SeV reprogramming was performed using the Cytotune reprogramming kit (Lifetech) following the manufacturer’s protocol. In brief: 80% confluent fibroblasts grown in fibroblast medium (10% FCS (Gemini), 1% L-glutamine, 1% sodium pyruvate and 1% MEM-NEAA in high-glucose DMEM (Lifetech)) in a 6-well plate were transduced with each of the four viruses at a multiplicity of infection (MOI) of 3. Cells were fed every other day, and 50 k, 100 k and 200 k cells were replated on day 7 onto 0.1% gelatin-coated, 10-cm dishes containing CF1 MEFs (Globalstem). Medium was switched to hESC medium (7 μl/liter 2ME (Sigma), 20% KOSR, 2× L-Glutamine, 1× MEM-NEAA, 10 ng/ml bFGF, in DMEM/F12 (Lifetech)). Colonies were picked by mechanical dissection, were transferred to fresh feeders, and were expanded using mechanical or enzymatic (Collagenase IV, Lifetech) clump passaging methods31 . Reprogramming efficiencies were determined by TRA-1-60 immunocytochemistry as described32 .
Cytotune reprogramming kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Cytotune reprogramming kit is a laboratory product designed for the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The kit provides the necessary components and protocols for this cellular reprogramming process.
Automatically generated - may contain errors
Lab products found in correlation
Human embryonic stem cell medium, by Thermo Fisher Scientific (2 mentions)
Matrigel coated dishes, by Corning (2 mentions)
Mtesr1 media, by STEMCELL (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Egf and fgf2, by Thermo Fisher Scientific (1 mentions)
Essential 8 media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Neural progenitor medium, by STEMCELL (1 mentions)
Matrigel, by Corning (1 mentions)
Accutase, by STEMCELL (1 mentions)
Stemmacs ips brew xf, by Miltenyi Biotec (1 mentions)
Matrigel coated plate, by Corning (1 mentions)
6 protocols using cytotune reprogramming kit
1
Fibroblast Reprogramming into iPSCs
2
Cortical Organoid Generation from Human iPSCs
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Human fibroblasts from neurotypic control 7545 were obtained from the Coriell Institute and reprogrammed into induced pluripotent stem cells by the laboratory of Mike McConnell (UVA) using a CytoTune Reprogramming Kit (Life Technologies) and maintained in Essential 8 media (Life Technologies). Cortical organoids were grown using a low-attachment protocol. Briefly, we used dispase to detach hIPSC colonies from matrigel dishes. These colonies were placed in ES/DMEM medium (GlobalStem) supplemented with 20% Knockout Serum Replacement (Life Technologies) and treated with 5 mM Dorsomorphin and 10 mM SB-431542 for the first 5 d. The ROCK inhibitor Y-27632 (10 mm) was added for the first 48 h. From day 6 to day 24, spheroids were fed with neuronal medium consisting of Neurobasal A with B27 without vitamin A, GlutaMax, and penicillin/streptomycin (Life Technologies) and supplemented with 20 ng/ml EGF and FGF2 (Peprotech). From day 25 to day 42, the growth factors were replaced with 20 ng/ml BDNF and NT3 (Peprotech). From day 43 onward, BDNF and NT3 were removed and organoids were grown solely in neuronal medium.
3
Fibroblast to iPSC Reprogramming
4
Modulation of miR-92a-2-5p in ASD NPCs
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The samples were collected from the foreskin tissue of ASD patients and children without ASD (Supplementary Table S1 ). hiPSCs were established using Cytotune Reprogramming kit (A16517, Thermo, Waltham, MA, USA) and plated onto Matrigel (354277, Corning, NY, USA) coated plates in Neural Progenitor Medium (05833, STEMCELL), and then were used to induce NPCs. NPCs were passaged at a ratio of 1:3 every three days using Accutase (7920, STEMCELL).
NPCs were transfected with miR-92a-2-5p mimic or mimic negative control at a final concentration of 50 nM, or inhibitor and inhibitor negative control at a final concentration of 100 nM, using Lipofectamine 3000 (Thermo, L3000008) for 8 h according to the manufacturer’s protocol. RNA and protein expression were determined at 24 h and 48 h after incubation.
NPCs were transfected with miR-92a-2-5p mimic or mimic negative control at a final concentration of 50 nM, or inhibitor and inhibitor negative control at a final concentration of 100 nM, using Lipofectamine 3000 (Thermo, L3000008) for 8 h according to the manufacturer’s protocol. RNA and protein expression were determined at 24 h and 48 h after incubation.
5
Genetic Engineering of Human Induced Pluripotent Stem Cells
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The genetic engineering was carried out on three hiPSC lines in parallel, obtained from healthy donors. Namely:
Cell line 802#7 was derived from skin fibroblasts collected at the University of Lübeck. Reprogramming method was non-integrating using episomal vectors.
For testing in silico designed sgRNAs we used HEK 293T cells expressing S. pyogenes Cas9 under control of a Tetracycline inducible promoter.
- 802#7
- SFC856-03-04 (STBCi063-A)
- SFC840-03-05 (STBCi026-C)
Cell line 802#7 was derived from skin fibroblasts collected at the University of Lübeck. Reprogramming method was non-integrating using episomal vectors.
| Cell line | Sex | Clinical status |
| 802#7 | F | healthy |
| SFC856-03-04 (STBCi063-A) | F | healthy |
| SFC840-03-05 (STBCi026-C) | F | healthy |
For testing in silico designed sgRNAs we used HEK 293T cells expressing S. pyogenes Cas9 under control of a Tetracycline inducible promoter.
6
Generating iPSCs from Skin Fibroblasts
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To generate iPSCs, skin fibroblasts were infected with Sendai virus vectors containing coding sequences of human OCT4, SOX2, KLF4, and c-MYC (Cytotune reprogramming kit, Thermo Fisher). Four days post-infection, fibroblasts were trypsinized to single cells, plated on the inactivated mouse embryonic fibroblast feeders, and cultured using a human embryonic stem cell medium (Gibco). After 3–4 weeks, iPSC clones were manually picked and propagated clonally on feeders. After 8–10 passages, iPSCs were transferred to a feeder-free system and grown on matrigel-coated dishes (Corning) in mTeSR1 media (StemCell Technologies). The cells were passaged by manually picking colonies.
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