現(xiàn)代技術(shù)課程相關(guān)

1、iPS細(xì)胞相關(guān)技術(shù)紀(jì)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在人類疾病研究中的作用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包裝RetroEfficient packaging of retroes by PLAT-E cells骨架質(zhì)粒：pMXs-Oct4

6、 / Sox2 / KLF4 / cMyc包裝質(zhì)粒：VSVG 和Gal-Pol包裝細(xì)胞：PlatE 或293TExperimental Hematology 31 (2003) 10071014Plasmid preparationRetropackage in PlatE轉(zhuǎn)染前48hMEF48hRetropackage in293TLeti骨架載體：pl-Oct4/Sox2/KLF4/cMycLetipacking system包裝質(zhì)粒：VSVG 和Gal-Pol包裝細(xì)胞：293TPLL-GFP 24h after transfectionDox inducible letisystemHU

7、MANMOUSEMaherali NCell stem cell 2008Adeno包裝細(xì)胞： 293AAdEasy SystemMultiplicity of Infection (MOI)Titer (tu / ml) ：number ofcellparticles per100000 (targetcells) x (% of GFP-positivecells) xdilution/ volume of supernatant (in ml).滴度測(cè)定（TCID50）1.用2FBS DMEM準(zhǔn)備20 mL105/ mL 293A細(xì)胞，加入2塊96孔板中， 每孔100 uL, 37, 5

8、% CO2 孵箱培養(yǎng)，使之過(guò)夜。2.以2FBS DMEM培養(yǎng)基連續(xù)10倍稀釋腺1010。儲(chǔ)存液，稀釋倍數(shù)1013.96孔板每排的前2孔做對(duì)照（只加100 uL 2FBS DMEM培養(yǎng)基），后10同一稀釋濃度的100 uL稀釋液，共10排；37, 5% CO2 孵箱培養(yǎng)10天。4. 倒置顯微鏡下觀察96孔板，有GFP表達(dá)或CPE現(xiàn)象陽(yáng)性。有效的對(duì)照生長(zhǎng)良好，無(wú)GFP表達(dá)或CPE現(xiàn)象，10倍稀釋滴度測(cè)定必須是度組10孔全部陽(yáng)性，而1010稀釋度組無(wú)一孔出現(xiàn)CPE。5.計(jì)算每一稀釋度陽(yáng)性加樣10孔的比率。如果有6個(gè)孔呈陽(yáng)性則陽(yáng)性比率為0.6。按照公式計(jì)算滴度：S代表10個(gè)稀釋度陽(yáng)性比率之和。Plaq

9、ue forming units （pfu)滴度10e（S+0.8）pfu/mL, 其中空斑形成實(shí)驗(yàn) （CPE）Episomal vectorsiPSC誘導(dǎo)小鼠iPSC誘導(dǎo)過(guò)程Episomal vectorsDowey SNNatrue protocol 2012iPSC多能性鑒定miPSC體內(nèi)外多能性的驗(yàn)證體外EB畸胎瘤Wang. Cell Research（2011）21:779-792.Oct4 啟動(dòng)子甲基化檢測(cè)Test for Pluripotency by Injection into 4-8-Cell EmbryosGTHuang.Stem Cel3l4s 08嵌合體小鼠鑒定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