- Email: [email protected]
Generation and characterization of twelve human induced pluripotent stem cell (iPSC) lines from four familial long QT syndrome type 1 (LQT1) patients carrying KCNQ1 c.1201dupC mutation
Stem Cell Research 41 (2019) 101650
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab Resource: Multiple Cell Lines
Generation and characterization of twelve human induced pluripotent stem cell (iPSC) lines from four familial long QT syndrome type 1 (LQT1) patients carrying KCNQ1 c.1201dupC mutation
T
Ning Gea, Min Liua,b, Yicheng Dinga, Janusz Krawczykc, Veronica McInerneyd, Joseph Galvine, ⁎ ⁎ ⁎ Sanbing Shena, , Terence Prendivillef, , Timothy O'Briena, a
Regenerative Medicine Institute, School of Medicine, National University of Ireland (NUI) Galway, Ireland Department of Physiology, College of Life Science, Hebei Normal University, Shijiazhuang, China Department of Haematology, Galway University Hospital, Ireland d HRB Clinical Research Facility, National University of Ireland (NUI) Galway, Ireland e Mater Misericordiae University Hospital, Eccles st., Dublin 7, Ireland f National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland b c
A B S T R A C T
In this study, we describe the generation and characterization of induced pluripotent stem cell (iPSC) lines from familial long QT syndrome type 1 (LQT1) patients carrying the KCNQ1 c.1201dupC (p.Arg401fs) frame shift mutation by using non-integrational Sendai reprogramming method. The patient-specific iPSC lines harboring the c.1201dupC mutation on KCNQ1 gene expressed pluripotency markers and had the capacity to differentiate into three germ layers.
Resource table
Unique stem cell line identifier
Alternative names of stem cell lines
Institution
⁎
NUIGi008-A NUIGi008-B NUIGi008-C NUIGi009-A NUIGi009-B NUIGi009-C NUIGi010-A NUIGi010-B NUIGi010-C NUIGi011-A NUIGi011-B NUIGi011-C LQTS007C1 (NUIGi008-A) LQTS007C2 (NUIGi008-B) LQTS007Cx (NUIGi008-C) LQTS008C1 (NUIGi009-A) LQTS008C6 (NUIGi009-B) LQTS008Cx (NUIGi009-C) LQTS009C2 (NUIGi010-A) LQTS009C3 (NUIGi010-B) LQTS009Cx (NUIGi010-C) LQTS010C3 (NUIGi011-A) LQTS010C4 (NUIGi011-B) LQTS010Cx (NUIGi011-C) Regenerative Medicine Institute, National University of Ireland Galway, H91 TK33 Galway, Ireland
Contact information of distributor Type of cell lines Origin Cell Source Clonality Method of reprogramming Multiline rationale Gene modification Type of modification Associated disease Gene/locus Method of modification Name of transgene or resistance Inducible/constitutive system Date archived/stock date Cell line repository/bank Ethical approval
Sanbing Shen [email protected] Induced pluripotent stem cells (iPSCs) Human Dermal fibroblasts Clonal or mixed CytoTune-iPSC 2.0 Sendai Reprogramming Kit Same disease non-isogenic cell lines Yes Congenital Long QT Syndrome type 1 (LQT1) c.1201dupC mutation on exon 9 of KCNQ1 gene N/A N/A N/A February 2018 Regenerative Medicine Institute, National University of Ireland Galway The study has been approved by the Ethics Committee of Galway University Hospitals (C.A.750). Patient written informed consent were obtained for skin biopsy procedure.
Resource utility These LQT1 patient-specific iPSC lines provide a valuable resource
Corresponding authors. E-mail addresses: [email protected] (S. Shen), [email protected] (T. Prendiville), [email protected] (T. O'Brien).
https://doi.org/10.1016/j.scr.2019.101650 Received 10 October 2019; Received in revised form 30 October 2019; Accepted 5 November 2019 Available online 14 November 2019 1873-5061/ © 2019 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/).
Stem Cell Research 41 (2019) 101650
N. Ge, et al.
maintained in culture with Essential 8™ Flex Medium in 5% CO2 at 37°C. IPSCs were passaged every 4–6 days using 1 in 4 splits by Gentle Cell Dissociation Reagent (STEMCELL).
to in vitro explore long QT syndrome disease pathogenesis and therapeutic interventions such as new drug screening and gene therapy. 1. Resource details
2.2. Pluripotent marker expression Long QT syndrome (LQTS), present in 1 per 2000 of the general population, is an inherited primary arrhythmia syndrome that presents with recurrent syncope or, rarely, sudden cardiac death secondary to malignant torsades de points. It is a recognized cause of Sudden Arrhythmic Death Syndrome (SADS) in the young and has an autosomal dominant inheritance pattern that can affect every second member of an extended family pedigree from an index case (Gajewski and Saul, 2010). LQTS is mechanistically due to a cardiac ion channelopathy that has a net effect of delayed repolarization at a myocyte level that manifests as a prolonged QT interval on surface electrocardiogram in the patient. Among the well described genetic etiologies, LQT1, encoding the Voltage-Gated Potassium Channel Kv7.1, is the most common and present in approximately 40–50% of all genotyped patients (Shimizu and Horie, 2011). The mechanistic effect of a pathogenic mutation is either to reduce the efficient trafficking of the channel protein to the cell membrane (haploinsufficiency) or to result in a dysfunctional channel with dominant-negative effect (Wu et al., 2016). This study was approved by the Clinical Research Ethics Committee of Galway University Hospital (C.A.750). Standard skin punch biopsy was collected from a 45-year-old female, a 63-year-old female, a 14year-old male and a 7-year-old female who are from the same family and all carry a c.1201dupC mutation on exon 9 of the KCNQ1 gene, incurring a frame shift mutation. Twelve induced pluripotent stem cell (iPSC) lines (8 clonal lines: NUIGi008-A, NUIGi008-B, NUIGi009-A, NUIGi009-B, NUIGi010-A, NUIGi010-B, NUIGi011-A, NUIGi011-B; and 4 mixed lines: NUIGi008-C, NUIGi009-C, NUIGi010-C, NUIGi011-C) were generated using non-integrating Sendai-virus vectors encoding four Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). All the iPSC lines showed typical ES-like morphology (Fig. 1A), expression of pluripotency markers and differentiation potentials. Molecular karyotyping analysis showed normal karyotype and no chromosomal disruption (Supplementary Fig. 1C). Short Tandem Repeat (STR) analysis for 6 polymorphic microsatellite loci showed identical profiles for all iPSC lines as parental fibroblasts. Mycoplasma test revealed no contamination in any iPSC line (Supplementary Fig. 1B). RT-PCR analysis showed that all iPSC lines were free of exogenous reprogramming vectors (Fig. 1E). Pluripotency was assessed by immunofluorescence staining for OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81 and SSEA4 (Fig. 1B), and qRT-PCR for endogenous pluripotency genes OCT4, SOX2 and NANOG (Fig. 1D). To validate the ability to differentiate into three germ lineages, in vitro embryonic body formation experiments were performed and immunofluorescence staining exhibited evidence for endodermal (α-fetoprotein, AFP), mesodermal (α-smooth muscle actin, α-SMA) and ectodermal (neuron-specific class III β-tubulin, TUJ1) markers (Fig. 1C). Lastly, Sanger sequencing was performed to verify the parental DNA mutation was retained in all iPSC lines (Supplementary Fig. 1A).
Expression of pluripotency markers OCT4, SOX2, NANOG, TRA-160, TRA-1-81 and SSEA4 (Cell Signaling Technology,Table 3) were verified by immunofluorescence staining. Briefly, cells were fixed with 4% paraformaldehyde [Room temperature (RT), 20 min], permeabilized with 0.3% Triton X-100 (RT, 15 min), and blocked with 1% BSA (RT, 1 h). Primary antibodies were incubated at 4°C overnight, and secondary antibodies and Hoechst (Nuclei counterstained) for 1 h (RT). All antibodies were diluted in 1% BSA. Images were acquired by inverted wide-field fluorescence system (Olympus IX81). 2.3. Pluripotency gene expression Total RNA was extracted using RNeasy Mini Kit (Qiagen). cDNA was synthesized using QuantiTect Rev. Transcription Kit (Qiagen). Pluripotency genes OCT4, SOX2 and NANOG were assessed by qPCR using Fast SYBR Green Master Mix (Applied Biosystems) with StepOnePlus™ Real-Time PCR System (Applied Biosystems). GAPDH was used as the ‘housekeeping gene’ control. LQT007 fibroblasts were used as the negative control, and the commercial iPSC line (NCRM1) was used as the positive control. All primers used were listed in Table 3. 2.4. Virus-free status of iPSC lines TopTaq® Master Mix (Qiagen) was used for PCR using Veriti Gradient Thermal Cycler (Applied Biosystems) under standard conditions [94°C 2 min (1X); 94°C 30 s, 55°C 30 s, 72°C 30 s (35X), 72°C 5 min (1X), 4°C (hold)]. PCR products were analyzed using agarose gel (1.5%) electrophoresis and were confirmed free of Sendai virus. 2.5. In vitro differentiation To validate the capacity to form three germ layers, 100k hiPSCs were spun in AggreWell 400 plate (STEMCELL) for 24 h to form embryonic bodies (EBs) and cultured in-suspension in EB medium, consisting of DMEM/F-12 medium, 20% KnockOut Serum Replacement, 1% L-glutamine, 1% Non-essential amino acids, 1% Penicillin-streptomycin and 0.2% β-mercaptoethanol. 7 days later, EBs were seeded onto Geltrex-coated chamber slides (ibidi) and cultured at 5% CO2 and 37°C for 4-weeks, and stained using antibodies against AFP, α-SMA and TUJ1. EB medium was used during the whole in vitro differentiation process. 2.6. Validation of KCNQ1 mutation To validate variants in KCNQ1 gene, genomic DNA was extracted by using DNeasy Blood and Tissue Kit (Qiagen) and amplified with primers (Table 3). Purified PCR products were submitted to Eurofins MWG Operon (Germany) for sequencing. The 2-read results from both directions were analyzed by Sequence Scanner Version (Applied Biosystems), which confirmed the c.1201dupC mutation.
2. Materials and methods 2.1. Fibroblast culture and iPSC reprogramming Skin fibroblasts were cultured in DMEM with Glutamax (Gibco) with 10% FBS (Sigma-Aldrich), 1% Antibiotic-Antimycotic (Gibco), and 1% Non-essential amino acids (Gibco) at 37°C in 5% CO2, and reprogramming performed using Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) (Takahashi et al., 2007). On day 7, transduced fibroblasts were plated onto Geltrex-coated plates with a density of 1 × 105/ 6-well in Essential 8™ Flex Medium (Gibco), and refreshed daily. Remaining cells were used as positive controls for SeV-specific RT-PCR. IPSC colonies were manually picked between day 21 and 27, and
3. SNP array IPSC (passage 20–30) genomic DNA was extracted for karyotyping (990k SNP array) at Beijing Hyslar Biotech (Beijing, China). Data were analyzed with Axiom Analysis Suite (ThermoFisher, USA), and Log2 ratio was generated to detect copy number variations. No gross chromosomal alterations were detected as a result of reprogramming. However, the limitations of molecular karyotyping are acknowledged including failure to detect a balanced translocation if it exists. 2
Stem Cell Research 41 (2019) 101650
N. Ge, et al.
Fig. 1. Generation and characterization of twelve human induced pluripotent stem cell (iPSC) lines from four familial long QT Syndrome Type 1 (LQT1) patients carrying KCNQ1 c.1201dupC mutation. Table 1 Summary of lines. iPSC line names
Abbreviation in figures
Gender
Age
Ethnicity
Genotype of locus
NUIGi008-A NUIGi008-B NUIGi008-C NUIGi009-A NUIGi009-B NUIGi009-C NUIGi010-A NUIGi010-B NUIGi010-C NUIGi011-A NUIGi011-B NUIGi011-C
LQTS007C1 LQTS007C2 LQTS007Cx LQTS008C1 LQTS008C6 LQTS008Cx LQTS009C2 LQTS009C3 LQTS009Cx LQTS010C3 LQTS010C4 LQTS010Cx
Female
45
Female
63
Male
14
Female
7
Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian
c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC
3.1. Fingerprinting
mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation
Disease on on on on on on on on on on on on
KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1
gene gene gene gene gene gene gene gene gene gene gene gene
Long Long Long Long Long Long Long Long Long Long Long Long
QT QT QT QT QT QT QT QT QT QT QT QT
Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome
type type type type type type type type type type type type
1 1 1 1 1 1 1 1 1 1 1 1
with PCR primers of six loci (Table 3) under condition [94°C 2 min (1X); 94°C 30 s, 55°C 30 s, 72°C 30 s (35X), 72°C 5 min (1X), 4°C (hold)]. PCR products were analyzed using 3% agarose gel by
Genomic DNA from parental fibroblasts and iPSCs were amplified 3
Stem Cell Research 41 (2019) 101650
N. Ge, et al.
Table 2 Characterization and validation. Classification
Test
Result
Data
Morphology Phenotype
Normal morphology Positive for SOX2, OCT4, NANOG, SSEA4, TRA-1-60 and TRA-1-81.
Fig. 1A Fig. 1B
Genotype Identity
Photography Qualitative analysis: Immunocytochemistry Quantitative analysis: qRT-PCR RT-PCR Single Nucleotide Polymorphism (SNP) Fingerprinting (STR analysis)
Fig. 1D Fig. 1E Supplementary Fig. 1C Available with the authors
Mutation analysis Microbiology and virology Differentiation potential
Sanger sequencing Mycoplasma In vitro embryonic body formation
Positive for SOX2, OCT4 and NANOG Negative for the presence of Sendai vectors No gross chromosomal alteration by reprogramming was detected Tested 6 sites (D1S1656,D3S1358, D7S796, D10S1214, D16S539, and D21S2055), all matched Heterozygous mutation DNA c.1201dupC on KCNQ1 gene Detection by PCR, negative α-fetoprotein (AFP) for endoderm, α-smooth muscle actin (α-SMA) for mesoderm, and β-III tubulin (TUJ1) for ectoderm
Supplementary Fig. 1A Supplementary Fig. 1B Fig. 1C
Table 3 Reagents details. Antibodies used for immunocytochemistry
Pluripotency markers
Differentiation markers
Secondary antibodies
Antibody Rabbit anti-OCT-4A (IgG) Rabbit anti-SOX2 Rabbit anti-NANOG Mouse anti-TRA-1-60 (IgM) Mouse anti-TRA-1-81 (IgM) Mouse anti-SSEA4 (IgG3) Mouse anti-α-Fetoprotein (AFP) Mouse anti-α-Actin, Smooth Muscle (α-SMA) Mouse anti-β3-Tubulin (TUJ1) (IgG) Goat anti-mouse IgG Fluor 555 Goat anti-rabbit IgG Fluor 488
Dilution 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:1000 1:1000
Company Cat# and RRID Cell Signaling Technology, Cat. #2840, RRID: AB_2167691 Cell Signaling Technology, Cat. #3579, RRID: AB_2195767 Cell Signaling Technology, Cat. #3580, RRID: AB_2150399 Cell Signaling Technology, Cat. #4746, RRID: AB_2119059 Cell Signaling Technology, Cat. #4745, RRID: AB_10829904 Cell Signaling Technology, Cat. #4755, RRID: AB_1264259 SIGMA, Cat. #A8452, RRID: AB_258392 Cell Marque Corp, Cat. #202M-95, RRID: AB_1157938 Cell Signaling Technology, Cat. #5568, RRID: AB_10692510 Cell Signaling Technology, Cat. #4409, RRID: AB_1904022 Cell Signaling Technology, Cat. #4412, RRID: AB_1904025
Target SeV/ 181 bp KOS/ 528 bp Klf4/ 410 bp c-Myc/ 532 bp OCT4/ 191 bp SOX2/ 187 bp NANOG/ 219 bp GAPDH/ 206 bp D1S1656 D3S1358 D7S796 D10S1214 D16S539 D21S2055 KCNQ1-Exon 9/ 401 bp
Forward/Reverse primer (5′−3′) GGA TCA CTA GGT GAT ATC GAG C/ ACC AGA CAA GAG TTT AAG AGA TAT GTA TC ATG CAC CGC TAC GAC GTG AGC GC/ ACC TTG ACA ATC CTG ATG TGG TTC CTG CAT GCC AGA GGA GCC C/ AAT GTA TCG AAG GTG CTC AA TAA CTG ACT AGC AGG CTT GTC G/ TCC ACA TAC AGT CCT GGA TGA TGA TG GGG GTT CTA TTT GGG AAG GTA T/ GTT CGC TTT CTC TTT CGG GC AGA CTT CAC ATG TCC CAG CAC T/ CGG GTT TTC TCC ATG CTG TTT C GGA TCC AGC TTG TCC CCA AA/ TGT TTG CCT TTG GGA CTG GT AGG GCT GCT TTT AAC TCT GGT/ CCC CAC TTG ATT TTG GAG GGA GTG TTG CTC AAG GGT CAA CT/ GAG AAA TAG AAT CAC TAG GGA ACC ACT GCA GTC CAA TCT GGG T/ ATG AAA TCA ACA GAG GCT TGC TTT TGG TAT TGG CCA TCC TA/ GAA AGG AAC AGA GAG ACA GGG TGC ATA AAA TAT TGC CCC AAA AC/ TTG AAG ACC AGT CTG GGA AG GAT CCC AAG CTC TTC CTC TT/ ACG TTT GTG TGT GCA TCT GT AAC AGA ACC AAT AGG CTA TCT ATC/ TAC AGT AAA TCA CTT GGT AGG AGA AGC TGT AGC TTC CAT AAG GGC/ CCT ACA TAC CCC CAA GTC GG
Primers Sendai Vectors
Pluripotency Markers
House-Keeping Genes Fingerprinting
Target mutation sequencing
and SS, the Chinese Scholarship Council and NUIG College Scholarship to NG, the Science Foundation Ireland for Grant 13/IA/1787 to SS and LG, 16/RC/3948 to FutureNeuro, for funding the project, and the Irish Government's Program for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 2007–2013 for the central facility.
electrophoresis. 3.2. Mycoplasma detection A standard PCR was routinely performed for mycoplasma test by using MycoSensor PCR Assay Kit (Agilent Technologies) according to manufacturer's instruction.
Supplementary materials Supplementary material associated with this article can be found, in the online version, at 10.1016/j.scr.2019.101650.
Declaration of Competing Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
References Gajewski, K.K., Saul, J.P., 2010. Sudden cardiac death in children and adolescents (excluding Sudden Infant Death Syndrome). Ann. Pediatr. Cardiol. 3, 107–112. Shimizu, W., Horie, M., 2011. Phenotypic manifestations of mutations in genes encoding subunits of cardiac potassium channels. Circ. Res. 109, 97–109. Wu, J., Ding, W.G., Horie, M., 2016. Molecular pathogenesis of long QT syndrome type 1. J. Arrhythm. 32, 381–388. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S., 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872.
Acknowledgments Authors wish to acknowledge Irish Research Council for Postdoctoral fellowship to ML, National Children's Research Centre (NCRC) for seed funding and innovation funding to TP, TOB, ML and SS, Galway University Foundation for equipment funding to TOB, ML 4
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab Resource: Multiple Cell Lines
Generation and characterization of twelve human induced pluripotent stem cell (iPSC) lines from four familial long QT syndrome type 1 (LQT1) patients carrying KCNQ1 c.1201dupC mutation
T
Ning Gea, Min Liua,b, Yicheng Dinga, Janusz Krawczykc, Veronica McInerneyd, Joseph Galvine, ⁎ ⁎ ⁎ Sanbing Shena, , Terence Prendivillef, , Timothy O'Briena, a
Regenerative Medicine Institute, School of Medicine, National University of Ireland (NUI) Galway, Ireland Department of Physiology, College of Life Science, Hebei Normal University, Shijiazhuang, China Department of Haematology, Galway University Hospital, Ireland d HRB Clinical Research Facility, National University of Ireland (NUI) Galway, Ireland e Mater Misericordiae University Hospital, Eccles st., Dublin 7, Ireland f National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland b c
A B S T R A C T
In this study, we describe the generation and characterization of induced pluripotent stem cell (iPSC) lines from familial long QT syndrome type 1 (LQT1) patients carrying the KCNQ1 c.1201dupC (p.Arg401fs) frame shift mutation by using non-integrational Sendai reprogramming method. The patient-specific iPSC lines harboring the c.1201dupC mutation on KCNQ1 gene expressed pluripotency markers and had the capacity to differentiate into three germ layers.
Resource table
Unique stem cell line identifier
Alternative names of stem cell lines
Institution
⁎
NUIGi008-A NUIGi008-B NUIGi008-C NUIGi009-A NUIGi009-B NUIGi009-C NUIGi010-A NUIGi010-B NUIGi010-C NUIGi011-A NUIGi011-B NUIGi011-C LQTS007C1 (NUIGi008-A) LQTS007C2 (NUIGi008-B) LQTS007Cx (NUIGi008-C) LQTS008C1 (NUIGi009-A) LQTS008C6 (NUIGi009-B) LQTS008Cx (NUIGi009-C) LQTS009C2 (NUIGi010-A) LQTS009C3 (NUIGi010-B) LQTS009Cx (NUIGi010-C) LQTS010C3 (NUIGi011-A) LQTS010C4 (NUIGi011-B) LQTS010Cx (NUIGi011-C) Regenerative Medicine Institute, National University of Ireland Galway, H91 TK33 Galway, Ireland
Contact information of distributor Type of cell lines Origin Cell Source Clonality Method of reprogramming Multiline rationale Gene modification Type of modification Associated disease Gene/locus Method of modification Name of transgene or resistance Inducible/constitutive system Date archived/stock date Cell line repository/bank Ethical approval
Sanbing Shen [email protected] Induced pluripotent stem cells (iPSCs) Human Dermal fibroblasts Clonal or mixed CytoTune-iPSC 2.0 Sendai Reprogramming Kit Same disease non-isogenic cell lines Yes Congenital Long QT Syndrome type 1 (LQT1) c.1201dupC mutation on exon 9 of KCNQ1 gene N/A N/A N/A February 2018 Regenerative Medicine Institute, National University of Ireland Galway The study has been approved by the Ethics Committee of Galway University Hospitals (C.A.750). Patient written informed consent were obtained for skin biopsy procedure.
Resource utility These LQT1 patient-specific iPSC lines provide a valuable resource
Corresponding authors. E-mail addresses: [email protected] (S. Shen), [email protected] (T. Prendiville), [email protected] (T. O'Brien).
https://doi.org/10.1016/j.scr.2019.101650 Received 10 October 2019; Received in revised form 30 October 2019; Accepted 5 November 2019 Available online 14 November 2019 1873-5061/ © 2019 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/).
Stem Cell Research 41 (2019) 101650
N. Ge, et al.
maintained in culture with Essential 8™ Flex Medium in 5% CO2 at 37°C. IPSCs were passaged every 4–6 days using 1 in 4 splits by Gentle Cell Dissociation Reagent (STEMCELL).
to in vitro explore long QT syndrome disease pathogenesis and therapeutic interventions such as new drug screening and gene therapy. 1. Resource details
2.2. Pluripotent marker expression Long QT syndrome (LQTS), present in 1 per 2000 of the general population, is an inherited primary arrhythmia syndrome that presents with recurrent syncope or, rarely, sudden cardiac death secondary to malignant torsades de points. It is a recognized cause of Sudden Arrhythmic Death Syndrome (SADS) in the young and has an autosomal dominant inheritance pattern that can affect every second member of an extended family pedigree from an index case (Gajewski and Saul, 2010). LQTS is mechanistically due to a cardiac ion channelopathy that has a net effect of delayed repolarization at a myocyte level that manifests as a prolonged QT interval on surface electrocardiogram in the patient. Among the well described genetic etiologies, LQT1, encoding the Voltage-Gated Potassium Channel Kv7.1, is the most common and present in approximately 40–50% of all genotyped patients (Shimizu and Horie, 2011). The mechanistic effect of a pathogenic mutation is either to reduce the efficient trafficking of the channel protein to the cell membrane (haploinsufficiency) or to result in a dysfunctional channel with dominant-negative effect (Wu et al., 2016). This study was approved by the Clinical Research Ethics Committee of Galway University Hospital (C.A.750). Standard skin punch biopsy was collected from a 45-year-old female, a 63-year-old female, a 14year-old male and a 7-year-old female who are from the same family and all carry a c.1201dupC mutation on exon 9 of the KCNQ1 gene, incurring a frame shift mutation. Twelve induced pluripotent stem cell (iPSC) lines (8 clonal lines: NUIGi008-A, NUIGi008-B, NUIGi009-A, NUIGi009-B, NUIGi010-A, NUIGi010-B, NUIGi011-A, NUIGi011-B; and 4 mixed lines: NUIGi008-C, NUIGi009-C, NUIGi010-C, NUIGi011-C) were generated using non-integrating Sendai-virus vectors encoding four Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). All the iPSC lines showed typical ES-like morphology (Fig. 1A), expression of pluripotency markers and differentiation potentials. Molecular karyotyping analysis showed normal karyotype and no chromosomal disruption (Supplementary Fig. 1C). Short Tandem Repeat (STR) analysis for 6 polymorphic microsatellite loci showed identical profiles for all iPSC lines as parental fibroblasts. Mycoplasma test revealed no contamination in any iPSC line (Supplementary Fig. 1B). RT-PCR analysis showed that all iPSC lines were free of exogenous reprogramming vectors (Fig. 1E). Pluripotency was assessed by immunofluorescence staining for OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81 and SSEA4 (Fig. 1B), and qRT-PCR for endogenous pluripotency genes OCT4, SOX2 and NANOG (Fig. 1D). To validate the ability to differentiate into three germ lineages, in vitro embryonic body formation experiments were performed and immunofluorescence staining exhibited evidence for endodermal (α-fetoprotein, AFP), mesodermal (α-smooth muscle actin, α-SMA) and ectodermal (neuron-specific class III β-tubulin, TUJ1) markers (Fig. 1C). Lastly, Sanger sequencing was performed to verify the parental DNA mutation was retained in all iPSC lines (Supplementary Fig. 1A).
Expression of pluripotency markers OCT4, SOX2, NANOG, TRA-160, TRA-1-81 and SSEA4 (Cell Signaling Technology,Table 3) were verified by immunofluorescence staining. Briefly, cells were fixed with 4% paraformaldehyde [Room temperature (RT), 20 min], permeabilized with 0.3% Triton X-100 (RT, 15 min), and blocked with 1% BSA (RT, 1 h). Primary antibodies were incubated at 4°C overnight, and secondary antibodies and Hoechst (Nuclei counterstained) for 1 h (RT). All antibodies were diluted in 1% BSA. Images were acquired by inverted wide-field fluorescence system (Olympus IX81). 2.3. Pluripotency gene expression Total RNA was extracted using RNeasy Mini Kit (Qiagen). cDNA was synthesized using QuantiTect Rev. Transcription Kit (Qiagen). Pluripotency genes OCT4, SOX2 and NANOG were assessed by qPCR using Fast SYBR Green Master Mix (Applied Biosystems) with StepOnePlus™ Real-Time PCR System (Applied Biosystems). GAPDH was used as the ‘housekeeping gene’ control. LQT007 fibroblasts were used as the negative control, and the commercial iPSC line (NCRM1) was used as the positive control. All primers used were listed in Table 3. 2.4. Virus-free status of iPSC lines TopTaq® Master Mix (Qiagen) was used for PCR using Veriti Gradient Thermal Cycler (Applied Biosystems) under standard conditions [94°C 2 min (1X); 94°C 30 s, 55°C 30 s, 72°C 30 s (35X), 72°C 5 min (1X), 4°C (hold)]. PCR products were analyzed using agarose gel (1.5%) electrophoresis and were confirmed free of Sendai virus. 2.5. In vitro differentiation To validate the capacity to form three germ layers, 100k hiPSCs were spun in AggreWell 400 plate (STEMCELL) for 24 h to form embryonic bodies (EBs) and cultured in-suspension in EB medium, consisting of DMEM/F-12 medium, 20% KnockOut Serum Replacement, 1% L-glutamine, 1% Non-essential amino acids, 1% Penicillin-streptomycin and 0.2% β-mercaptoethanol. 7 days later, EBs were seeded onto Geltrex-coated chamber slides (ibidi) and cultured at 5% CO2 and 37°C for 4-weeks, and stained using antibodies against AFP, α-SMA and TUJ1. EB medium was used during the whole in vitro differentiation process. 2.6. Validation of KCNQ1 mutation To validate variants in KCNQ1 gene, genomic DNA was extracted by using DNeasy Blood and Tissue Kit (Qiagen) and amplified with primers (Table 3). Purified PCR products were submitted to Eurofins MWG Operon (Germany) for sequencing. The 2-read results from both directions were analyzed by Sequence Scanner Version (Applied Biosystems), which confirmed the c.1201dupC mutation.
2. Materials and methods 2.1. Fibroblast culture and iPSC reprogramming Skin fibroblasts were cultured in DMEM with Glutamax (Gibco) with 10% FBS (Sigma-Aldrich), 1% Antibiotic-Antimycotic (Gibco), and 1% Non-essential amino acids (Gibco) at 37°C in 5% CO2, and reprogramming performed using Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) (Takahashi et al., 2007). On day 7, transduced fibroblasts were plated onto Geltrex-coated plates with a density of 1 × 105/ 6-well in Essential 8™ Flex Medium (Gibco), and refreshed daily. Remaining cells were used as positive controls for SeV-specific RT-PCR. IPSC colonies were manually picked between day 21 and 27, and
3. SNP array IPSC (passage 20–30) genomic DNA was extracted for karyotyping (990k SNP array) at Beijing Hyslar Biotech (Beijing, China). Data were analyzed with Axiom Analysis Suite (ThermoFisher, USA), and Log2 ratio was generated to detect copy number variations. No gross chromosomal alterations were detected as a result of reprogramming. However, the limitations of molecular karyotyping are acknowledged including failure to detect a balanced translocation if it exists. 2
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Fig. 1. Generation and characterization of twelve human induced pluripotent stem cell (iPSC) lines from four familial long QT Syndrome Type 1 (LQT1) patients carrying KCNQ1 c.1201dupC mutation. Table 1 Summary of lines. iPSC line names
Abbreviation in figures
Gender
Age
Ethnicity
Genotype of locus
NUIGi008-A NUIGi008-B NUIGi008-C NUIGi009-A NUIGi009-B NUIGi009-C NUIGi010-A NUIGi010-B NUIGi010-C NUIGi011-A NUIGi011-B NUIGi011-C
LQTS007C1 LQTS007C2 LQTS007Cx LQTS008C1 LQTS008C6 LQTS008Cx LQTS009C2 LQTS009C3 LQTS009Cx LQTS010C3 LQTS010C4 LQTS010Cx
Female
45
Female
63
Male
14
Female
7
Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian
c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC c.1201dupC
3.1. Fingerprinting
mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation
Disease on on on on on on on on on on on on
KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1 KCNQ1
gene gene gene gene gene gene gene gene gene gene gene gene
Long Long Long Long Long Long Long Long Long Long Long Long
QT QT QT QT QT QT QT QT QT QT QT QT
Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome Syndrome
type type type type type type type type type type type type
1 1 1 1 1 1 1 1 1 1 1 1
with PCR primers of six loci (Table 3) under condition [94°C 2 min (1X); 94°C 30 s, 55°C 30 s, 72°C 30 s (35X), 72°C 5 min (1X), 4°C (hold)]. PCR products were analyzed using 3% agarose gel by
Genomic DNA from parental fibroblasts and iPSCs were amplified 3
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Table 2 Characterization and validation. Classification
Test
Result
Data
Morphology Phenotype
Normal morphology Positive for SOX2, OCT4, NANOG, SSEA4, TRA-1-60 and TRA-1-81.
Fig. 1A Fig. 1B
Genotype Identity
Photography Qualitative analysis: Immunocytochemistry Quantitative analysis: qRT-PCR RT-PCR Single Nucleotide Polymorphism (SNP) Fingerprinting (STR analysis)
Fig. 1D Fig. 1E Supplementary Fig. 1C Available with the authors
Mutation analysis Microbiology and virology Differentiation potential
Sanger sequencing Mycoplasma In vitro embryonic body formation
Positive for SOX2, OCT4 and NANOG Negative for the presence of Sendai vectors No gross chromosomal alteration by reprogramming was detected Tested 6 sites (D1S1656,D3S1358, D7S796, D10S1214, D16S539, and D21S2055), all matched Heterozygous mutation DNA c.1201dupC on KCNQ1 gene Detection by PCR, negative α-fetoprotein (AFP) for endoderm, α-smooth muscle actin (α-SMA) for mesoderm, and β-III tubulin (TUJ1) for ectoderm
Supplementary Fig. 1A Supplementary Fig. 1B Fig. 1C
Table 3 Reagents details. Antibodies used for immunocytochemistry
Pluripotency markers
Differentiation markers
Secondary antibodies
Antibody Rabbit anti-OCT-4A (IgG) Rabbit anti-SOX2 Rabbit anti-NANOG Mouse anti-TRA-1-60 (IgM) Mouse anti-TRA-1-81 (IgM) Mouse anti-SSEA4 (IgG3) Mouse anti-α-Fetoprotein (AFP) Mouse anti-α-Actin, Smooth Muscle (α-SMA) Mouse anti-β3-Tubulin (TUJ1) (IgG) Goat anti-mouse IgG Fluor 555 Goat anti-rabbit IgG Fluor 488
Dilution 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:500 1:1000 1:1000
Company Cat# and RRID Cell Signaling Technology, Cat. #2840, RRID: AB_2167691 Cell Signaling Technology, Cat. #3579, RRID: AB_2195767 Cell Signaling Technology, Cat. #3580, RRID: AB_2150399 Cell Signaling Technology, Cat. #4746, RRID: AB_2119059 Cell Signaling Technology, Cat. #4745, RRID: AB_10829904 Cell Signaling Technology, Cat. #4755, RRID: AB_1264259 SIGMA, Cat. #A8452, RRID: AB_258392 Cell Marque Corp, Cat. #202M-95, RRID: AB_1157938 Cell Signaling Technology, Cat. #5568, RRID: AB_10692510 Cell Signaling Technology, Cat. #4409, RRID: AB_1904022 Cell Signaling Technology, Cat. #4412, RRID: AB_1904025
Target SeV/ 181 bp KOS/ 528 bp Klf4/ 410 bp c-Myc/ 532 bp OCT4/ 191 bp SOX2/ 187 bp NANOG/ 219 bp GAPDH/ 206 bp D1S1656 D3S1358 D7S796 D10S1214 D16S539 D21S2055 KCNQ1-Exon 9/ 401 bp
Forward/Reverse primer (5′−3′) GGA TCA CTA GGT GAT ATC GAG C/ ACC AGA CAA GAG TTT AAG AGA TAT GTA TC ATG CAC CGC TAC GAC GTG AGC GC/ ACC TTG ACA ATC CTG ATG TGG TTC CTG CAT GCC AGA GGA GCC C/ AAT GTA TCG AAG GTG CTC AA TAA CTG ACT AGC AGG CTT GTC G/ TCC ACA TAC AGT CCT GGA TGA TGA TG GGG GTT CTA TTT GGG AAG GTA T/ GTT CGC TTT CTC TTT CGG GC AGA CTT CAC ATG TCC CAG CAC T/ CGG GTT TTC TCC ATG CTG TTT C GGA TCC AGC TTG TCC CCA AA/ TGT TTG CCT TTG GGA CTG GT AGG GCT GCT TTT AAC TCT GGT/ CCC CAC TTG ATT TTG GAG GGA GTG TTG CTC AAG GGT CAA CT/ GAG AAA TAG AAT CAC TAG GGA ACC ACT GCA GTC CAA TCT GGG T/ ATG AAA TCA ACA GAG GCT TGC TTT TGG TAT TGG CCA TCC TA/ GAA AGG AAC AGA GAG ACA GGG TGC ATA AAA TAT TGC CCC AAA AC/ TTG AAG ACC AGT CTG GGA AG GAT CCC AAG CTC TTC CTC TT/ ACG TTT GTG TGT GCA TCT GT AAC AGA ACC AAT AGG CTA TCT ATC/ TAC AGT AAA TCA CTT GGT AGG AGA AGC TGT AGC TTC CAT AAG GGC/ CCT ACA TAC CCC CAA GTC GG
Primers Sendai Vectors
Pluripotency Markers
House-Keeping Genes Fingerprinting
Target mutation sequencing
and SS, the Chinese Scholarship Council and NUIG College Scholarship to NG, the Science Foundation Ireland for Grant 13/IA/1787 to SS and LG, 16/RC/3948 to FutureNeuro, for funding the project, and the Irish Government's Program for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 2007–2013 for the central facility.
electrophoresis. 3.2. Mycoplasma detection A standard PCR was routinely performed for mycoplasma test by using MycoSensor PCR Assay Kit (Agilent Technologies) according to manufacturer's instruction.
Supplementary materials Supplementary material associated with this article can be found, in the online version, at 10.1016/j.scr.2019.101650.
Declaration of Competing Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
References Gajewski, K.K., Saul, J.P., 2010. Sudden cardiac death in children and adolescents (excluding Sudden Infant Death Syndrome). Ann. Pediatr. Cardiol. 3, 107–112. Shimizu, W., Horie, M., 2011. Phenotypic manifestations of mutations in genes encoding subunits of cardiac potassium channels. Circ. Res. 109, 97–109. Wu, J., Ding, W.G., Horie, M., 2016. Molecular pathogenesis of long QT syndrome type 1. J. Arrhythm. 32, 381–388. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S., 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872.
Acknowledgments Authors wish to acknowledge Irish Research Council for Postdoctoral fellowship to ML, National Children's Research Centre (NCRC) for seed funding and innovation funding to TP, TOB, ML and SS, Galway University Foundation for equipment funding to TOB, ML 4