finally, we sent the samples for Sanger sequencing to Source Biosciences (UK), and analysed the data using SnapGene software. 4.6. Chromosomal microarray Using genomic DNA of the iPSC lines, Thermo Scientific (USA) per-formed the KaryoStat assay (Thermo Scientific, USA), an array comparative genomic hybridization (CGH). 4.7. Short tandem repeat (STR) profiling The Cell Services, a Science Technology Platform (STP) within the Francis Crick Institute, performed the STR profiling on DNAs from the parental sample and iPSC line using the Powerplex 16 HS System (Promega). All lines were sent regularly for STR profiling (every 3 pas-sages) since reprogramming started. 4.8. Mycoplasma detection test The Cell Services (STP) confirmed the absence of mycoplasma Fig. 1.  L.G. Devito et al.                                                                                                                                                                                                                                Stem Cell Research 69 (2023) 1030794contamination using PCR amplification using the Universal Mycoplasma Detection Kit (ATCC 30–1012 K) for PCR amplification. Cells were regularly sent for Mycoplasma testing (every 3 passages) since reprog-ramming started. Declaration of Competing Interest The authors declare the following financial interests/personal re-lationships which may be considered as potential competing interests: Liani G Devito reports financial support was provided by The Francis Crick Institute. Acknowledgments The authors wish to thank the patient’s family, Mr Pierpaolo Ala (MRC Centre Neuromuscular Biobank Research Coordinator) and the MRC Centre for Neuromuscular Diseases Biobank London, supported by the National Institute for Health Research Biomedical Research Centres at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London Hospitals NHS Foundation Trust and UCL, for providing fibroblasts. We thank the Francis Crick Institute Genomics Equipment Park, Cell Services STPs and HESCU LRS Ilenia D’Angelo. This work was supported by: The Francis Crick Institute, which receives its funding from Cancer Research UK (CC0199), the UK Medical Research Council (CC0199) and the Wellcome Trust (CC0199). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. VML and FST acknowledge funding by AFM-Telethon (23782). FST acknowledges support by the European Research Council (759108 – HISTOID) and the NIHR (CL-2018-18-008; the views expressed are those of the authors and not necessarily those of the Na-tional Health Service, the NIHR, or the Department of Health). References Bachmann, C., Jungbluth, H., Muntoni, F., Manzur, A.Y., Zorzato, F., Treves, S., 2017. Cellular, biochemical and molecular changes in muscles from patients with X-linked myotubular myopathy due to MTM1 mutations. Human Mol. Genetics 26, 2. Devito, L.G., Healy, L., Mohammed, S., Guillemot, F., Dias, C., 2021. Generation of an iPSC line (CRICKi001-A) from an individual with a germline SMARCA4 missense mutation and autism spectrum disorder. Stem Cell Res. 53, 102304. Table 2 Reagents details.  Antibodies used for immunocytochemistry/flow-cytometry   Antibody Dilution/Amount of staining Company Cat # RRID Pluripotency Markers BD PharmingenTM Alexa Fluor® 647 Mouse anti- SSEA-4 20 μL per sample (5x105 to 1x 06 cells) BD #560477 AB_2869350 BD PharmingenTM PE Mouse anti-SSEA-1 20 μL per sample (5x105 to 1x 06 cells) BD #560477 AB_2869350 BD PharmingenTM PerCP-CyTM5.5 Mouse anti- Oct3/4 20 μL per sample (5x105 to 1x 06 cells) BD #560477 AB_2869350 Differentiation Markers Rabbit anti-GATA-4 1:500 Cell Signaling Technology #36966 AB_2799108 Mouse anti-βIII-tubulin 1:100 Invitrogen # MA1-118 AB_2536829 Mouse anti smooth muscle actin SMA 1:100 Sigma #A5228 AB_262054 Donkey anti-goat FITC IgG 1:100 Jackson Immunoresearch #705-095- 147 AB_2340401 Donkey anti-rabbit Alexa Fluor 1:500 Thermofisher #A-21206 AB_2535792 Goat anti-rabbit Alexa Fluor 1:500 Thermofisher #A-11029 AB_2534088  Primers Target Forward/Reverse primer (5′-3′) Size of Band Targeted mutation analysis MTM1 (F) CCC TGT GGT CAA AGA GGC AT 300 bp MTM1 (R) TCC AAA AGG ACC GTT TCT CTA AGT  L.G. Devito et al.                                                                                                                                                                                                                                

Generation of an MTM1-mutant iPSC line (CRICKi008-A) from an individual with X-linked myotubular myopathy (XLMTM)

https://discovery.ucl.ac.uk/10168055/1/1-s2.0-S187350612300065X-main.pdf

Generation of an MTM1-mutant iPSC line (CRICKi008-A) from an individual with X-linked myotubular myopathy (XLMTM)

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