IPS课件InducedPluripotentStemCells(iPSCs).ppt

Induced Pluripotent Stem Cells(iPSCs) a candidate of Embryonic stem cells,武栋成 武汉大学基础医学院生化教研室 海外实验室,Bone Marrow Transplant,造血干细胞,造血干细胞是指骨髓中的干细胞，具有自我更新能力，能发育生成各种类型的血细胞。造血干细胞可以救助很多患有血液病的人们，最常见的就是白血病。,脐带血干细胞,间充质干细胞,是属于中胚层的一类多能干细胞，主要存在于结缔组织和器 官间质中。,骨髓间充质干细胞不仅可分化为造血细胞，还具有分化为肌 细胞、肝细胞、成骨细胞、软骨细胞、基质细胞等多种细胞 的能力，也可分化为各种神经细胞。,脐带间充质干细胞来源广泛，易获得，表达多种胚胎干细胞 的特有分子标志，并具有间充质细胞的所有特性。,100,间充质干细胞,神经干细胞,神经干细胞是神经系统形成和发育的源泉。,我们培养的神经干细胞（P1）,我们培养的神经干细胞（P4）,成体干细胞,存在于发育成熟机体器官组织中的具有高度自我 更新和增殖潜能的未分化细胞，可以分化成为组 成该组织的特定细胞类型，并可横向分化为至少 23种以上其他的组织细胞。,干细胞,自我复制,产生大量细胞,增殖,多向分化,任何类型体细胞,诱导、分化,自我复制,多向分化潜能,干细胞,胚胎干细胞,造血干细胞,脐带间充质干细胞,脐带血干细胞,成体干细胞,骨髓间充质干细胞,神经干细胞,干细胞分类,诱导多功能 干细胞(iPS),胚胎干细胞,胚胎干细胞是指当受精卵分裂发育成囊胚 时内细胞团的细胞，它具有体外培养无限 增殖、自我更新和多向分化的特性。胚胎 干细胞是最理想的全能干细胞。,Embryonic stem cells (ESCs),self-renewal,plenty of cells,proliferation,pluripotency,Any kind of somatic cells,induction,differentiation,Embryonic stem cells (ESCs),Human ES cells might be used to treat a host of diseases, such as Parkinsons disease, spinal cord injury, and diabetes,In January 2009, the US Food and Drug Administration (FDA) approved the first clinical trial for using human ES cells to treat patients with spinal cord injury,Embryonic Stem Cells,inner cell mass,feeder layer,Set up ES system Store forever，numerous copies,Embryonic stem cells (ESCs),However,Clinical limitation,ethical difficulties,potential tumor formation,tissue rejection,One way to solve these issues is the generation of pluripotent cells directly from the patients own cells.,Reprogramming Strategies,Reprogramming： Somatic cells can be reprogrammed to a state of pluripotency,Hypothesis,The Third Reprogramming Strategies,Induction of Pluripotent Stem Cells from mouse fibroblats by defined factors,The factors are expressed in ES cells ，but not in somatic cells.,Cell 2006,introduce each of the 24 candidate genes into mouse embryonic fibroblasts(MEFs) from Fbx15geo/geo embryos ( retroviral transduction),(a bgeo cassette (a fusion of the b-galactosidase and neomycin resistance genes) into the mouse Fbx15 gene by homologous recombination,Fbx15geo/geo inactivated,no G418-resistant colonies,Strategy to test candidate factors,Schematic drawing representing the strategy for reprogramming,-4 day,0 day,3 day,5 day,Retrovirus transduction,Plate on Feeder,ES medium + bFGF,iPS generation,Somatic cells,5*104,Day 1:细胞准备 When human adult fibroblast (HAF) cells have reached 80% confluence, aspirate medium, wash once with PBS, cover cells with 0.05% trypsin, incubate for 5 min at 37 C. Inactivate trypsin with HAF medium, collect cells in a 50-ml conical tube. Centrifuge the cells at 200g at room temperature for 4 min and discard the supernatant.,从体细胞到干细胞的重编程,Day 1:细胞准备 Resuspend the cells in 1 ml HAF medium and determine cell number using hemacytometer. Dilute cell suspension with HAF medium to 1 104 cells ml-1. Transfer 1 ml HAF suspension (totally 1 104 cells) per well of 12-well plate (matrigel coated). Incubate at 37 C, 5% CO2, for 24 h.,Day 2: 病毒感染细胞 Aspirate the medium, replace with 1ml fresh HAF medium ,add polybrene(final 8ug/ml). Add OCT4, SOX2, Nanog, Lin28 lentivirus 100ul (one T75 flask produced virus has been resuspended in 1ml medium). Incubate at 37 C, 5% CO2, for 24 h.,Day 3: 弃病毒 Aspirate medium, wash cells three times with 3 ml PBS, add 1 ml iPS medium.,100,初始细胞形态/Fibroblast,Day 4day21: 培养 Change medium everyday for three weeks. At this stage, you could see different colonies, which show different cell morphology compared with HAF.,Day 22: 挑取ES样细胞克隆/传代 Mechanical pick up single clone and seed to the single wells which containing iMEFs. 48 hours, change medium every two days. One weeks later, the exactly iPS colonies will appear. live staining by Tra1-60 will be positive.,24 factors,22 G418-resistant colonies,whether clones possessed have ES cell-like morphology and proliferation properties,doubling time of iPS was equivalent to that of ES cells,ES cell markers (RT-PCR),RT minus: negativecontrol , add everything except reverse transcriptase,The promoters of Fbx15 and Nanog(Bisulfite genomic sequencing),black points indicate methylated CpGs; white points indicate unmethylated CpG dinucleotides,which of the 24 candidates were critical/important,method:the effect of withdrawal/deletion of individual factors from 24 genes on the formation of G418-resistant colonies.,result:identified 10 factors(3, 4, 5, 11, 14, 15, 18, 20, 21, and 22),Combination of these 10 genes alone produced more ES cell-like colonies than transduction of all 24 genes did,Effect of the removal of individual factors from the selected 10 factors on the formation of G418-resistant colonies,Combination of 4 factors,identify 4 factors,Combination of 3 factors,Combination of 2 factors,Four factors,Induced Induced Pluripotent Stem Cells,whether clones possessed have ES cell-like morphology and express ES marker genes(RT-PCR),expressed the majority of marker genes, with the exception of Ecat1 .,Sox2 was only expressed in iPS-MEF10-6,the promoters of Oct3/4 and Nanog(Chromatin immunoprecipitation analyses),the promoters of Oct3/4 and Nanog increased acetylation of histone H3 and decreased dimethylation of lysine 9 of histone H3,CpG dinucleotides in these promoters remained partially methylated in iPS cells,alkaline phosphatase and SSEA-1,DNA Microarrays,Red indicates increased expression,green means decreased expression,the expression level of iPS is similar to ES,Genes upregulated in both ES and iPS cells, Genes in ES cells are upregulated more,Pluripotency of iPS Cells Derived from MEFs,teratoma Formation (in vivo),differentiation into neural and muscle tissues (immunostaining),subcutaneous injection into nude mice,iPS-MEF10, iPS-MEF4, and iPS-MEF3 cells formed embryoid bodies ( in vitro),embryoid bodies from iPS-MEF3 cells remained undifferentiated,pluripotency of iPS-MEF10 and iPS-MEF4 and nullipotency of iPS-MEF3 in vitro,a-smooth muscle actin (mesoderm marker), a-fetoprotein (endoderm marker), III tubulin (ectoderm marker),Differentiation,So induced pluripotent stem cells from mouse embryonic fibroblasts(MEFs) have ES-like morphology ， express ES marker genes and own pluripotency as well as ES cells,Can Oct3/4, Sox2, c-Myc, and Klf4 induce stem cells from human somatic cells ?,Cell 2007,Induced Pluripotent Stem Cells,Skin fibroblast,Patient,Reprogramming,Unknown genetic and epigenetic processes,Pluripotent stem cell,Disease Models (chemical + genetic screening),“Personalized” Regenerative Medicine (no rejection),Nanog,Sox 2,Oct 3/4,1.Optimize transduction method,GFP,Amphochroic retrovirus,HDF,Less than 20% cells expressed GFP,GFP,Ecotropic retrovirus,MEF,More than 60% cells expressed GFP,Slc7a-1:,Lentitrovirus,HDF,HDF-Slc7a-1,GFP,Ecotropic retrovirus,About 80% cells expressed GFP,HDF-Slc7a-1,GFP： green fluorescent protein HDF：human dermal fibroblasts MEF：mouse embryonic fibroblasts,mouse receptor for retroviruses,2. Generation of iPS cells from Adult HDF,Facial dermis,HDF,Slc7a-1,HDF-Slc7a-1,Oct3/4,Sox2,c-Myc,Klf4,Day6,SNL Feeder Cells DMEM+10%FBS,Replace the medium: Primate ES culture +bFGF,Day7,Day21,Non-ES cell like colony,Day25,hES-like colony,B,c,D,Day30,Pick up ES-like cells and mechanically disaggregate,SNL Feeder Cells Primate ES culture+bFGF,ES-like colony,1. Tightly packed and flat colonies,2. Large nuclei and scant cytoplasm,3. Spontaneous differentiation in the colonies center,1. Tightly packed and flat colonies,3. Spontaneous differentiation in the colonies center,2. Large nuclei and scant cytoplasm,3.Human iPS Express hES Markers,3.1 iPS cells express hES cell-specific surface antigen,3.2 iPS Express hES Markers,retroviral silencing.,Efficient silencing of all the four retroviruses,4. High Telomerase Activity and Exponential Growth of Human iPS Cells,5.Embryoid Body-Mediated Differentiation of Human iPS Cells,embroid body -various kind of cells,Neuron-like,Epithelial,Cobblestone -like,Ectoderm,Mesoderm,Endoderm,Endoderm,Mesoderm,Ectoderm,differentiate into three germ layers in vitro,Direct differention into Neural Cells,Neuronal Structure cells,immunochemistry,Marker gene expression,markerof dopaminergic neuron,Marker of astrocyte,Marker of Ectoderm and neuron,GFAP: Glial fibrillary acidic protein MAP2: microtubule-associated protein 2,Direct differention into Cardiac Cells,Phase-contrast image of iPS cells differentiated into cardiomyocytes.,6.Teratoma Formation from Human iPS Cells,three germ layer cells （HE staining）,The established human iPS cells are similar to hES cells in many aspects, including morphology, proliferation, feeder dependence, surface markers, gene expression, promoter activities, telomerase activities, in vitro differentiation, and teratoma formation.,Any help