Among the known causative genes of familial ALS, SOD1 mutation is one of the most common. It encodes for the ubiquitous detoxifying copper/zinc binding SOD1 enzyme, whose mutations selectively cause motor neuron death, although the mechanisms are not as yet clear. What is known is that mutant-mediated toxicity is not caused by loss of its detoxifying activity but by a gain-of-function. In order to better understand the pathogenic mechanisms of SOD1 mutation, a human induced pluripotent stem cell (hiPSC) line was generated from the somatic cells of a female patient carrying a missense variation in SOD1 (L145F)

Alice Di Pierro

Angela D'Anzi

Angelo Luigi Vescovi

Daniela Ferrari

Elisa Perciballi

Fabiola De Marchi

Filomena Altieri

Jessica Rosati

Laura Bernardini

Letizia Mazzini

Marina Goldoni

Maurizio Gelati

Sandra D'Alfonso

English

Among the known causative genes of familial ALS, SOD1mutation is one of the most common. It encodes for the ubiquitous detoxifying copper/zinc binding SOD1 enzyme, whose mutations selectively cause motor neuron death, although the mechanisms are not as yet clear. What is known is that mutant-mediated toxicity is not caused by loss of its detoxifying activity but by a gain-of-function. In order to better understand the pathogenic mechanisms of SOD1 mutation, a human induced pluripotent stem cell (hiPSC) line was generated from the somatic cells of a female patient carrying a missense variation in SOD1 (L145F)

D'Anzi, Angela

Altieri, Filomena

Perciballi, Elisa

Ferrari, Daniela

Bernardini, Laura

Goldoni, Marina

Mazzini, Letizia

De Marchi, Fabiola

Di Pierro, Alice

D'Alfonso, Sandra

Gelati, Maurizio

Vescovi, Angelo Luigi

Rosati, Jessica

Archivio Istituzionale della Ricerca- Università del Piemonte Orientale

Generation of an induced pluripotent stem cell line, CSSi011-A (6534), from an Amyotrophic lateral sclerosis patient with heterozygous L145F mutation in SOD1 gene

Open Access Repository

Contents lists available at ScienceDirectStem Cell Researchjournal homepage: www.elsevier.com/locate/scrGeneration of an induced pluripotent stem cell line, CSSi011-A (6534), froman Amyotrophic lateral sclerosis patient with heterozygous L145F mutationin SOD1 geneAngela D'Anzia, Filomena Altieria, Elisa Perciballia, Daniela Ferrarib, Laura Bernardinic,Marina Goldonic, Letizia Mazzinid, Fabiola De Marchid, Alice Di Pierroe, Sandra D'Alfonsoe,Maurizio Gelatif, Angelo Luigi Vescovia,b,⁎, Jessica Rosatia,⁎a Fondazione IRCCS Casa Sollievo della Sofferenza, Cellular Reprogramming Unit - Viale dei Cappuccini, 71013 San Giovanni Rotondo, Foggia, ItalybUniversity of Milano Bicocca, Department of Biotechnology and Biosciences, Piazza della Scienza 2, 20126 Milan, Italyc Fondazione IRCCS Casa Sollievo della Sofferenza, Medical Genetics Unit - Viale dei Cappuccini, 71013 San Giovanni Rotondo, Foggia, ItalydALS Centre Maggiore della Carità hospital and Università del Piemonte Orientale, Novara, Italye Department of Health Sciences, Center on Autoimmune and Allergic Diseases (CAAD), UPO, University of Eastern Piedmont, A. Avogadro University, Novara, Italyf Fondazione IRCCS Casa Sollievo della Sofferenza, UPTA Unit – Viale dei Cappuccini, 71013 San Giovanni Rotondo, Foggia, ItalyA B S T R A C TAmong the known causative genes of familial ALS, SOD1 mutation is one of the most common. It encodes for the ubiquitous detoxifying copper/zinc binding SOD1enzyme, whose mutations selectively cause motor neuron death, although the mechanisms are not as yet clear. What is known is that mutant-mediated toxicity is notcaused by loss of its detoxifying activity but by a gain-of-function. In order to better understand the pathogenic mechanisms of SOD1 mutation, a human inducedpluripotent stem cell (hiPSC) line was generated from the somatic cells of a female patient carrying a missense variation in SOD1 (L145F).Unique stem cell line id-entifierCSSi011-A (6534)Alternative name(s) ofstem cell lineSLA PZ.2 cl1Institution Casa Sollievo della Sofferenza – Viale dei Cappuccini,71013 San Giovanni Rotondo, Foggia, ItalyContact information ofdistributorJessica ROSATI, j.rosati@css-mendel.itType of cell line iPSCOrigin humanAdditional origin info Age:48 yrsSex: femaleEthnicity if known: Caucasian/AlbanianCell Source Dermal fibroblastsClonality ClonalMethod of reprogram-mingNon integrating episomal vectorsGenetic Modification YesType of Modification SpontaneousAssociated disease Amyotrophic lateral sclerosisGene/locus SOD1:c.435G>CMethod of modification HereditaryName of transgene or r-esistanceN/AInducible/constitutive s-ystemN/ADate archived/stock date January 2019Cell line repository/bank HPSC registryEthical approval Comitato Etico Interaziendale Novara: CE 54/171. Resource utilityThe iPSC line was derived from an individual carrying the L145FSOD1 mutation, typical of Mediterranean countries, sharing peculiarclinical features such as lower-limb onset with slow evolution andcognitive involvement, uncommon in SOD1-ALS patients. This patient-derived iPSC line will be a useful cellular system for modelling SOD1pathogenetic mechanisms (see Table 1).2. Resource detailsAmyotrophic lateral sclerosis (ALS) is a terminal motor neurondisease, characterized by motor neuron degeneration in the primarymotor cortex, brainstem and spinal cord, leading to the paralysis ofvoluntary muscles. The paralysis begins focally and disseminates in apattern that suggests that degeneration is spreading among contiguouspools of motor neurons. Respiratory failure causes death, which typi-cally occurs within five years of developing this debilitating condition(Pasinelli and Brown, 2006). In about 10% of patients, the disease ishttps://doi.org/10.1016/j.scr.2020.101924Received 3 June 2020; Received in revised form 23 June 2020; Accepted 19 July 2020⁎ Corresponding authors.E-mail addresses: vescovia@gmail.com (A.L. Vescovi), j.rosati@css-mendel.it (J. Rosati).Stem Cell Research 47 (2020) 101924Available online 25 July 20201873-5061/ © 2020 The Authors. 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/).Tfamilial (FALS), while the majority of patients are sporadic cases (SALS)(Chia et al., 2018). Mutations in at least 25 genes are implicated in ALSpathogenesis, with C9orf72 SOD1, FUS and TARDBP representing themost common mutated genes, accounting for 60% of FALS and 10% ofSALS cases (Chia et al., 2018). This study concerned a female patientwith a family history of ALS, carrying a missense variation in the SOD1gene (L145F). The patient presented progressive weakness in the rightfoot, followed by mild muscle hypotrophy and weakness in the entireright lower limb, also reporting upper right limb weakness. Fibroblastsderived from a skin biopsy were reprogrammed into iPSCs, using threenon-integrative episomal vectors containing the reprogramming factorsOCT3/4, SOX2, L-MYC, KLF4, LIN28, sh-p53 (Okita et al., 2011). iPSCcolonies displayed typical iPSC morphology, with well-defined edgesand normal growth behaviour (Fig. 1A). The expanded colonies werestained for the endogenous TRA-1–60, a protein expressed on the sur-face of iPSCs, and for the homeodomain transcription factor OCT-3/4,demonstrating iPSC pluripotency (Fig. 1B), further confirmed throughqRT-PCR (Fig. 1C). We examined the presence of episomal plasmidDNA in this cell line, using primers specific to a common sequencepresent in all three reprogramming plasmids. We observed that, after 10passages, the iPSCs were devoid of vector sequences, as shown by RT-qPCR, using the fibroblasts one week from episomal nucleofection aspositive controls (Fig. 1D). Genomic stability of the iPSCs was con-firmed through karyotype, which provided a normal diploid 46, XXchromosome arrangement without any detectable abnormalities(Fig. 1E). The differentiation potential was demonstrated in vitro by theformation of embryoid bodies (EBs) (Fig. 1F) and in vivo by teratomaformation (Fig. 1G). The image showed: ectodermal layer representedby primitive epidermal tissue composed by cells characteristically linedup to form palisade-like structures bordered by loose connective layer(typical organization of epithelial tissue), mesodermal layer showed bythe presence of loose connective tissue, typically composed by abun-dant extracellular matrix (pink, eosin stained) with few cellular ele-ments (dark violet, hematoxylin stained) and endodermal layer, re-presented by a pluri-stratified epithelium organized in villi-likestructures characterized by batiprismatic cellular elements. qRT-PCRanalysis showed endogenous expression of the three germ layer markersin the embroyoid bodies (Fig. 1H). PCR-based detection tests confirmedthe absence of Mycoplasma contamination at this stage (SupplementaryFig. 1). In addition, Short Tandem Repeat (STR) profiling confirmedthat these iPSC lines had the same genetic identity as the donor's fi-broblasts (data available from the authors). The presence of the het-erozygous c.435G > C (NM_000454.4) mutation harboured by par-ental fibroblasts was confirmed in the generated IPS cell line throughSanger sequencing (Fig. 1I).3. Materials and methodsFibroblasts derived from skin biopsy were cultured in DMEM highglucose, 20% FBS, 2mML-glutamine and 1% penicillin–streptomycin(all reagents from Sigma Aldrich) at 37 °C, 5% CO2. Subsequently,3 × 105 fibroblasts were nucleofected, using the Nucleofector programnamed “FF113”, with 3 μg 1:1:1 mix of the episomal plasmids pCXLE-hUL (Addgene #27080), pCXLEhSK (Addgene #27078) and pCXLE-hOCT4-shp53 (Addgene #27077). On day 7, the nucleofected fibro-blasts were plated on Matrigel (1:100) (BD Biosciences) and cultured inNutristemXF medium (Biological Industries). The hiPSC colonies werepicked and expanded under feeder-free conditions and passagedthrough manual picking in NutristemXF medium. Absence of myco-plasma contamination was verified using N-Garde Mycoplasma PCR kit(EuroClone). For amplification, the kit provides a reaction mixturecontaining all ingredients necessary for PCR, including the positivecontrol. 1 Kb was used as ladder in the running. After ten passages,clearance of the exogenous reprogramming factors was confirmed byqRT-PCR.4. Embryoid body formationMechanically detached iPSCs were plated in Petri dishes inNutristemXF medium, which was substituted with differentiationmedium: DMEM/F12, 20% KOSR (Gibco), 0,1mM NEAA, 0.1 mM β-mercaptoethanol, 1% Pen/Strep the following day. Fourteen days later,the EBs thus obtained were pelletted and RNAs were extracted for qRT-PCR analysis.5. Teratoma formationiPSCs derived from six well plates (approximately 6x106 cells)combined with a Matrigel substrate (Corning, Inc., USA) were injectedinto the right flank of nude mice. After 1 month, tumors were collectedfor histological analysis to check their in vivo differentiation capacityinto derivatives of all three germ layers.6. Real-Time PCR analysisTotal RNAs were extracted using Trizol reagent (Life Technology)and cDNA synthesized using the High capacity cDNA RT(LifeTechnology) following manufacturer’s recommendations. qPCRanalysis was performed in three minimum independent biological ex-periments with TaqMan primers (Table 2) for three germ layers(Thermo Fischer Scientific) and SyBr green primers (Table 2) forstemness markers according to the manufacturer's protocol. The ex-pression ratio of the target genes was calculated by using the 2 − ΔCtTable 1Characterization and validation.Classification Test Result DataMorphology Photography Normal Fig. 1APhenotype Immunocytochemistry Staining of pluripotency markers: Oct4; Tra-1–60. Fig. 1BqRT-PCR Expression of pluripotency markers: OCT4, LIN28, L-MYC,KLF4, SOX2Fig. 1CGenotype Karyotype (G-banding) and resolution 46 XX, Resolution 450–500 Fig. 1EIdentity STR analysis 19 sites tested, all matched With AuthorsMutation analysis (IF APPLICABLE) Sequencing Heterozygous mutation Fig. 1IMicrobiology and virology Mycoplasma Mycoplasma tested by N-Garde Mycoplasma PCR kit(EuroClone) is Negative.Supplementary Fig.1Differentiation potential Embryoid body formation and TeratomaformationGenes expressed in embryoid bodies: SOX1, NESTIN, PAX6,EOMES, T, GATA4, FOXA2, SOX17Proof of three germ layers formation.Fig. 1F, 1G, 1HDonor screening (OPTIONAL) HIV 1 + 2 Hepatitis B, Hepatitis C N/AGenotype additional info(OPTIONAL)Blood group genotyping N/AHLA tissue typing N/AA. D'Anzi, et al. Stem Cell Research 47 (2020) 1019242Fig. 1. Characterization of CSSi011-A (6534) A. Phase contrast image of the iPSC morphology. B. Representative immunofluorescent images of iPSCs marked for stemcell markers, OCT4 (green) and TRA-1-60 (red). Nucleus is labelled with Hoechst 33342 (blue). C. Expression analysis of stemness markers D. qRT-PCR analysis oftransgene expression showing the loss of episomal vectors during iPSC amplification. E. Cytogenetic analysis showing a normal karyotype (46, XX). F. Phase contrastimage of embryoid bodies. G. Teratoma showing a normal ectodermal, mesodermal and endodermal differentiation. H. Expression analysis of differentiation markers.I. DNA sequencing analysis of (SOD1):c.435G>C mutation. Blue arrows indicate mutation site.A. D'Anzi, et al. Stem Cell Research 47 (2020) 1019243method, considering 18S as reference gene.7. SequencingGenomic DNA was extracted from iPSC and parental fibroblastsusing ReliaPrep™ Blood gDNA Miniprep System. SOD1 exon 5 was am-plified by PCR using the following primers: Forward: 5′-TTGTTGGGAGGAGGTAGTG-3′, Reverse: 5′-AAAGCAACTCTGAAAAAGT-3′. Theamplicon was sequenced by BigDye terminator v.3.1 Cycle Sequencingkit on ABI 3130XL Genetic Analyzer.8. Immunofluorescence stainingCells were fixed using 4% paraformaldehyde and stained. The cellswere incubated with Blocking Buffer (PBS containing 20% Normal GoatSerum, 0.1% Triton X-100) for 30 min at room temperature. Next,primary antibodies, listed in Table 2, diluted in blocking buffer wereadded and incubated O/N at 4 °C. After extensive washing, Alexa Fluor594- and/or Alexa Fluor 488-conjugated secondary antibodies wereadded 1 h at room temperature. Cellular nuclei were counterstainedwith DAPI. Microphotograps were taken using a Nikon C2 fluorescencemicroscope and NIS Elements 1.49 software.9. STR analysisFibroblasts and iPSCs DNA was extracted by Dneasy blood andtissue kit (QIAGEN). PCR amplification of 19 distinct STRs (D13S252,D13S305, D13S634, D13S800, D13S628, D18S819, D18S535,D18S978, D18S386, D18S390, D21S11, D21S1437, D21S1409,D21S1442, D21S1435, D21S1446, DXS6803, XHPRT, DXS1187) wascarried out using the QST*Rplusv2 kit (Elucigene Diagnostics), PCRproducts were separated on an ABI Prism 3130 DNA sequencer andanalyzed by GeneMapper version 4.0 (Applied Biosystems).10. KaryotypingiPSCs were cultured in chamber slides (Thermo Fisher Scientific)coated with Matrigel (1:100) in NutristemXF medium for 2–3 days.Cells were blocked in metaphase by adding 0.1 μg/ml of COLCEMIDsolution (Thermo Fisher Scientific) to culture medium for 60 min at37 °C and by adding hypotonic KCl solution (30 mM) in 10%FBS at37 °C for 6 min. Cells were fixed in a cold fresh-made 3:1 ethanol:aceticacid solution. Karyotype analysis was carried out on GTG-banded me-taphases. Fifteen metaphases were counted and three karyotypes ana-lyzed. Only clonal aberrations were considered, following ISCN re-commendations.Declaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared toTable 2Reagents details. RRID Requirement for antibodies: use http://antibodyregistry.org/ to retrieve RRID for antibodies and include ID in table as shown in examples.Antibodies used for immunocytochemistry/flow-citometryAntibody Dilution Company Cat # and RRIDPluripotency Markers Rabbit anti-OCT4 Mouse anti-TRA-1-60 1:100 1:100 Life technologies (A13998)RRID: AB_2534182 Life technologies (411000)RRID: AB_2533494Secondary antibodies anti-Rabbit AlexaFluor 488 anti-Mouse AlexaFluor 555 1:10000 1:10000 Invitrogen (A11034)RRID: AB_2576217 Invitrogen (A21422)RRID: AB_2535844SyBr green Primers used for qPCR Target Forward/Reverse sequence (5′-3′) (411000)Episomal genes eOCT4 Fwd: CAT TCA AAC TGA GGT AAG GGRev: TAG CGT AAA AGG AGC AAC ATA GeKLF4 Fwd: CCA CCT CGC CTT ACA CAT GAA GARev: TAG CGT AAA AGG AGC AAC ATA GeLIN28 Fwd: AGC CAT ATG GTA GCC TCA TGT CCG CRev: TAG CGT AAA AGG AGC AAC ATA GeL-MYC Fwd: GGC TGA GAA GAG GAT GGC TACRev: TTT GTT TGA CAG GAG CGA CAA TeSOX2 Fwd: TTC ACA TGT CCC AGC ACT ACC AGARev: TTT GTT TGA CAG GAG CGA CAA TPluripotency markers OCT4 Fwd: CCC CAG GGC CCC ATT TTG GTA CCRev: ACC TCA GTT TGA ATG CAT GGG AGA GCLIN28 Fwd: CCC CAG GGC CCC ATT TTG GTA CCRev: ACC TCA GTT TGA ATG CAT GGG AGA GCL-MYC Fwd: GCG AAC CCA AGA CCC AGG CCT GCT CCRev: CAG GGG GTC TGC TCG CAC CGT GAT GSOX2 Fwd: TTC ACA TGT CCC AGC ACT ACC AGARev: TCA CAT GTG TGA GAG GGG CAG TGT GCHouse-Keeping Gene β-ACTIN Fwd: GGC ATC CTC ACC CTG AAG TARev: GGG GTG TTG AAG GTC TCA AATaqMan primers used for qPCR Target ProbeDifferentation markers SOX1 Hs01057642_s1NESTIN Hs04187831_g1PAX6 Hs00240871_m1T Hs00610080_m1EOMES Hs00172872_m1GATA4 Hs00171403_m1FOXA2 Hs00232764_m1SOX17 Hs00751752_s1β-ACTIN Hs 99999903_m1A. D'Anzi, et al. Stem Cell Research 47 (2020) 1019244influence the work reported in this paper.AcknowledgmentsThis work was supported by grants from the Italian Ministry ofHealth: Ricerca Corrente 2019-2020 to JR, from the Italian Ministry ofResearch and University, grant N. 201534HNXC to SD, from Universitàdel Piemonte Orientale, AGING Project for Department of Excellence atthe Department of Translational Medicine (DIMET) to LM and SD.Appendix A. Supplementary dataSupplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2020.101924.ReferencesPasinelli, P., Brown, R., 2006. Molecular biology of amyotrophic lateral sclerosis: insightsfrom genetics. Nat. Rev. Neurosci. 7, 710–723.Chia, R., Chiò, A., Traynor, B.J., 2018. Novel genes associated with amyotrophic lateralsclerosis: diagnostic and clinical implications. Lancet Neurol. 17 (1), 94–102.Okita, K., Matsumura, Y., Sato, Y., Okada, A., Morizane, A., Okamoto, S., Hong, H.,Nakagawa, M., Tanabe, K., TezukaK, S.T., Kunisada, T., Takahashi, M., Takahashi, J.,Saji, H., Yamanaka, S., 2011. A more efficient method togenerate integration-freehuman iPS cells. Nat. Methods 8 (5), 409–412.A. D'Anzi, et al. Stem Cell Research 47 (2020) 1019245

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Generation of an induced pluripotent stem cell line, CSSi011-A (6534), from an Amyotrophic lateral sclerosis patient with heterozygous L145F mutation in SOD1 gene

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