Mononuclear cells were isolated from patient blood by Ficoll gradient centrifugation and cultured for 10 days in serum-free medium containing SCF, IL-3, EPO and transferrin (Chou et al., 2015 (link)). At the end of expansion, the cells were transfected with plasmids MOS (expressing OCT4 and SOX2, addgene plasmid #64120), MMK (expressing c-MYC and KLF4, addgene plasmid #64121) and GBX (expressing BCL-XL, addgene plasmid #64123) using 4D Nucleofector (Lonza) (Chou et al., 2015 (link)). The cells were then cultured in the same erythroblast expansion condition for two days before being plated onto vitronectin-coated (Life Technologies) plate in DMEM medium containing 10% FBS. Essential 8 medium was used to replace the serum-containing medium the next day and was used throughout the reprogramming process and for continued iPSC culture. Colonies with iPSC morphology were picked at day 14 and expanded as previously described (Chou et al., 2015 (link)).
Vitronectin coated
Manufactured by Thermo Fisher Scientific
Vitronectin-coated is a type of lab equipment used in cell culture applications. It provides a surface coating that promotes cell attachment and growth. The core function of this product is to facilitate the adhesion and proliferation of cells in a controlled in vitro environment.
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4 protocols using vitronectin coated
1
Generation of patient-derived iPSCs
2
Generation of patient-derived iPSCs
3
Xeno-free human iPSC and ESC culture
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De-identified human iPSC lines YH10, BJ4, and 1323–2 were obtained from Dr Bruce Conklin (Gladstone Institutes, described previously in [27 (link)]). The H1 ESCs were obtained from Dr Paul Tesar (Case Western Reserve University). Human ESCs and iPSCs were used between passages 20–40, maintained in xeno-free conditions in E8 medium on Vitronectin-coated (Life Technologies) plates, and passaged every 3–5 days to prevent differentiation. Unless otherwise specified, reagents were purchased from Gibco.
4
Generation of Huntington's Disease iPSCs
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The HD-hiPSC “109Q” line ND42222 (XX, 109 CAG, passage 42) was obtained from Coriell repository. This line is heterozygous for HTT p.Gln18[109] and thus has 109 CAG repeats in one of the two alleles of HTT. Human iPSC amplification, neuronal cell generation, and terminal differentiation were performed as previously described [29 ]. 109Q iPS cells were maintained on vitronectin-coated (Life Technology) plates in mTeSRplus medium (STEMCELL Technologies). Cultures were fed every other day and passaged via manual dissociation using 0.02% EDTA (0.25 mM; pH 7.2; Merck Sigma-Aldrich) every 4 to 5 days. The human embryonic stem cells lines were cultured as previously described for each line (WT-hiPSC: i90cl16 XX, passage 44 and WT-hESC: RC9 (WT, XY, passage 20–60, RoslinCells) [30 (link)]; HD-hESC: SIVF018 (XX, 46 CAG, passages 18–30, Sydney IVF Stem Cells) and SI187 (XY, 51 CAG, passages 12–25, Stemride, USA) [31 (link)]; HD-iPSC: HD-71Q (ND42228), XX 71 CAG, passages 30–35, Coriell repository) [32 (link)].
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