iPS Cell Induction from Human Non-T, B cells from Peripheral Blood
用人外周血液中的非T淋巴细胞、B细胞诱导生成全能干细胞
发布: 2013年09月20日第3卷第18期 DOI: 10.21769/BioProtoc.907 浏览次数: 9024
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2011
Abstract
The generation of iPS cells gives an opportunity to use patient-specific somatic cells which are a valuable source for disease modeling and drug discovery. To promote these studies, it is important to make iPS cells from easily accessible and less invasive tissues like blood. Here, we describe the basic method to generate human iPS cells from adult peripheral blood. After the isolation of mononuclear cells, a combination of cytokines stimulates the expansion of hematopoietic stem/progenitor population, which is the main target of this protocol. The cells are transduced with plasmid mixture encoding reprogramming factors. In most cases, the plasmids are lost during the establishment of iPS clones.
Materials and Reagents
- Fresh anti-coagulated blood (~10 ml)
- PBS without Ca2+ and Mg2+ (Nacalai tesque, catalog number: 14249-95 )
- Ficoll-paque Plus (GE Healthcare, catalog number: 17-1440-02 )
- StemSpan H3000 (STEMCELL Technologies, catalog number: 0 9800 )
- Recombinant human Interleukin (IL)-6 (100 μg/ml) (PeproTech, catalog number: AF-200-06 )
- Recombinant human Stem Cell Factor (SCF) (300 μg/ml) (PeproTech, catalog number: AF-300-07 )
- Recombinant human Thrombopoietin (TPO) (300 μg/ml) (PeproTech, catalog number: AF-300-18 )
- Recombinant human Flt3 ligand (300 μg/ml) (PeproTech, catalog number: AF-300-19 )
- Recombinant human Interleukin (IL)-3 (10 μg/ml) (PeproTech, catalog number: AF-200-03 )
- Recombinant human basic Fibroblast Growth Factor (bFGF) (10 μg/ml) (Wako, catalog number: 064-04541 )
- Amaxa Human CD34+ cell Nucleofector Kit (Lonza, catalog number: VPA-1003 )
- MEF feeder (Repro Cell, catalog number: RCHEFC003 )
- Matrigel growth factor reduced (BD Biosciences, catalog number: 356231 )
- Primate ES Cell Medium (Repro Cell, catalog number: RCHEMD001 )
- Essential 6 (Life Technologies, catalog number: A1516401 )
- Gelatin (Sigma-Aldrich, catalog number: G1890 )
- Dulbecco’s Modified Eagle’s Medium (DMEM) High glucose with stable L-glutamine (Nacalai tesque, catalog number: 08459-35 )
- Fetal Bovine Serum (FBS) (Life Technologies, catalog number: 10437-028 )
- Plasmid set 2 (Life Technologies, catalog number: A15960 )
- Blood medium (see Recipes)
- Plasmid mixture (see Recipes)
- Transfection mixture (see Recipes)
- iPS medium 1 (see Recipes)
- iPS medium 2 (see Recipes)
- 0.1% Gelatin solution (see Recipes)
- MEF medium (see Recipes)
Equipment
- Nucleofector 2b (Lonza, catalog number: AAB-1001 )
- 6-well tissue culture plate (BD Biosciences, Falcon®, catalog number: 353046 )
- 15 ml conical tube (BD Biosciences, Falcon®, catalog number: 352196 )
- 50 ml conical tube (BD Biosciences, Falcon®, catalog number: 352070 )
- 37 °C 5% CO2 Cell culture incubator
- Microscope
- Centrifuge
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Okita, K. (2013). iPS Cell Induction from Human Non-T, B cells from Peripheral Blood. Bio-protocol 3(18): e907. DOI: 10.21769/BioProtoc.907.
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分类
干细胞 > 多能干细胞 > 细胞诱导
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