PBMCs were isolated using tubes containing EDTA and Ficoll and cultured using the Stanford Biobank protocol as previously described (16 (link), 30 (link)). PBMCs were cultured in STEMPro034 SFM (Gibco) medium with cytokines at a density of 2 × 105 cells/mL, for 7 days. The cells were transduced by Sendai virus using the CytoTune-iPS 2.0 Sendai Virus Reprogramming Kit (Thermo Fisher Scientific), delivered at an MOI of 5-5-3 (KOS MOI = 5, hc-Myc MOI = 5, hKlf4 MOI = 3) without polybrene, to a final total volume between 0.4 and 1 mL. Cells and viruses were centrifuged at 300g for 15 minutes at room temperature. Each PBMC pellet was resuspended in complete SFEM II (STEMCELL Technologies) with cytokines and transferred to a well of a 24-well plate. After 3–4 weeks, the colonies were visualized and manually picked. The iPSC colonies were maintained on Matrigel-coated wells in mTeSR1. For karyotyping, iPSCs at passage 4 were analyzed by G-band karyotyping at WiCell Research Institute.
Cytotune ips 2.0 sendai virus reprogramming kit
Manufactured by Thermo Fisher Scientific
The CytoTune-iPS 2.0 Sendai Virus Reprogramming Kit is a laboratory tool designed for the generation of induced pluripotent stem cells (iPSCs) from various cell types. The kit utilizes Sendai virus vectors to deliver the transcription factors necessary for cellular reprogramming.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Essential 8 media, by Thermo Fisher Scientific (2 mentions)
Advanced dmem, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacer, by Thermo Fisher Scientific (1 mentions)
Coating matrix, by Thermo Fisher Scientific (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Mtesr plus medium, by STEMCELL (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
5 protocols using cytotune ips 2.0 sendai virus reprogramming kit
1
Generation of iPSCs from PBMCs
2
Reprogramming Fibroblasts into iPSCs
3
Reprogramming Fibroblasts into iPSCs
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Although all iPSC lines were not generated at the same time, disease-control pairs from the same source repository were established and reprogrammed simultaneously. All fibroblasts were reprogrammed using the CytoTune-iPS 2.0 Sendai virus reprogramming kit (ThermoFisher), following the manufacturer protocol. Briefly, fibroblasts were transduced with a Sendai virus cocktail containing four transcription factors (hOct3/4, hSOX2, hKlf4, hc-Myc) and maintained in Fibroblast medium (DMEM, GlutaMAX, FBS; ThermoFisher) for one week. Transduced fibroblasts were replated onto Vitronectin coated dishes in Essential-8 media (ThermoFisher) and individual iPSC colonies began to emerge within two weeks. We manually isolated clonal iPSC colonies from each culture and expanded two clones per iPSC line for characterization.
4
Keratinocyte-Derived Induced Pluripotent Stem Cells
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Thawed keratinocytes at passage 2 were seeded 1.84 × 104/cm2 in 2 wells of a 4-well dish coated with Coating Matrix (Gibco). Cells were reprogrammed 2 days post-seeding, using the CytoTune-iPS 2.0 Sendai Virus Reprogramming Kit (ThermoFisher) according to the manufacturer’s instructions, with modifications (Re et al., 2018 ). Cells cultured in EpiLife supplemented with HKGS and 5 mM Y-27362 were transduced with the viral vectors at MOIs reported in (Re et al., 2018 ) and placed in a humidified incubator at 37°C, 5% CO2. The next day the media was replaced with media without viral vectors. Six days post-infection cells were passaged onto previously inactivated MEFs and transferred to a hypoxic incubator at 37 °C, 5% CO2, 5% O2. The medium was progressively switched over 4 days from day 13 to 100% KSR medium (Advanced DMEM(Gibco)/Knock-Out Serum Replacer (Gibco)/Glutamax (Gibco)/2-Mercaptoethanol (Gibco)/4ng/ml FGF 2 (Gibco)) with 10µM Y-27362.
After the emergence of iPSC-like colonies, those with appropriate morphology were manually picked on day 28 and transferred to Matrigel coated 6-well plates with mTeSR1 medium (StemCell Technologies) containing 10µM Y-27362. Medium was changed after 24 hours. Colonies were expanded by splitting at 1:3 to 1:6 ratio every 4-6 days and maintained in a hypoxic incubator at 37 °C, 5% CO2, 5% O2.
After the emergence of iPSC-like colonies, those with appropriate morphology were manually picked on day 28 and transferred to Matrigel coated 6-well plates with mTeSR1 medium (StemCell Technologies) containing 10µM Y-27362. Medium was changed after 24 hours. Colonies were expanded by splitting at 1:3 to 1:6 ratio every 4-6 days and maintained in a hypoxic incubator at 37 °C, 5% CO2, 5% O2.
5
Generation of iPSCs from T-ALL Patients
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The peripheral blood mononuclear cells (PBMC) from the T-ALL patients and healthy donors were infected with the Cyto Tune-iPS 2.0 Sendai virus reprogramming Kit (Thermo Fisher). The obtained ESClike clones were identi ed for pluripotency, using methods like alkaline phosphatase staining [13] (link), ow cytometry (2.4), and immuno uorescence staining [13] (link) for the pluripotency markers, and teratoma formation [14] (link). The T-ALL-iPSCs were cultured with mTeSR plus medium (STEMCELL Technologies).
To prepare cell lysates, the iPSCs derived from the healthy cells (H-iPSCs) (1×107/ml) and T-ALL-iPSCs (1×107/ml) were frozen in liquid nitrogen and disrupted by three freeze-thaw cycles. The cell lysates were then spun at 13000g for 20 min, and the supernatants were collected as iPSC antigens. The total protein concentration of the cell lysates was quanti ed using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China).
To prepare cell lysates, the iPSCs derived from the healthy cells (H-iPSCs) (1×107/ml) and T-ALL-iPSCs (1×107/ml) were frozen in liquid nitrogen and disrupted by three freeze-thaw cycles. The cell lysates were then spun at 13000g for 20 min, and the supernatants were collected as iPSC antigens. The total protein concentration of the cell lysates was quanti ed using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China).
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