现代技术课程相关

1、iPS细胞相关技术纪光臻2013.1.11ContentPart I History of iPSCsPartII Induction and characterization of iPSCsPart IIIFuture prospectPart IHistory of iPSCsHuman DevelopmentThe journey towards iPSCJohn Gurdon,1962, frog Ian Wilmt ,1996,sheepShoukhrat Mitalipov,2013,humanFibroblast to muscle progenitor cells by tr

2、ansfection with MyoDGeneration of iPS cells from MEF cultures via 24 factorsYamanaka factors:Oct4, Sox2, Klf4 and MycTakahashi K and Yamanaka S 2006 Cell.iPSC在人类疾病研究中的作用Togarrat .Int J Hematol (2012) 95:617623Viral versus nonviral methods for induction of iPS cellsiPS cell induction strategiesWu YL.

3、 2013 CellDifferent reprogramming methods for teration of iPS cellsBlood system and iPSCsGeneration of induced pluripotent stem cells from human blood.2009 BloodDifferentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells.Nature genetics 200

4、9Reprogramming of T Cells from Human Peripheral Blood.2010 Cell stem cellDirect conversion of human fibroblasts to multilineage blood progenitors.2010 NatureGeneration human iPSC from blood with episomalChou.cell research 2011 Mar;21(3):518-29.promotorBCL-XLEpigenetic memory in iPSCsEpigenetic memor

5、y in induced pluripotent stem cellsKim K 2010 NatureCell type origin influences the molecular and function properties of mouse induced pluripotent stem cellsPolo JM 2010 Nature BiotecnologyPart IIInduction and characterizationofiPSCs包装RetroEfficient packaging of retroes by PLAT-E cells骨架质粒：pMXs-Oct4

6、 / Sox2 / KLF4 / cMyc包装质粒：VSVG 和Gal-Pol包装细胞：PlatE 或293TExperimental Hematology 31 (2003) 10071014Plasmid preparationRetropackage in PlatE转染前48hMEF48hRetropackage in293TLeti骨架载体：pl-Oct4/Sox2/KLF4/cMycLetipacking system包装质粒：VSVG 和Gal-Pol包装细胞：293TPLL-GFP 24h after transfectionDox inducible letisystemHU

7、MANMOUSEMaherali NCell stem cell 2008Adeno包装细胞： 293AAdEasy SystemMultiplicity of Infection (MOI)Titer (tu / ml) ：number ofcellparticles per100000 (targetcells) x (% of GFP-positivecells) xdilution/ volume of supernatant (in ml).滴度测定（TCID50）1.用2FBS DMEM准备20 mL105/ mL 293A细胞，加入2块96孔板中， 每孔100 uL, 37, 5

8、% CO2 孵箱培养，使之过夜。2.以2FBS DMEM培养基连续10倍稀释腺1010。储存液，稀释倍数1013.96孔板每排的前2孔做对照（只加100 uL 2FBS DMEM培养基），后10同一稀释浓度的100 uL稀释液，共10排；37, 5% CO2 孵箱培养10天。4. 倒置显微镜下观察96孔板，有GFP表达或CPE现象阳性。有效的对照生长良好，无GFP表达或CPE现象，10倍稀释滴度测定必须是度组10孔全部阳性，而1010稀释度组无一孔出现CPE。5.计算每一稀释度阳性加样10孔的比率。如果有6个孔呈阳性则阳性比率为0.6。按照公式计算滴度：S代表10个稀释度阳性比率之和。Plaq

9、ue forming units （pfu)滴度10e（S+0.8）pfu/mL, 其中空斑形成实验 （CPE）Episomal vectorsiPSC诱导小鼠iPSC诱导过程Episomal vectorsDowey SNNatrue protocol 2012iPSC多能性鉴定miPSC体内外多能性的验证体外EB畸胎瘤Wang. Cell Research（2011）21:779-792.Oct4 启动子甲基化检测Test for Pluripotency by Injection into 4-8-Cell EmbryosGTHuang.Stem Cel3l4s 08嵌合体小鼠鉴定Tot

10、ipotency of iPSCsKang LZhao XY2009 Cell stem cell. 2009 NatureSchematic for generating tetraploid complementation mice from MEFsHuman iPSC characterizationPart III Future prospectHuman Embryonic Stem cellsH9 hESCThomson JA 1998ScienceComparison of pluripotency levels of iPS cells from mouse, human a

11、nd monkeyAccessing rodent na¨ve pluripotency through three different routesAttempts to generate na¨ve human pluripotent stem cells through direct reprogramming or reversion of hESCs/hiPSCsCapturing the ground state of human naive pluripotencyGafni O. Nature. 2013.Robust generation of cross-species chimaeric human