Sendai virus

Manufactured by Thermo Fisher Scientific

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The Sendai virus is a laboratory equipment used for the transfer of genetic material into cells. It functions as a vector, facilitating the introduction of foreign genetic information into target cells.

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19 protocols using sendai virus

iPSC Generation from Erythroblasts

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Once the erythoblast population had been expanded from the patient’s PBMCs, we used a Sendai virus (Life Technologies) for cell reprogramming.
In brief, we incubated 150,000 erythroblasts in StemSpan containing erythroid cytokines for 24 h. Sendai viruses encoding OCT3/4, SOX2, KLF4, and C-MYC pluripotency factors were added, in accordance with the manufacturer’s instructions, in the presence of StemSpan medium containing erythroid expansion factors. Two days later, the cells were used to seed cultures on MEF feeder cells in the presence of one volume of StemSpan medium with erythroid cytokines and 2 volumes of iPS medium supplemented with bFGF, or to seed one volume of StemSpan medium with erythroid cytokines and 2 volumes of ReproTeSR in Matrigel-coated plates. The medium was progressively replaced, ending with 100% iPSC medium supplemented with bFGF, or 100% ReproTeSR medium for cells on Matrigel. The generation of iPSC colonies was monitored daily, by checking for morphological changes. Colonies began to appear two weeks after transduction and were picked during the 3^rd and 4^th weeks.

Mouka A., Izard V., Tachdjian G., Brisset S., Yates F., Mayeur A., Drévillon L., Jarray R., Leboulch P., Maouche-Chrétien L, & Tosca L. (2017). Induced pluripotent stem cell generation from a man carrying a complex chromosomal rearrangement as a genetic model for infertility studies. Scientific Reports, 7, 39760.

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Induced Neural Stem Cell Generation

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Three million PBMNCs were suspended in SCF medium and then infected by Sendai virus (Life Technologies, Carlsbad, USA) encoding OCT3/4, SOX2, KLF4, and c-MYC (OSKM) (MOI=10). Two days later, these cells were plated at a density of 2×10⁵/well on poly-D-lysine/laminin-coated 12-well plates in NSC medium, consisting of DMEM/F12:Neurobasal (1:1), 1×N2, 1×B27, 2 mM GlutaMAX, 1% NEAA (Life Technologies), 10 ng/mL recombinant human leukemia inhibitory factor (rhLIF, Millpore, Billerica, USA), 3 μM CHIR99021 and 2 μM SB431542 (both from Gene Operation, Michigan, USA). The medium was changed every other day. Ten days later, epithelium-like clones appeared in the culture. On days 32 to 35, the clones were large enough to be picked up and transferred onto PDL/laminin-coated 96-well plates for expansion. To inactivate residual Sendai virus, the incubator temperature was raised from 37 °C to 39 °C for one week when iNSCs were at passage number three. Culture for one week at higher temperature resulted in death of around half of the iNSCs, and the surviving cells were passaged in PDL/laminin-coated 24-well plates, and then onto coated 6-well plates when the cell population was sufficiently large.

Yuan Y., Tang X., Bai Y.F., Wang S., An J., Wu Y., Xu Z.Q., Zhang Y.A, & Chen Z. (2018). Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson's disease model. Theranostics, 8(17), 4679-4694.

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Efficient Generation of Human iNSCs

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Human induced neural stem cells were generated as previously reported (Yuan et al., 2018). Briefly, human PBMCs were isolated from the blood of two healthy men (age 25 and 28 years; 30 mL blood each) under informed consent. Cultured PBMCs were then infected by Sendai virus (Life Technologies, Carlsbad, CA, USA) encoding OCT3/4, SOX2, KLF4, and c-MYC (OSKM) (multiplicity of infection [MOI] = 10). After 2 days of infection, cells were changed into iNSC proliferation medium consisting of DMEM/F12 (Gibco, Carlsbad, CA, USA), Neurobasal-A (Gibco), N2 (50×, Gibco), B27 (100×, Gibco), GlutaMAX (100×, Gibco), NEAA (100×, Gibco), CHIR99021 (final concentration 3 μM), SB431542 (final concentration 2 μM), and leukemia inhibitory factor (LIF) (final concentration 10 ng/mL). Equal volumes of DMEM/F12 and Neurobasal-A were used. The medium was prepared and stored at 4°C for 1 week. Prior to changing, the medium was placed at room temperature instead of 37°C. The medium was half-changed every 2 days and passaged every 4–6 days. Prior to cell transplantation, iNSCs were dissociated into single cells with Accutase (Gibco) at 37°C for 5–20 minutes, followed by centrifugation at 250 × g for 5 minutes, and then mixed with the biomaterial (described in “Animal surgery and transplantation”).

Liu S., Liu B., Li Q., Zheng T., Liu B., Li M, & Chen Z. (2023). Transplantation of fibrin-thrombin encapsulated human induced neural stem cells promotes functional recovery of spinal cord injury rats through modulation of the microenvironment. Neural Regeneration Research, 19(2), 440-446.

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Generation of iPSCs from CMT4A Patients

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iPSC lines from two CMT4A patients and one healthy donor were generated with Sendai virus (CytoTune, DNAVEC Corporation) coding for POU5F1, SOX2, KLF4 and MYC. All subjects gave written informed consent prior and the study was approved by the local ethics committee at the Universities of Warsaw (patient #1), Düsseldorf (patient #2) and Bonn (healthy donor). In brief, Sendai virus-infected primary fibroblasts were immediately centrifuged for 45 min at 32 °C with 1500 g (spinfection) and cultivated in Advanced DMEM containing 5% fetal calf serum (FCS) and 1% GlutaMAX™ (all from Life Technologies). On the following day the virus-containing medium was replaced with fresh culture medium. Five days post infection (d5), transduced fibroblasts were trypsinized and seeded onto mouse feeder-coated dishes in DMEM/F-12 containing 10% KnockOut™ Serum Replacement, 1% nonessential amino acids (NEAA), 1% GlutaMAX™, 1% pyruvate, 0.1 mM β-mercaptoethanol and 10 ng/ml basic fibroblast growth factor (bFGF) (all from Life Technologies). Medium was changed every other day until clonal iPSC colonies were manually picked and adapted to feeder-free culture conditions. Several clonal lines were subjected to SNP genotyping in order to identify iPSC clones with normal karyotype.

Wolf C., Pouya A., Bitar S., Pfeiffer A., Bueno D., Rojas-Charry L., Arndt S., Gomez-Zepeda D., Tenzer S., Bello F.D., Vianello C., Ritz S., Schwirz J., Dobrindt K., Peitz M., Hanschmann E.M., Mencke P., Boussaad I., Silies M., Brüstle O., Giacomello M., Krüger R, & Methner A. (2022). GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton. Communications Biology, 5, 541.

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Generation of iPSCs from SHED

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iPSCs were generated from Human Exfoliated Deciduous Teeth Stem Cells (SHED), from control donors recruited through the Tooth Fairy Project initiative (USP, Ethics Committee Protocol 1001). Reprogramming using the Sendai virus (Life Technologies) carried Oct4, Sox2, Kfl4 and c-Myc (Takahashi and Yamanaka, 2006 (link)). SHED were transduced and transferred two days later to a feeder layer condition (murine embryonic fibroblasts, MEFs (Millipore)) and maintained in DMEM/F12 (Life Technologies), 20% KSR (Invitrogen), 1% NEAA, and 100 μM beta-mercaptoethanol. The iPSCs colonies were identified after 3 weeks and transferred to Matrigel (BD Biosciences) coated plates and maintained in mTeSR medium (Stem Cell Technologies), changed daily.

Correa Leite P.E., de Araujo Portes J., Pereira M.R., Russo F.B., Martins-Duarte E.S., Almeida dos Santos N., Attias M., Barrantes F.J., Baleeiro Beltrão-Braga P.C, & de Souza W. (2020). Morphological and biochemical repercussions of Toxoplasma gondii infection in a 3D human brain neurospheres model. Brain, Behavior, & Immunity - Health, 11, 100190.

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Monkey Fibroblast to XF-iPSC Reprogramming

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Monkey fibroblast cells were isolated from skin and further cultured in fibroblast culture medium, which contained DMEM (Thermo Fisher Scientific) plus 15% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% minimum essential medium (MEM) non-essential amino acids solution (Thermo Fisher Scientific).
To establish XF-iPSC lines, monkey fibroblast cells were seeded at 1 × 10⁵ cells in 6-well plates and further cultured for 1–2 days in a fibroblast culture medium. The Sendai virus (Thermo Fisher Scientific) was applied to reprogram monkey fibroblast cells. Sendai virus transduction was performed according to the user’s manual. 7 days post transduction, infected monkey fibroblast cells were replated onto Vitronectin XF-coated 6-well plates at 1 × 10⁵ cells/cm². The cultured medium was refreshed to XF-PSC medium. From 7 to 14 days post transduction onwards, XF-iPSC colonies emerged and could be picked up. Colonies were enzymatically dissociated into single cells using Accutase.

Wu J., Kang Y., Luo X., Dai S., Shi Y., Li Z., Tang Z., Chen Z., Zhu R., Yang P., Li Z., Wang H., Chen X., Zhao Z., Ji W, & Niu Y. (2023). Long-term in vivo chimeric cells tracking in non-human primate. Protein & Cell, 15(3), 207-222.

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Culturing Human Cell Lines and Primary Neurons

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Human neuroblastoma SK-N-BE cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 2 g/l glucose, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (all from Gibco).
Human embryonic kidney (HEK) 293T cells were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.
Human inducent pluripotent stem cells (iPSC) were obtained by Sendai virus (Thermo Fisher Scientific) reprogramming of fibroblasts from three healthy donors after informed consent, according to the local ethics committee. iPSCs were maintained in E8 essential medium (Thermo Fisher Scientific) in Matrigel-coated wells (Corning) and characterized for the expression of pluripotency markers (Supplementary Fig. 3).
Primary cortical neurons were isolated from E.15 wild-type mouse embryos dissociated in 0.05% trypsin-EDTA (Thermo Fisher Scientific) at 37 °C for 12 min. 400,000 cells/well were plated on glass coverslips coated with 0.125 mg/ml of poly d-Lysine (Sigma) and 5 μg/ml of laminin (Corning) in 6-well plates and grown in Neurobasal medium (Thermo Fisher Scientific) supplemented with 2% B-27 (Gibco), 2 mM GlutaMAX (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin for 4–7 days in vitro (DIV).

Gumina V., Colombrita C., Fallini C., Bossolasco P., Maraschi A.M., Landers J.E., Silani V, & Ratti A. (2019). TDP-43 and NOVA-1 RNA-binding proteins as competitive splicing regulators of the schizophrenia-associated TNIK gene. Biochimica et biophysica acta. Gene regulatory mechanisms, 1862(9), 194413.

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Characterization of Human Pluripotent Stem Cells

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The human PSC lines used in this study are described in Supplementary Table S1. HES3 NKX2-5^eGFP/w was kindly donated to us by Dr. David Elliot (Elliott et al., 2011 (link)). The other cells lines were generated in our laboratory from peripheral blood mononuclear cells using Sendai virus (Thermo Scientific) (Mesquita et al., 2019 (link); Cruvinel et al., 2020 (link)) (Kasai-Brunswick et al., 2018 (link)). PSC were cultured in mouse embryonic fibroblast feeder layers under standard conditions (Thomson et al., 1998 (link)).

Carvalho A.B., Coutinho K.C., Barbosa R.A., de Campos D.B., Leitão I.D., Pinto R.S., Dos Santos D.S., Farjun B., De Araújo D.D., Mesquita F.C., Monnerat-Cahli G., Medei E.H., Kasai-Brunswick T.H, & De Carvalho A.C. (2022). Action potential variability in human pluripotent stem cell-derived cardiomyocytes obtained from healthy donors. Frontiers in Physiology, 13, 1077069.

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Generation of hiPSC Clones from Cells

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hiPSCs were generated using the method of Ju et al. [5 (link)]. The cells were detached with trypsin/EDTA and 2 × 10⁵ cells were obtained per well. The cells were resuspended in 20% DMEM and seeded into a 6-well plate. The next day, pre-aliquoted Sendai virus (Thermo Fisher Scientific, Carlsbad, CA, USA) was added to the cells and incubated in the presence of 5% CO₂ at 37 °C for 48 h. The virus-containing media was removed after 48 h. Cell media was changed every other day for six days. On day 7, the media was changed to Essential 8 (E8, Thermo Fisher Scientific) media and cultured until colonies appeared. The media was changed daily after it transitioned to E8 media. Three clones of iPSCs were generated from each individual.

Rim Y.A., Nam Y., Park N., Lee K., Jung H., Jung S.M., Lee J, & Ju J.H. (2021). Characterization of Early-Onset Finger Osteoarthritis-Like Condition Using Patient-Derived Induced Pluripotent Stem Cells. Cells, 10(2), 317.

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Macaque Fibroblast Reprogramming to iPSCs

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As described previously [16 (link)], macaque skin fibroblasts were transfected with a Sendai virus (Thermo Fisher Scientific) mixture at multiplicities of infection (MOIs) of 5, 5, and 3 for KOS (OCT4/SOX2/KLF4), hc-Myc, and hKLF4, respectively, at 37 °C in air containing 5% CO₂ for 40 min. The cells were then plated in Matrigel-coated 6-well plates and cultured in DMEM with 10% FBS and 1 mM valproic acid for 3–4 days until significant morphological changes were observed. Then, the cells were trypsinised and re-plated onto fresh mitomycin C-treated mouse embryonic fibroblastic cells (MEFs) at a 1:6 ratio. MEFs pre-treated with mitomycin C served as feeder cells. The medium was replaced with knockout serum replacement (KSR) medium containing 85% DMEM/F12 (DF12), 15% KSR (Thermo Fisher Scientific), 1 mM l-glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, and 5 ng/ml basic fibroblast growth factor (bFGF, Thermo Fisher Scientific). The medium was changed every day until the colonies grew sufficiently large to be picked up.
The clone-like macaque iPSCs were passaged every 4–6 days with collagenase IV and re-plated onto mitomycin C– pre-treated MEFs. The induced cells were demonstrated as iPSCs by alkaline phosphatase (AP) staining, quantitative PCR and immunohistochemical staining for pluripotency markers, and teratoma generation assay using cells of passage 10.

Lu M., Peng L., Ming X., Wang X., Cui A., Li Y., Wang X., Meng D., Sun N., Xiang M, & Chen S. (2019). Enhanced wound healing promotion by immune response-free monkey autologous iPSCs and exosomes vs. their allogeneic counterparts. EBioMedicine, 42, 443-457.

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