Cytotune sendai reprogramming kit

Manufactured by Thermo Fisher Scientific

Sourced in France

The Cytotune Sendai Reprogramming Kit is a laboratory product designed for the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The kit contains Sendai virus vectors that carry the reprogramming factors necessary to induce pluripotency in target cells.

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CytoTune-iPS 2.0 Sendai Reprogramming Kit (250 citations) CytoTune-iPS Sendai Reprogramming Kit (46 citations) CytoTune™-iPSC 2.0 Sendai Reprogramming Kit (25 citations) CytoTune (22 citations) CytoTune®-iPSC Sendai Reprogramming Kit (9 citations) Cytotune reprogramming kit (6 citations) CytoTune-iPS 2.0 Sendai Virus Reprogramming Kit (5 citations) CytoTune 2.0 Sendai Reprogramming Kit (4 citations) CytoTune kits (2 citations) OSKM CytoTune-iPS 2.0 Sendai Reprogramming kit viral particle factors (2 citations)

13 protocols using cytotune sendai reprogramming kit

Generation and Characterization of CMT1A hiPSCs

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Fibroblasts from four CMT1A patients were obtained from the Coriell Biorepository (GM05146, GM05148, GM05165, GM05167). Fibroblasts were reprogrammed into induced pluripotent stem cells using previously reported methods^{45 (link)} and the Cytotune Sendai Reprogramming Kit (LifeTech A1378001). After 2 weeks of reprogramming, IPS cells were identified by their colony morphology and replated onto mouse embryonic fibroblast (MEF) feeder layers. They were maintained on MEF feeder layers through weekly passaging for up to 50 passages. Lines were regularly assessed to be free of mycoplasma contamination. The 18 hiPSC clones initially generated were screened for expression of the genes HHLA1 and LincROR, which were shown by Yamanaka’s group to predict clones would be differentiation-defective versus differentiation competent^{46 (link)}, and microRNA 371 cluster expression, which was shown to predict differentiation towards neuronal lineages.^{47 (link)} Based on the results of our screens (data not shown), five of the 18 CMT1A-hiPSC clones were chosen for further studies. Due to variability in PMP22 expression levels, matched pairs of CMT1A hiPSC-Schwann cell precursors and controls hiPSC-SCPs with at least a PMP22 upregulation of 1.5x were used for subsequent experiments to best model CMT1A physiology.

Mukherjee-Clavin B., Mi R., Kern B., Choi I.Y., Lim H., Oh Y., Lannon B., Kim K.J., Bell S., Hur J.K., Hwang W., Hyun Che Y., Habib O., Baloh R.H., Eggan K., Brandacher G., Hoke A., Studer L., Jun Kim Y, & Lee G. (2019). Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder. Nature biomedical engineering, 3(7), 571-582.

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Fibroblast-Derived iPSC Generation

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Fibroblasts were derived from a skin biopsy and cultured in fibroblast medium: DMEM supplemented with 10% FBS (all Life Technologies). In total, 1 × 10⁶ fibroblasts were reprogrammed using CytoTune Sendai reprogramming Kit (Life Technologies A16518), transduced with the recommended multiplicity of infection (MOI) for each vector (KOS MOI = 5, hc-Myc MOI = 5, hKlf4 MOI = 3) for 24 h and seeded on mouse embryonic fibroblast (MEF) feeder, switched to human embryonic stem cells (hESC) medium 2 days post transduction: DMEM/F12 with GlutaMAX, 10% KnockOut Serum Replacement, 1× nonessential amino acids (Life Technologies), 0.1 mM 2-mercaptoethanol (Sigma), and 10 ng/mL bFGF (Waisman Center). Picked induced pluripotent stem cell (iPSC) clones were cultured on MEF feeder/hESC medium first and subsequently adapted in feeder-free conditions on matrigel (Corning) in the presence of E8 medium (Life Technologies). iPSC lines were routinely passaged using 0.5 mM EDTA and thawed in the presence of ROCK inhibitor (Y-27632, Sigma) 24 h after thawing.

Norrie J.L., Nityanandam A., Lai K., Chen X., Wilson M., Stewart E., Griffiths L., Jin H., Wu G., Orr B., Tran Q., Allen S., Reilly C., Zhou X., Zhang J., Newman K., Johnson D., Brennan R, & Dyer M.A. (2021). Retinoblastoma from human stem cell-derived retinal organoids. Nature Communications, 12, 4535.

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Reprogramming PBMCs to iPSCs

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Peripheral blood mononuclear cells (PBMCs) were isolated from a remotely collected blood sample using a Vacutainer CPT cell preparation tube with sodium citrate (BD Biosciences), separated by centrifugation, and cultured in PBMC medium: RPMI-1640 plus 10% FBS, GlutaMAX and sodium pyruvate (all Life Technologies). In all, 1 × 10⁶ PBMCs were reprogrammed using CytoTune Sendai reprogramming Kit (Life Technologies A16518), transduced with the recommended MOI for each vector (KOS MOI = 5, hc-Myc MOI = 5, hKlf4 MOI = 3) for 24 h, and seeded on MEF feeder, 2 days post transduction switched to hESC medium. hESC medium: DMEM/F12 with GlutaMAX (Life Technologies), 10% KnockOut Serum Replacement (Life Technologies), 0.1 mM 2-mercaptoethanol (Life Technologies), 1× nonessential amino acids (Life Technologies), and 10 ng/mL bFGF. Picked iPSC clones were cultured on MEF/hESC medium first and subsequently adapted and cultured in a feeder-free condition on matrigel (Corning) in the presence of E8 medium (Life Technologies). iPSC lines were routinely passaged using 0.5 mM EDTA and thawed in the presence of ROCK inhibitor (Y-27632, Sigma) 24 h after thawing.

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Reprogramming of HEF into hiPSCs for Neuronal Differentiation

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The HEF were reprogrammed into human-induced pluripotent stem cells (hiPSC) using a CytoTune™ Sendai Reprogramming Kit (Life Technologies, Thermo-Fisher, Illkirch-Graffenstaden, France), according to the manufacturer’s instructions. The HEF and hiPSCs were declared with the number DC-2020-3895 onto the CODECOH platform “https://appliweb.dgri.education.fr/appli_web/codecoh/IdentCodec.jsp (accessed on 18 May 2020)”. The cells were maintained in mTeSR1 (StemCell Technologies, Saint Egreve, France) on a matrigel coating (Corning, Thermo-Fisher, Illkirch-Graffenstaden, France), and dissociated with dispase (StemCell Technologies), according to the manufacturer’s instructions. Two independent isolates were used. These cells were subsequently differentiated into neurons, astrocytes, and oligodendrocytes, according to the protocol reported below.

Lacote S., Tamietti C., Chabert M., Confort M.P., Conquet L., Pulido C., Aurine N., Baquerre C., Thiesson A., Pain B., De Las Heras M., Flamand M., Montagutelli X., Marianneau P., Ratinier M, & Arnaud F. (2022). Intranasal Exposure to Rift Valley Fever Virus Live-Attenuated Strains Leads to High Mortality Rate in Immunocompetent Mice. Viruses, 14(11), 2470.

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Derivation of Human iPSC Lines

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HEL139 clones were derived from adult female skin fibroblasts (F72) using GG-EBNA-OMKSL-PP, EBNA-EEA-5guides-PGK-Puro, and an additional GG-EBNA-KM-PP plasmid (KLF4 and MYC five guides each). HEL140 was derived from neonatal male skin fibroblasts (HFFs) with GG-EBNA-OMKSL-PP, EBNA-EEA-5guides-PGK-Puro, and GG-EBNA-KM-PP plasmids. HEL141 was derived from neonatal male skin fibroblasts (HFFs) with GG-EBNA-EEA-5guides-PGK-Puro, GG-EBNA-KM-PP, and GG-EBNA-OS-PP plasmids (OCT4 and SOX2 five guides each). The above cell lines were derived using pCXLE-dCas9VPH-T2A-GFP-shP53 activator plasmid. HEL144 was derived from neonatal male skin fibroblasts (HFFs) with inducible PiggyBac vectors using PB-tight-DDdCas9VP192-GFP-IRES-Neo activator and PB-EEA-5g-OSK2M2L1-PGK-Puro guides. Control cell line HEL46.11 was derived from HFFs using CytoTune Sendai Reprogramming Kit (Life Technologies) according to manufacturer’s instructions.

Weltner J., Balboa D., Katayama S., Bespalov M., Krjutškov K., Jouhilahti E.M., Trokovic R., Kere J, & Otonkoski T. (2018). Human pluripotent reprogramming with CRISPR activators. Nature Communications, 9, 2643.

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Generation and Characterization of CMT1A hiPSCs

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Generating NGLY1-deficiency hPSC Models

Cited 2 times

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WA09-C3, -C4, and -C6 human embryonic stem cells (hESCs) were generated through CRISPR-Cas9-mediated editing of the NGLY1 gene in WA09 hESCs followed by single-cell cloning [16 (link)]. Human-induced pluripotent stem cells (hiPSCs) were established from NGLY1-deficiency patients’ fibroblasts (GM25990 and GM26607; Coriell Institute for Medical Research) using CytoTune Sendai Reprogramming Kit (Thermo Fisher Scientific). Except using TeSR-E8 medium (Stemcell Technologies) and 2 mM EDTA passaging solution (Thermo Fisher Scientific), we generally followed the reported method [31 (link)] to culture undifferentiated hPSCs in a feeder cell-free condition. All the hPSCs were routinely subcultured when cell density reached 80% to 90%. The passage numbers of the hESCs and hiPSCs used in our studies spanned across 65–95 and 35–65, respectively. Additional information relevant to the cells was summarized in Table S1. The experiments using hPSCs were performed in compliance with the guidelines and approval of the institutional biosafety committee and embryonic stem cell research oversight committee. All cells were periodically tested using the MycoAlert mycoplasma detection kit (Lonza) and free of mycoplasma.

Lin V.J., Hu J., Zolekar A., Salick M.R., Mittal P., Bird J.T., Hoffmann P., Kaykas A., Byrum S.D, & Wang Y.C. (2022). Deficiency of N-glycanase 1 perturbs neurogenesis and cerebral development modeled by human organoids. Cell Death & Disease, 13(3), 262.

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Cardiomyocyte Differentiation from CPVT iPSCs

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CPVT patients with pathogenic RYR2 mutations provided informed consent to participate in this study, under protocols approved by the Boston Children’s Hospital Institutional Review Board (IRB). Peripheral blood mononuclear cells were reprogrammed to pluripotency using the CytoTune Sendai reprogramming kit (ThermoFisher). iPSCs were differentiated to iPSC-CMs as shown in Supplemental Figure 1A. iPSC-CM purity was assessed by flow cytometry (Supplemental Figure 1B).

Bezzerides V.J., Caballero A., Wang S., Ai Y., Hylind R.J., Lu F., Heims-Waldron D.A., Chambers K.D., Zhang D., Abrams D.J, & Pu W.T. (2019). Gene therapy for catecholaminergic polymorphic ventricular tachycardia by inhibition of Ca2+/calmodulin-dependent kinase II. Circulation, 140(5), 405-419.

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Generation of SCA3 Patient-Derived iPSCs

Cited 1 time

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The study was approved by the University of Florida Institutional Review Board. The SCA3 subject was provided with the approved informed consent. Skin biopsy was performed by punch biopsy (6 mm in diameter) under local anesthesia. The skin specimens were placed in sterile DMEM medium supplemented with 20% FBS and 1% P/S at room temperature for transport to the lab. Biopsy specimens were processed into 0.5-mm cubes and placed into duplicate 25 cm² flasks. The explants were air-dried for 30 min and 12 ml of primary culture medium (DMEM with 20% FBS) was added to the flask. The flasks were placed in a 37 °C 5% CO₂ incubator. The medium was replenished after 7 days. When fibroblasts from adjacent explants started to merge, the flasks were treated with 0.05% Trypsin/EDTA and passed to a 75 cm² flask. These cells were designated as passage 1. Passage 3 fibroblasts were used for reprogramming.
Reprogramming was performed by a non-integrating method using CytoTune Sendai Reprogramming Kit (Thermo Fisher Scientific) on the fibroblasts according to the manufacturer’s protocol as previously published [50 (link)]. Isolated iPSC clones were cultured on either vitronectin- or Matrigel-coated plates in TeSR-E8 medium (STEMCELL Technologies).

Zhang N., Bewick B., Schultz J., Tiwari A., Krencik R., Zhang A., Adachi K., Xia G., Yun K., Sarkar P, & Ashizawa T. (2021). DNAzyme Cleavage of CAG Repeat RNA in Polyglutamine Diseases. Neurotherapeutics, 18(3), 1710-1728.

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Generation of Integration-free iPSCs

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IPSCs were generated as described^{15 (link)}. Cyto-Tune Sendai reprogramming kit (ThermoFisher) was used according to the manufacturer’s instructions and colonies were expanded on Matrigel-coated (BD Biosciences) plates in mTeSR1 medium (Stemcell Technologies). Lack of genomic integration of Sendai virus into iPSCs was confirmed by PCR and karyotype analysis confirmed the expected chromosomal copy numbers^{16 (link)}. Every 7–8 days, iPSCs were treated with collagenase, and passaged. To verify pluripotency, cells were characterized for Nanog and Tra1–60^{17 (link)}.

Mishra H.K., Ying N.M., Luis A., Wei H., Nguyen M., Nakhla T., Vandenburgh S., Alda M., Berrettini W.H., Brennand K.J., Calabrese J.R., Coryell W.H., Frye M.A., Gage F.H., Gershon E.S., McInnis M.G., Nievergelt C.M., Nurnberger J.I., Shilling P.D., Oedegaard K.J., Zandi P.P., Kelsoe J.R., Welsh D.K, & McCarthy M.J. (2021). Circadian rhythms in bipolar disorder patient-derived neurons predict lithium response: Preliminary studies. Molecular psychiatry, 26(7), 3383-3394.

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