- Email: [email protected]
Generation, genome edition and characterization of iPSC lines from a patient with coenzyme Q10 deficiency harboring a heterozygous mutation in COQ4 gene
Generation, genome edition and characterization of iPSC lines from a patient with coenzyme Q 10 deficiency harboring a heterozygous mutation in COQ4 gene Dami`a Romero-Moya, Julio Casta˜no, Carlos Santos-Oca˜na, Pl´acido Navas, Pablo Menendez PII: DOI: Reference:
S1873-5061(16)30122-2 doi:10.1016/j.scr.2016.09.007 SCR 826
To appear in:
Stem Cell Research
Received date: Accepted date:
4 September 2016 14 September 2016
Please cite this article as: Romero-Moya, Dami`a, Casta˜ no, Julio, Santos-Oca˜ na, Carlos, Navas, Pl´ acido, Menendez, Pablo, Generation, genome edition and characterization of iPSC lines from a patient with coenzyme Q10 deficiency harboring a heterozygous mutation in COQ4 gene, Stem Cell Research (2016), doi:10.1016/j.scr.2016.09.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Stem Cell Research: Lab Resource
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Title: Generation, genome edition and characterization of iPSC lines from a patient with
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coenzyme Q10 deficiency harboring a heterozygous mutation in COQ4 gene.
Authors: Damià Romero-Moya1, Julio Castaño 1, Carlos Santos-Ocaña2, Plácido Navas2,
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Pablo Menendez P1,3.
Affiliations: 1Josep Carreras Leukemia Research Institute. Department of Biomedicine, School
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of Medicine. University of Barcelona. Barcelona. Spain. 2Centro Andaluz de Biología del Desarrollo. Universidad Pablo Olavide, Sevilla, Spain. 3Instituciò Catalana de recerca I Estudis
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Avançats (ICREA), Barcelona, Spain.
Abstract: We report the generation, CRISPR/Cas9-edition and characterization of induced
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pluripotent stem cell (iPSC) lines from a patient with coenzyme Q10 deficiency harboring the heterozygous mutation c.483G>C in the COQ4 gene. iPSCs were generated using nonintegrative Sendai Viruses containing the reprogramming factors OCT4, SOX2, KLF4 and C-
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MYC. The iPSC lines carried the c.483G>C COQ4 mutation, silenced the OKSM expression and were mycoplasma-free. They were bona fide pluripotent cells as characterized by morphology, immunophenotype/gene expression for pluripotent-associated markers/genes, NANOG and OCT4 promoter demethylation, karyotype and teratoma formation. The COQ4 mutation was CRISPR/Cas9 edited resulting in isogenic, diploid and off-target free COQ4corrected iPSCs.
ACCEPTED MANUSCRIPT Resource Table: CQ4-iPSC
Institution
Josep Carreras Leukemia Research Institute (IJC)
Person who created resource
Damia Romero-Moya, Julio Castaño, Pablo Menéndez
Contact person and email
Pablo Menéndez, [email protected]
Date archived/stock date
May, 2015
Origin
Human dermal fibroblasts
Type of resource
Biological Reagent: human COQ4-mutated iPSC line and its
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Name of Stem Cell line
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CRISPR/Cas9-genome corrected isogenic iPSC line.
Sub-type
Cell line
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Link to related literature
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Authentication
OCT4, SOX2, KLF4, C-MYC
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Key transcription factors
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Information in public databases
Ethics
Identity of the iPSC line confirmed by fingerprinting and PCR http://www.ncbi.nlm.nih.gov/pubmed/22368301 http://www.ncbi.nlm.nih.gov/pubmed/26185144 http://www.ncbi.nlm.nih.gov/pubmed/25658047 http://www.ncbi.nlm.nih.gov/pubmed/26850912 http://www.ncbi.nlm.nih.gov/pubmed/26500142 http://www.isciii.es/ISCIII/es/contenidos/fd-el-instituto/fdorganizacion/fd-estructura-directiva/fd-subdireccion-generalinvestigacion-terapia-celular-medicina-regenerativa/fd-centrosunidades/fd-banco-nacional-lineas-celulares/fd-lineascelulares-disponibles/lineas-de-celulas-iPS.shtml Ethics Review Board-competent authority approval obtained
ACCEPTED MANUSCRIPT Resource Details:
A 4-year-old girl was diagnosed in our Institution with minor mental retardation and lethal
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rhabdomyolysis associated to Coenzyme Q10 deficiency (Trevisson et al., 2011) Genetic
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analysis identified a heterozygous c.483 G>C mutation in the COQ4, leading to the amino acid
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change E161D. COQ4-mutated dermal fibroblasts were reprogrammed into iPSCs. Patient and control fibroblasts were transduced with OSKM-expressing Sendai virus (SeV, CytoTuneTM-iPS
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Reprogramming System, Life Technologies) as previously described (Bueno et al., 2016; Munoz-Lopez et al., 2016). iPSC colonies emerged 16-18 days after OSKM transduction and
MA
were picked for further characterization. iPSCs revealed an embryonic stem cell (ESC)-like morphology and alkaline phosphatase activity (Fig 1A). Short tandem repeat analysis
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confirmed iPSC identity (Fig 2). PCR analysis and Sanger sequencing confirmed the c.483
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G>C mutation in all CQ4-iPSC clones (Fig 1B). After 8-10 passages, iPSC lines were OSKM transgene independent as revealed by immunostaining and qRT-PCR analysis for SeV (Fig
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1C). Endogenous expression of NANOG and OCT4 was accompanied by the extensive loss of CpG methylation in their promoters (Fig 1D). CQ4-iPSCs were CRISPR/Cas9 edited in c.483
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and introduced a Taqα1 restriction site with a synonymous change of nucleotides (Fig 1E). The top off-target predicted beforehand, GRID2, was sequenced in CQ4-edited iPSC (CQ4ed-iPSC) lines with no alteration (Fig 1F). Importantly, mutated and edited iPSCs remained diploid after cell reprogramming and further CRISPR/Cas9 edition (Fig 1G). In addition, the endogenous
pluripotency-associated genes OCT4, SOX2, REX1, NANOG and CRIPTO were expressed at comparable levels to human ESCs in both, CQ4- and CQ4ed-iPSCs by qRT-PCR (Fig 1H). Similarly, immunostaining and flow cytometry revealed bona fide expression of OCT4, NANOG,
TRA-1-60, TRA-1-81, SSEA3 and SSEA4 in both CQ4- and CQ4ed-iPSCs (Fig 1I). In vivo differentiation capacity was demonstrated by teratoma formation in immunodeficient mice comprising tissue representing all three germ layers (Fig 1J).
ACCEPTED MANUSCRIPT Materials and Methods iPSC generation and maintenance
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Fibroblasts were maintained in low glucose DMEM supplemented with 20% FBS and 1%
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penicillin/streptomycin (P/S). The iPSCs were generated using the CytoTune®-iPS Sendai
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Reprogramming Kit (Life Technologies). iPSC clones were fully characterized and maintained undifferentiated on irradiated mouse embryonic fibroblasts (iMEF) in hESC media (KO-DMEM supplemented with 20% KO Serum Replacement, 1% GlutaMAX, 1% P/S, 1% NEAA and 0.1
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mM β-mercaptoethanol (all from Life Technologies)) and 10 ng/ml basic fibroblast growth factor
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(bFGF; Miltenyi), or on Matrigel (BD) with MEF-conditioned media (MEF-CM) (Montes et al., 2009; Ramos-Mejia et al., 2012). iPSCs were passaged when confluent with 0.1% collagenase
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iPSC Characterization
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IV/dispase or 0.05% trypsin.
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Established iPSC were fully characterized as previously described (Bueno et al., 2016). SeV elimination was determined by qRT-PCR. Expression of pluripotency markers was performed by immunostaining (alkaline phosphatase, OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, TRA-1-
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81) and qRT-PCR (OCT-4, NANOG, SOX2, REX1, CRIPTO) using the primers and antibodies described in Table 1 and Table 2. G-banding karyotype was performed as previously described (Catalina et al., 2008). For teratoma assays, the different iPSC clones were collected through enzymatic dissociation using collagenase IV, and 2 million cells were re-suspended and injected with 250 µl DMEM and 50µl Matrigel subcutaneously in the back of the NSG mice (Gutierrez-Aranda et al., 2010). Animal experimentation was approved by the Animal Care Committee of the University of Barcelona. Tumors generally developed within 6-8 weeks. Animals were sacrificed for teratoma dissection. After sectioning, the presence of cells from the three germ layer was assessed following Hematoxylin & Eosin staining (Gutierrez-Aranda et al., 2010).
ACCEPTED MANUSCRIPT DNA and RNA extraction and (qRT)-PCR DNA was obtained using the Maxwell 16 Blood DNA Purification kit (Promega). Isolation of total
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RNA was performed using RNAqueous-Micro kit (Ambion). The cDNA synthesized by using
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SuperScript III Reverse Trascriptase kit (Invitrogen). qRT-PCR was done using SyberGreen
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(Applied) and values were normalized to β-ACTIN or GAPDH. PCR conditions were 95ºC for 10min, followed by 40 cycles of 95ºC for 15s and 60ºC for 60s.
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Immunophenotyping
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Cells were fixed with 2-4% PFA in PBS for 20 min at RT and washed 3 times with PBS. Antigen blocking was done with 6% donkey FBS with 0.5% Triton X-100. Cells were then washed and
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incubated at 4ºC overnight with the primary antibody diluted in blocking solution with 0.1%
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Triton X-100. After washing, cells were incubated with the secondary antibody for 2h at RT. To check by flow cytometry the pluripotency-associated markers the iPSCs were dissociated by
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incubation with trypsin 0.05% for 40s. Then, were resuspended in FACS Buffer (5%FBS, 2mM EDTA in PBS) and incubated with the specific antibody in 200µl for 15 min in the darkness.
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Next, the cells were washed twice and stained with 7-aminoactinomycin D (7-AAD) (BD Bioscience) for 5 min. Stained cells were analyzed using FACS Canto II (BD). COQ4 mutation detection For genomic detection of the COQ4 mutation, a touchdown PCR was performed with 100ng of gDNA. PCR conditions were 1 cycle of 5 min at 95°C; 24 cycles of 30 sec at 95°C, 30 sec at 62°C (-0.5°C per cycle), 1 min at 72°C; 6 cycles of 30 sec at 95°C, 30 sec at 50°C, 1 min at 72°C; 1 cycle of 10 min at 72°C. PCRs were resolved in 1% agarose gels. Primers used to confirm the mutation on patient’s fibroblast and iPSC as well as primers used for Sanger sequencing are described in Table 1.
ACCEPTED MANUSCRIPT Promoter Demethylation Bisulfite pyrosequencing of OCT4 and NANOG promoters was done as described (Bueno et
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al., 2016). Briefly, bisulfite modification of genomic DNA was performed with the EZ DNA
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Methylation-Gold kit (Zymo Research) following the manufacturer's instructions. The set of
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primers for PCR amplification and sequencing of NANOG and OCT4 were designed using the
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software PyroMark Assay Design and are detailed elsewhere (Bueno et al., 2016).
ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1: iPSC generation, genome edition and characterization: (A) Representative
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morphology and alkaline phosphatase staining of CQ4-iPSC. (B) PCR and Sanger sequencing
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confirming the presence of COQ4-mutated allele in several representative CQ4-iPSC clones.
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(C) Immunostaining confirming high-level infection with SeV 48 h post-transduction and SeV elimination on all iPSC clones at p8-10 (left panel). qPCR reflecting complete absence of SeV
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at p8-10 (right panel). (D) Pyrosequencing revealing demethylation of NANOG and OCT4 promoters in COQ4-mutated iPSCs as compared to original fibroblasts. (E) Sanger DNA
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sequencing confirming the gene edition. (F) Sanger DNA sequencing of the major predicted offtarget GRID2 gene. (G) Diploid karyotype of CQ4-iPSC and CQ4ed-iPSC. (H) qRT-PCR for the
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pluripotency genes OCT4, SOX2, REX1, NANOG, and CRIPTO in hESC and CQ4-mutated
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and CQ4-edited iPSCs. (I) Representative immunostaining (top panels) for the pluripotency markers OCT4, SSEA-4, NANOG, TRA-1-60 and representative flow cytometry plots (bottom
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panel) for the pluripotency markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81. (J) Representative teratoma analysis revealing three germ layer differentiation of iPSC. CQ4-iPSC: COQ4 mutated
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iPSC; Ctrl-iPSC: Control iPSC; CQ4ed-iPSC: COQ4-edited iPSC. Figure 2: Cell identity confirmed by fingerprinting analysis of patient fibroblasts and iPSCs.
ACCEPTED MANUSCRIPT
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GATGGCCACGGCTGCTT AGGACTCCATGCCCAGGAA ACCACAGTCCATGCCATCAC TCCACCACCCTGTTGCTGT GGATCACTAGGTGATATCGAGC ACCAGACAAGAGTTTAAGAGATATGTATC GGGTTTTTGGGATTAAGTTCTTCA GCCCCCACCCTTTGTGTT CCTGCAGGCGGAAATAGAAC GCACACATAGCCATCACATAAGG ACAACTGGCCGAAGAATAGCA GGTTCCCAGTCGGGTTCAC CAAAAATGGCCATGCAGGTT AGTTGGGATCGAACAAAAGCTATT CTGGGAACCATCAGGAAGG CCAAGAGTGAACAGAAGGGAG ATTCAGCGGTACCGGGAC CCAGACACCCGAGCACCC AGAAGGGAGTAGGGGATGGA GTGCAGATTCAACCAGCCAT CTCGGTAAGGACTGCCATGT
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β –ACTIN-FW β –ACTIN-RV GAPDH-FW GAPDH-RV SeV-Fw SeV-RV OCT4-FW OCT4-RV REX1-FW REX1-RV NANOG-FW NANOG-RV SOX2-FW SOX2-RV COQ4-FW COQ4-RV COQ4-Mut-FW SEQ-COQ4-FW SEQ-COQ4-RV GRID2-FW GRID2-RV
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Table 1: Primers used in this study
Table 2: Antibodies used in this study
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Antibody OCT-4 SSEA-4 NANOG TRA-1-60 Alexa Fluor® 488 α-Rabbit Alexa Fluor® 555 α-Mouse SSEA-3-PE SSEA-4-V450 TRA-1-60-BV510 TRA-1-81-AlexaFlour647®
Supplier ABCAM ABCAM ABCAM ABCAM Jackson ImmunoResearch Life Technologies BD Bioscience BD Bioscience BD Bioscience BD Bioscience
Reference AB19857 AB16287 AB21624 AB16288 711-545-152 A31570 560237 561156 563188 560793
Dilution 1:500 1:50 1:50 1:50 1:500 1:500 1:100 1:100 1:100 1:100
ACCEPTED MANUSCRIPT References
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Bueno, C., Sardina, J. L., Di Stefano, B., Romero-Moya, D., Munoz-Lopez, A., Ariza, L., Chillon, M. C., Balanzategui, A., Castano, J., Herreros, A., Fraga, M. F., Fernandez, A., Granada, I., Quintana-Bustamante, O., Segovia, J. C., Nishimura, K., Ohtaka, M., Nakanishi, M., Graf, T., & Menendez, P. (2016). Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPalpha. Leukemia, 30(3), 674682. Catalina, P., Montes, R., Ligero, G., Sanchez, L., de la Cueva, T., Bueno, C., Leone, P. E., & Menendez, P. (2008). Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties? Mol Cancer, 7, 76. Gutierrez-Aranda, I., Ramos-Mejia, V., Bueno, C., Munoz-Lopez, M., Real, P. J., Macia, A., Sanchez, L., Ligero, G., Garcia-Parez, J. L., & Menendez, P. (2010). Human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem Cells, 28(9), 1568-1570. Montes, R., Ligero, G., Sanchez, L., Catalina, P., de la Cueva, T., Nieto, A., Melen, G. J., Rubio, R., Garcia-Castro, J., Bueno, C., & Menendez, P. (2009). Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGFbeta and IGF-II. Cell Res, 19(6), 698-709. Munoz-Lopez, A., van Roon, E. H., Romero-Moya, D., Lopez-Millan, B., Stam, R. W., Colomer, D., Nakanishi, M., Bueno, C., & Menendez, P. (2016). Cellular Ontogeny and Hierarchy Influence the Reprogramming Efficiency of Human B Cells into Induced Pluripotent Stem Cells. Stem Cells, 34(3), 581-587. Ramos-Mejia, V., Bueno, C., Roldan, M., Sanchez, L., Ligero, G., Menendez, P., & Martin, M. (2012). The adaptation of human embryonic stem cells to different feeder-free culture conditions is accompanied by a mitochondrial response. Stem Cells Dev, 21(7), 1145-1155. Trevisson, E., DiMauro, S., Navas, P., & Salviati, L. (2011). Coenzyme Q deficiency in muscle. Curr Opin Neurol, 24(5), 449-456.
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Figure 1
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Figure 2
S1873-5061(16)30122-2 doi:10.1016/j.scr.2016.09.007 SCR 826
To appear in:
Stem Cell Research
Received date: Accepted date:
4 September 2016 14 September 2016
Please cite this article as: Romero-Moya, Dami`a, Casta˜ no, Julio, Santos-Oca˜ na, Carlos, Navas, Pl´ acido, Menendez, Pablo, Generation, genome edition and characterization of iPSC lines from a patient with coenzyme Q10 deficiency harboring a heterozygous mutation in COQ4 gene, Stem Cell Research (2016), doi:10.1016/j.scr.2016.09.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Stem Cell Research: Lab Resource
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Title: Generation, genome edition and characterization of iPSC lines from a patient with
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coenzyme Q10 deficiency harboring a heterozygous mutation in COQ4 gene.
Authors: Damià Romero-Moya1, Julio Castaño 1, Carlos Santos-Ocaña2, Plácido Navas2,
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Pablo Menendez P1,3.
Affiliations: 1Josep Carreras Leukemia Research Institute. Department of Biomedicine, School
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of Medicine. University of Barcelona. Barcelona. Spain. 2Centro Andaluz de Biología del Desarrollo. Universidad Pablo Olavide, Sevilla, Spain. 3Instituciò Catalana de recerca I Estudis
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Avançats (ICREA), Barcelona, Spain.
Abstract: We report the generation, CRISPR/Cas9-edition and characterization of induced
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pluripotent stem cell (iPSC) lines from a patient with coenzyme Q10 deficiency harboring the heterozygous mutation c.483G>C in the COQ4 gene. iPSCs were generated using nonintegrative Sendai Viruses containing the reprogramming factors OCT4, SOX2, KLF4 and C-
AC
MYC. The iPSC lines carried the c.483G>C COQ4 mutation, silenced the OKSM expression and were mycoplasma-free. They were bona fide pluripotent cells as characterized by morphology, immunophenotype/gene expression for pluripotent-associated markers/genes, NANOG and OCT4 promoter demethylation, karyotype and teratoma formation. The COQ4 mutation was CRISPR/Cas9 edited resulting in isogenic, diploid and off-target free COQ4corrected iPSCs.
ACCEPTED MANUSCRIPT Resource Table: CQ4-iPSC
Institution
Josep Carreras Leukemia Research Institute (IJC)
Person who created resource
Damia Romero-Moya, Julio Castaño, Pablo Menéndez
Contact person and email
Pablo Menéndez, [email protected]
Date archived/stock date
May, 2015
Origin
Human dermal fibroblasts
Type of resource
Biological Reagent: human COQ4-mutated iPSC line and its
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Name of Stem Cell line
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CRISPR/Cas9-genome corrected isogenic iPSC line.
Sub-type
Cell line
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Link to related literature
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Authentication
OCT4, SOX2, KLF4, C-MYC
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Key transcription factors
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Information in public databases
Ethics
Identity of the iPSC line confirmed by fingerprinting and PCR http://www.ncbi.nlm.nih.gov/pubmed/22368301 http://www.ncbi.nlm.nih.gov/pubmed/26185144 http://www.ncbi.nlm.nih.gov/pubmed/25658047 http://www.ncbi.nlm.nih.gov/pubmed/26850912 http://www.ncbi.nlm.nih.gov/pubmed/26500142 http://www.isciii.es/ISCIII/es/contenidos/fd-el-instituto/fdorganizacion/fd-estructura-directiva/fd-subdireccion-generalinvestigacion-terapia-celular-medicina-regenerativa/fd-centrosunidades/fd-banco-nacional-lineas-celulares/fd-lineascelulares-disponibles/lineas-de-celulas-iPS.shtml Ethics Review Board-competent authority approval obtained
ACCEPTED MANUSCRIPT Resource Details:
A 4-year-old girl was diagnosed in our Institution with minor mental retardation and lethal
T
rhabdomyolysis associated to Coenzyme Q10 deficiency (Trevisson et al., 2011) Genetic
IP
analysis identified a heterozygous c.483 G>C mutation in the COQ4, leading to the amino acid
SC R
change E161D. COQ4-mutated dermal fibroblasts were reprogrammed into iPSCs. Patient and control fibroblasts were transduced with OSKM-expressing Sendai virus (SeV, CytoTuneTM-iPS
NU
Reprogramming System, Life Technologies) as previously described (Bueno et al., 2016; Munoz-Lopez et al., 2016). iPSC colonies emerged 16-18 days after OSKM transduction and
MA
were picked for further characterization. iPSCs revealed an embryonic stem cell (ESC)-like morphology and alkaline phosphatase activity (Fig 1A). Short tandem repeat analysis
D
confirmed iPSC identity (Fig 2). PCR analysis and Sanger sequencing confirmed the c.483
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G>C mutation in all CQ4-iPSC clones (Fig 1B). After 8-10 passages, iPSC lines were OSKM transgene independent as revealed by immunostaining and qRT-PCR analysis for SeV (Fig
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1C). Endogenous expression of NANOG and OCT4 was accompanied by the extensive loss of CpG methylation in their promoters (Fig 1D). CQ4-iPSCs were CRISPR/Cas9 edited in c.483
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and introduced a Taqα1 restriction site with a synonymous change of nucleotides (Fig 1E). The top off-target predicted beforehand, GRID2, was sequenced in CQ4-edited iPSC (CQ4ed-iPSC) lines with no alteration (Fig 1F). Importantly, mutated and edited iPSCs remained diploid after cell reprogramming and further CRISPR/Cas9 edition (Fig 1G). In addition, the endogenous
pluripotency-associated genes OCT4, SOX2, REX1, NANOG and CRIPTO were expressed at comparable levels to human ESCs in both, CQ4- and CQ4ed-iPSCs by qRT-PCR (Fig 1H). Similarly, immunostaining and flow cytometry revealed bona fide expression of OCT4, NANOG,
TRA-1-60, TRA-1-81, SSEA3 and SSEA4 in both CQ4- and CQ4ed-iPSCs (Fig 1I). In vivo differentiation capacity was demonstrated by teratoma formation in immunodeficient mice comprising tissue representing all three germ layers (Fig 1J).
ACCEPTED MANUSCRIPT Materials and Methods iPSC generation and maintenance
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Fibroblasts were maintained in low glucose DMEM supplemented with 20% FBS and 1%
IP
penicillin/streptomycin (P/S). The iPSCs were generated using the CytoTune®-iPS Sendai
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Reprogramming Kit (Life Technologies). iPSC clones were fully characterized and maintained undifferentiated on irradiated mouse embryonic fibroblasts (iMEF) in hESC media (KO-DMEM supplemented with 20% KO Serum Replacement, 1% GlutaMAX, 1% P/S, 1% NEAA and 0.1
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mM β-mercaptoethanol (all from Life Technologies)) and 10 ng/ml basic fibroblast growth factor
MA
(bFGF; Miltenyi), or on Matrigel (BD) with MEF-conditioned media (MEF-CM) (Montes et al., 2009; Ramos-Mejia et al., 2012). iPSCs were passaged when confluent with 0.1% collagenase
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iPSC Characterization
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IV/dispase or 0.05% trypsin.
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Established iPSC were fully characterized as previously described (Bueno et al., 2016). SeV elimination was determined by qRT-PCR. Expression of pluripotency markers was performed by immunostaining (alkaline phosphatase, OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, TRA-1-
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81) and qRT-PCR (OCT-4, NANOG, SOX2, REX1, CRIPTO) using the primers and antibodies described in Table 1 and Table 2. G-banding karyotype was performed as previously described (Catalina et al., 2008). For teratoma assays, the different iPSC clones were collected through enzymatic dissociation using collagenase IV, and 2 million cells were re-suspended and injected with 250 µl DMEM and 50µl Matrigel subcutaneously in the back of the NSG mice (Gutierrez-Aranda et al., 2010). Animal experimentation was approved by the Animal Care Committee of the University of Barcelona. Tumors generally developed within 6-8 weeks. Animals were sacrificed for teratoma dissection. After sectioning, the presence of cells from the three germ layer was assessed following Hematoxylin & Eosin staining (Gutierrez-Aranda et al., 2010).
ACCEPTED MANUSCRIPT DNA and RNA extraction and (qRT)-PCR DNA was obtained using the Maxwell 16 Blood DNA Purification kit (Promega). Isolation of total
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RNA was performed using RNAqueous-Micro kit (Ambion). The cDNA synthesized by using
IP
SuperScript III Reverse Trascriptase kit (Invitrogen). qRT-PCR was done using SyberGreen
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(Applied) and values were normalized to β-ACTIN or GAPDH. PCR conditions were 95ºC for 10min, followed by 40 cycles of 95ºC for 15s and 60ºC for 60s.
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Immunophenotyping
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Cells were fixed with 2-4% PFA in PBS for 20 min at RT and washed 3 times with PBS. Antigen blocking was done with 6% donkey FBS with 0.5% Triton X-100. Cells were then washed and
D
incubated at 4ºC overnight with the primary antibody diluted in blocking solution with 0.1%
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Triton X-100. After washing, cells were incubated with the secondary antibody for 2h at RT. To check by flow cytometry the pluripotency-associated markers the iPSCs were dissociated by
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incubation with trypsin 0.05% for 40s. Then, were resuspended in FACS Buffer (5%FBS, 2mM EDTA in PBS) and incubated with the specific antibody in 200µl for 15 min in the darkness.
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Next, the cells were washed twice and stained with 7-aminoactinomycin D (7-AAD) (BD Bioscience) for 5 min. Stained cells were analyzed using FACS Canto II (BD). COQ4 mutation detection For genomic detection of the COQ4 mutation, a touchdown PCR was performed with 100ng of gDNA. PCR conditions were 1 cycle of 5 min at 95°C; 24 cycles of 30 sec at 95°C, 30 sec at 62°C (-0.5°C per cycle), 1 min at 72°C; 6 cycles of 30 sec at 95°C, 30 sec at 50°C, 1 min at 72°C; 1 cycle of 10 min at 72°C. PCRs were resolved in 1% agarose gels. Primers used to confirm the mutation on patient’s fibroblast and iPSC as well as primers used for Sanger sequencing are described in Table 1.
ACCEPTED MANUSCRIPT Promoter Demethylation Bisulfite pyrosequencing of OCT4 and NANOG promoters was done as described (Bueno et
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al., 2016). Briefly, bisulfite modification of genomic DNA was performed with the EZ DNA
IP
Methylation-Gold kit (Zymo Research) following the manufacturer's instructions. The set of
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primers for PCR amplification and sequencing of NANOG and OCT4 were designed using the
AC
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D
MA
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software PyroMark Assay Design and are detailed elsewhere (Bueno et al., 2016).
ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1: iPSC generation, genome edition and characterization: (A) Representative
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morphology and alkaline phosphatase staining of CQ4-iPSC. (B) PCR and Sanger sequencing
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confirming the presence of COQ4-mutated allele in several representative CQ4-iPSC clones.
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(C) Immunostaining confirming high-level infection with SeV 48 h post-transduction and SeV elimination on all iPSC clones at p8-10 (left panel). qPCR reflecting complete absence of SeV
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at p8-10 (right panel). (D) Pyrosequencing revealing demethylation of NANOG and OCT4 promoters in COQ4-mutated iPSCs as compared to original fibroblasts. (E) Sanger DNA
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sequencing confirming the gene edition. (F) Sanger DNA sequencing of the major predicted offtarget GRID2 gene. (G) Diploid karyotype of CQ4-iPSC and CQ4ed-iPSC. (H) qRT-PCR for the
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pluripotency genes OCT4, SOX2, REX1, NANOG, and CRIPTO in hESC and CQ4-mutated
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and CQ4-edited iPSCs. (I) Representative immunostaining (top panels) for the pluripotency markers OCT4, SSEA-4, NANOG, TRA-1-60 and representative flow cytometry plots (bottom
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panel) for the pluripotency markers SSEA-3, SSEA-4, TRA-1-60, TRA-1-81. (J) Representative teratoma analysis revealing three germ layer differentiation of iPSC. CQ4-iPSC: COQ4 mutated
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iPSC; Ctrl-iPSC: Control iPSC; CQ4ed-iPSC: COQ4-edited iPSC. Figure 2: Cell identity confirmed by fingerprinting analysis of patient fibroblasts and iPSCs.
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GATGGCCACGGCTGCTT AGGACTCCATGCCCAGGAA ACCACAGTCCATGCCATCAC TCCACCACCCTGTTGCTGT GGATCACTAGGTGATATCGAGC ACCAGACAAGAGTTTAAGAGATATGTATC GGGTTTTTGGGATTAAGTTCTTCA GCCCCCACCCTTTGTGTT CCTGCAGGCGGAAATAGAAC GCACACATAGCCATCACATAAGG ACAACTGGCCGAAGAATAGCA GGTTCCCAGTCGGGTTCAC CAAAAATGGCCATGCAGGTT AGTTGGGATCGAACAAAAGCTATT CTGGGAACCATCAGGAAGG CCAAGAGTGAACAGAAGGGAG ATTCAGCGGTACCGGGAC CCAGACACCCGAGCACCC AGAAGGGAGTAGGGGATGGA GTGCAGATTCAACCAGCCAT CTCGGTAAGGACTGCCATGT
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β –ACTIN-FW β –ACTIN-RV GAPDH-FW GAPDH-RV SeV-Fw SeV-RV OCT4-FW OCT4-RV REX1-FW REX1-RV NANOG-FW NANOG-RV SOX2-FW SOX2-RV COQ4-FW COQ4-RV COQ4-Mut-FW SEQ-COQ4-FW SEQ-COQ4-RV GRID2-FW GRID2-RV
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Table 1: Primers used in this study
Table 2: Antibodies used in this study
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Antibody OCT-4 SSEA-4 NANOG TRA-1-60 Alexa Fluor® 488 α-Rabbit Alexa Fluor® 555 α-Mouse SSEA-3-PE SSEA-4-V450 TRA-1-60-BV510 TRA-1-81-AlexaFlour647®
Supplier ABCAM ABCAM ABCAM ABCAM Jackson ImmunoResearch Life Technologies BD Bioscience BD Bioscience BD Bioscience BD Bioscience
Reference AB19857 AB16287 AB21624 AB16288 711-545-152 A31570 560237 561156 563188 560793
Dilution 1:500 1:50 1:50 1:50 1:500 1:500 1:100 1:100 1:100 1:100
ACCEPTED MANUSCRIPT References
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Bueno, C., Sardina, J. L., Di Stefano, B., Romero-Moya, D., Munoz-Lopez, A., Ariza, L., Chillon, M. C., Balanzategui, A., Castano, J., Herreros, A., Fraga, M. F., Fernandez, A., Granada, I., Quintana-Bustamante, O., Segovia, J. C., Nishimura, K., Ohtaka, M., Nakanishi, M., Graf, T., & Menendez, P. (2016). Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPalpha. Leukemia, 30(3), 674682. Catalina, P., Montes, R., Ligero, G., Sanchez, L., de la Cueva, T., Bueno, C., Leone, P. E., & Menendez, P. (2008). Human ESCs predisposition to karyotypic instability: Is a matter of culture adaptation or differential vulnerability among hESC lines due to inherent properties? Mol Cancer, 7, 76. Gutierrez-Aranda, I., Ramos-Mejia, V., Bueno, C., Munoz-Lopez, M., Real, P. J., Macia, A., Sanchez, L., Ligero, G., Garcia-Parez, J. L., & Menendez, P. (2010). Human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem Cells, 28(9), 1568-1570. Montes, R., Ligero, G., Sanchez, L., Catalina, P., de la Cueva, T., Nieto, A., Melen, G. J., Rubio, R., Garcia-Castro, J., Bueno, C., & Menendez, P. (2009). Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGFbeta and IGF-II. Cell Res, 19(6), 698-709. Munoz-Lopez, A., van Roon, E. H., Romero-Moya, D., Lopez-Millan, B., Stam, R. W., Colomer, D., Nakanishi, M., Bueno, C., & Menendez, P. (2016). Cellular Ontogeny and Hierarchy Influence the Reprogramming Efficiency of Human B Cells into Induced Pluripotent Stem Cells. Stem Cells, 34(3), 581-587. Ramos-Mejia, V., Bueno, C., Roldan, M., Sanchez, L., Ligero, G., Menendez, P., & Martin, M. (2012). The adaptation of human embryonic stem cells to different feeder-free culture conditions is accompanied by a mitochondrial response. Stem Cells Dev, 21(7), 1145-1155. Trevisson, E., DiMauro, S., Navas, P., & Salviati, L. (2011). Coenzyme Q deficiency in muscle. Curr Opin Neurol, 24(5), 449-456.
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Figure 1
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Figure 2