实用生物医学实验技术-细胞重编程.ppt

1. Choose an appropriate cell source For mouse: embryonic fibroblasts (MEFs)---isolate from mouse embryos neural progenitor cells---isolate from brain adipose stromal cells---isolate from fat tissue tail tip fibroblasts---cut 1cm tail tip For human: skin fibroblast---skin biopsy, ~3mm punch of circular skin adipose stromal cells---isolate from fat tissue blood mononuclear cells---isolate from blood, ~5-10ml Adipose stromal cells for reprogramming Sugii et al., 2011 Adipose stromal cells for reprogramming Sun et al., 2009 Reprogramming with skin fibroblasts Most used cell types in current studies Reprogramming with peripheral blood mononuclear cells Seki et al., Nat Protoc, 2012 2. Culture of the isolated somatic cells Fibroblasts: DMEM 10% FBS + anti-anti and anti-mycoplasma reagent Neural progenitors: N2B27 Medium + anti-anti and anti-mycoplasma reagent Adipose stromal cells: ASC specific medium, e.g. StemPro MSC SFM (Lonza) Blood mononuclear cells: mononuclear specific medium Freeze down when cells were amplified (usually P2-P3 depending on cell number) 2. Culture of the isolated somatic cells---continue prepare cells for reprogramming---usually ~10,000 cells needed For fibroblasts reprogramming, we can seed different number of cells in 12- or 24-well plates, ~70-80% confluence, well-distributed 5000 10000 20000 2000 Cell quality is critical for reprogramming, better to start reprogramming in 24-48h 3. Choose a delivery method for reprogramming plasmids Oct4, Sox2, Klf4, c-MYC, either individual plasmids or a 4-in-1 plasmid Viral method: retrovirus, lentivirus, sendai virus, adenovirus, does not care about genomic integration or use of virus, high reprogramming efficiency Non-viral: episomal plasmids only, available through Addgene, clinical grade iPSCs requires integration-free and virus-free methods Reprogramming efficiency of different methods Transduction or transfection of the cells Viral method: Virus transduction u