无异条件下多原代羊水和膜细胞重新编程
1Mitchell Cancer Institute,University of South Alabama, 2College of Medicine,University of South Alabama, 3Institute for Regenerative Medicine,University of Zurich, 4Department of Dermatology,University Hospital Zurich, 5Center for Applied Biotechnology and Molecular Medicine (CABMM),University of Zurich – Irchel Campus
Summary
该协议描述了在完全化学定义的条件下, 使用 non-integrating episomal 方法将原代羊水和膜间充质干细胞重新编程为诱导多能干细胞。详细介绍了通过严格的方法提取、培养、重新编程和表征所产生的诱导多能干细胞的过程。
Abstract
随着诱导多能干细胞的引入, 自体细胞疗法在现实中得到了更进一步的研究。胚胎干细胞, 如羊水和膜间充质干细胞, 代表了一种独特的组织工程中的未分化细胞, 并为未来的儿科干预和干细胞银行重组为 iPSC。这里介绍的协议描述了一个优化的过程, 以提取和培养初级羊水和膜间充质干细胞和产生 episomal 诱导多能干细胞从这些细胞在完全化学定义的文化利用人类重组受精和 E8 培养基的条件。本文还描述了采用严格的方法-流式细胞仪、共聚焦成像、畸胎瘤形成和转录分析-的新品系的特性。新生成的 Oct3/4A、Nanog、Sox2、TRA-1-60、TRA-1-81、SSEA-4 等胚胎干细胞的标记, 同时对 SSEA-1 标记呈阴性。干细胞系形成畸胎瘤在免疫缺陷-米色小鼠6-8 周和畸胎瘤包含组织代表的所有三种细菌层。通过将全球表达微阵列数据提交到生物多评估算法中的线转录分析认为所有的线多能性, 因此, 这种方法是一个诱人的替代动物试验。新的 iPSC 线可以很容易地用于下游实验, 涉及优化分化和组织工程。
Introduction
诱导多能干细胞技术 (iPSC) 带来了潜在的细胞置换疗法, 疾病和发育模型, 药物和毒理学筛选1,2,3。在概念上可以通过细胞注射、体外分化组织 (如心脏贴片) 或通过组织工程进行引导再生来实现替代疗法。羊水 (AFSC) 和膜干细胞 (AMSC) 是一种极好的细胞源, 这些干预措施要么直接4,5,6,7 ,要么作为重新编程的起始单元格填充进入多8,9,10,11,12。
早期的方法使用未定义的区域性系统或重新编程的方法, 需要构建9、10、11、12。最近的一项研究采用了无异的培养基, 即使使用了较不明确的基底膜附着基质 (钢), 从羊水上皮细胞中生成 iPSC。然而, 畸胎瘤的形成方法并没有包括在研究中, 以及大量的体外和分子数据。羊水上皮细胞与新生儿成纤维细胞的13相比, 具有大约8倍的高重编程效率。在另一项研究中, 羊水中的间充质干细胞也被发现被重新编程成 iPSC, 效率更高12。
多能干细胞可以分化为所有3种细菌层的组织代表, 因此具有最广泛的潜能。儿科患者可以受益于他们的自体羊水干细胞产前和羊膜干细胞产期的收获, 重新编程和组织工程。此外, 相对较低的胚胎干细胞分化水平 (低于成人干细胞14,15) 可以从理论上帮助解决从 iPSC16中的源细胞观察到的表观遗传偏见的保留。
在这里, 我们提出了一个协议, 以重新编程羊水和膜干细胞多在化学定义的无异 E8 培养基上的重组受精17 (VTN) 使用 episomal 质粒18。羊水和膜细胞作为细胞重新编程的主要优势在于它们的可用性和产期, 因此这种方法主要有利于儿科组织工程的研究。
Protocol
该议定书遵循人类研究道德委员会的机构准则。病人的书面同意获得了使用羊水进行研究。 该议定书遵循南阿拉巴马大学机构动物保育和使用委员会的政策。 1. 原代羊膜间充质干细胞的分离培养 羊膜液细胞的电镀 在医生的羊膜穿刺过程中获得至少2.5 毫升的羊水。注: 所有活细胞和组织的处理必须在无菌组织培养柜中进行,…
Representative Results
在为遗传学测试目的采集羊水之前, 患者获得了知情的书面同意, 并为研究提供了一小分。由于胎盘代表医疗废物, 在研究中使用羊膜是不需要同意的。羊水和膜干细胞表现出典型的间质性, 其细胞形态呈纺锤形, 相亮。在重新编程时, 细胞经历 mesenchymal-to-epithelial (MET) 过渡, 并获得鹅卵石样的形态学和空间组织的殖民地, 表明上皮的性质。这一过程是启动早在 48-72 h 后引进重…
Discussion
胚胎干细胞 iPSC 生成的初始阶段需要从胎儿组织中提取源细胞, 它们的培养、扩展和 episomal 重编程质粒的引入。在第一个完全重新编程的殖民地可以扩展之前, 这一阶段的文化周期大约14-18 天。最后阶段是 iPSC 无性系的成熟。羊膜干细胞的初步提取是通过羊膜的联合机械和酶消化的方法来实现的。我们发现, 潜伏期为30分钟, 产生的细胞数量最多, 提取的存活率最高。消化过程可以产生小块的组织和…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了全 Medizinische Forschung 在苏黎世大学的 Forschungskredit, 苏黎世大学, absciex NMSCh在研究金10.216 和 12.176, 瑞士国立心脏病学会, 瑞士国家科学依据授予 [320030-122273] 并且 [310030-143992], 第七框架节目, 生活阀门, 欧共体根据授予 [242008], 奥尔加 Mayenfisch 基础, EMDO 基础, 启动的津贴2012大学医院苏黎世和米切尔癌症研究所的内部资金。
Materials
| Tumor Dissociation Kit, human | Miltenyi Biotec | 130-095-929 | tissue dissociation system, reagent kit, includes tissue dissociation tubes and tissue dissociation enzymes |
| gentleMACS Dissociator | Miltenyi Biotec | 130-093-235 | tissue dissociation system, dissociator |
| Thermo Scientific™ Shandon™ Disposable Scalpel No. 10, Sterile, Individually Wrapped, 5.75 (14.6cm) | Thermo-Fisher | 3120032 | |
| 70 µm cell strainers | Corning | 10054-456 | |
| RPMI 1640 medium | Thermo-Fisher | 32404014 | |
| rocking platform | VWR | 40000-300 | |
| 50 ml centrifuge tubes | Thermo-Fisher | 339652 | |
| 15 ml centrifuge tubes | Thermo-Fisher | 339650 | |
| EBM-2 basal medium | Lonza | CC-3156 | basal medium for AFMC medium |
| FGF 2 Human (expressed in E. coli, non-glycosylated) | Prospec Bio | CYT-218 | bFGF, supplement for AFMC medium |
| EGF Human, Pichia | Prospec Bio | CYT-332 | EGF, supplement for AFMC medium |
| LR3 Insulin Like Growth Factor-1 Human Recombinant | Prospec Bio | CYT-022 | IGF, supplement for AFMC medium |
| Fetal Bovine Serum, embryonic stem cell-qualified | Thermo-Fisher | 10439024 | FBS |
| Antibiotic-Antimycotic (100X) | Thermo-Fisher | 15240062 | for primary AFSC/AMSC, for routine AFSC/AMSC it should not be necessary, do not use in medium for transfected cells! |
| Accutase cell detachment solution | StemCell Technologies | 07920 | cell detachment enzyme |
| CryoStor™ CS10 | StemCell Technologies | 07930 | complete freezing medium |
| PBS, pH 7.4 | Thermo-Fisher Scientific | 10010023 | |
| EndoFree Plasmid Maxi Kit (10) | Qiagen | 12362 | for plasmid isolation |
| pEP4 E02S EN2K | Addgene | 20925 | EN2K, reprogramming factors Oct4+Sox2, Nanog+Klf4 |
| pEP4 E02S ET2K | Addgene | 20927 | ET2K, reprogramming factors Oct4+Sox2, SV40LT+Klf4 |
| pCEP4-M2L | Addgene | 20926 | M2L, reprogramming factors c-Myc+LIN28 |
| NanoDrop 2000c UV-Vis Spectrophotometer | Thermo-Fisher | ND-2000C | spectrophotometer |
| Neon® Transfection System | Thermo-Fisher | MPK5000 | transfection system, components: Neon pipette – transfection pipette Neon device – transfection device |
| Neon® Transfection System 10 µL Kit | Thermo-Fisher | MPK1025 | consumables kit for the Neon Transfection System, it contains: Neon tip – transfection tip Neon tube – transfection tube buffer R – resuspension buffer buffer E – electrolytic buffer |
| Stemolecule™ Sodium Butyrate | StemGent | 04-0005 | small molecule enhancer of reprogramming |
| TeSR-E8 | StemCell Technologies | 05940 | E8 medium |
| Vitronectin XF™ | StemCell Technologies | 07180 | VTN, stock concentration 250 µg/ml, used for coating at 1 µg/cm2 in vitronectin dilution (CellAdhere) buffer |
| CellAdhere™ Dilution Buffer | StemCell Technologies | 07183 | vitronectin dilution buffer |
| UltraPure™ 0.5M EDTA, pH 8.0 | Thermo-Fisher | 15575020 | dilute with PBS to 0.5 mM before use |
| EVOS® FL Imaging System | Thermo-Fisher Scientific | AMF4300 | LCD imaging microscope system |
| CKX53 Inverted Microscope | Olympus | phase contrast cell culture microscope | |
| Pierce™ 16% Formaldehyde (w/v), Methanol-free | Thermo-Fisher | 28908 | dilute to 4% with PBS before use, diluted can be stored at 2-8 °C for 1 week |
| Perm Buffer III | BD Biosciences | 558050 | permeabilization buffer, chill to -20 °C before use |
| Mouse IgG1, κ Isotype Control, Alexa Fluor® 488 | BD Biosciences | 557782 | isotype control for Oct3/4A, Nanog |
| Mouse IgG1, κ Isotype Control, Alexa Fluor® 647 | BD Biosciences | 557783 | isotype control for Sox2 |
| Mouse anti-human Oct3/4 (Human Isoform A), Alexa Fluor® 488 | BD Biosciences | 561628 | |
| Mouse anti-human Nanog, Alexa Fluor® 488 | BD Biosciences | 560791 | |
| Mouse anti-human Sox-2, Alexa Fluor® 647 | BD Biosciences | 562139 | |
| Mouse IgGM, κ Isotype Control, Alexa Fluor® 488 | BD Biosciences | 401617 | isotype control for TRA-1-60 |
| Mouse IgGM, κ Isotype Control, Alexa Fluor® 647 | BD Biosciences | 401618 | isotype control for TRA-1-81 |
| Mouse anti-human TRA-1-60, Alexa Fluor® 488 | BD Biosciences | 330613 | |
| Mouse anti-human TRA-1-81, Alexa Fluor® 647 | BD Biosciences | 330705 | |
| Mouse IgG1, κ Isotype Control, Alexa Fluor® 488 | BD Biosciences | 400129 | isotype control for SSEA-1 |
| Mouse IgG3, κ Isotype Control, Alexa Fluor® 647 | BD Biosciences | 401321 | isotype control for SSEA-4 |
| Mouse anti-human SSEA-1, Alexa Fluor® 488 | BD Biosciences | 323010 | |
| Mouse anti-human SSEA-4, Alexa Fluor® 647 | BD Biosciences | 330407 | |
| Affinipure F(ab')2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor® 488 | Jackson Immunoresearch | 115-606-068 | use at a dilution of 1:600 or further optimize |
| Affinipure F(ab')2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor® 647 | Jackson Immunoresearch | 115-546-068 | use at a dilution of 1:600 or further optimize |
| DAPI | Thermo-Fisher Scientific | D21490 | stock solution 10 mM, further dilute to 1:12.000 for a working solution |
| Corning® Matrigel® Growth Factor Reduced, Phenol Red-Free | Corning | 356231 | basement membrane matrix (BMM) |
| scid-beige mice, female | Taconic | CBSCBG-F | |
| RNeasy Plus Mini Kit (50) | Qiagen | 74134 | RNA isolation kit |
| T-25 flasks, tissue culture-treated | Thermo-Fisher | 156367 | |
| T-75 flasks, tissue culture-treated | Thermo-Fisher | 156499 | |
| Nunc™ tissue-culture dish | Thermo-Fisher | 12-567-650 | 10 cm tissue culture dish |
| 6-well plates, tissue-culture treated | Thermo-Fisher | 140675 | |
| Neubauer counting chamber (hemacytometer) | VWR | 15170-173 | |
| Mr. Frosty™ Freezing Container | Thermo-Fisher | 5100-0001 | freezing container |
| FACS tubes, Round Bottom Polystyrene Test Tube, 5ml | Corning | 352058 | 5 ml polystyrene tubes |
| Eppendorf tubes, 1.5 ml | Thermo-Fisher | 05-402-96 | 1.5 ml microcentrifuge tubes |
| PCR tubes, 200 µl | Thermo-Fisher | 14-222-262 | |
| pipette tips, 100 to 1250 µl | Thermo-Fisher | 02-707-407 | narrow-bore 1 mL tips |
| pipette tips, 5 to 300 µl | Thermo-Fisher | 02-707-410 | |
| pipette tips, 0.1 to 10 µl | Thermo-Fisher | 02-707-437 | |
| wide-bore pipette tips, 1000 µl | VWR | 89049-166 | wide-bore 1 mL tips |
| glass Pasteur pipettes | Thermo-Fisher | 13-678-20A | |
| ethanol, 200 proof | Thermo-Fisher | 04-355-451 | |
| vortex mixer | VWR | 10153-842 | |
| chambered coverglass, 8-well, 1.5mm borosilicate glass | Thermo-Fisher | 155409 | glass-bottom confocal-grade cultureware |
| 22G needles | VWR | 82002-366 | |
| insulin syringes | Thermo-Fisher | 22-253-260 | |
| Formalin solution, neutral buffered, 10% | Sigma-Aldrich | HT501128-4L | fixation of explanted teratomas |
| Illumina HT-12 v4 Expression BeachChip | Illumina | BD-103-0204 | expression microarray, supported by PluriTest, discontinued by manufacturer |
| PrimeView Human Genome U219 Array Plate | Thermo-Fisher | 901605 | expression microarray (formerly Affymetrix brand), soon to be supported by PluriTest |
| GeneChip™ Human Genome U133 Plus 2.0 Array | Thermo-Fisher | 902482 | expression microarray (formerly Affymetrix brand), supported by CellNet, soon to be supported by PluriTest |
| PluriTest® | Coriell Institute | www.pluritest.org, free service for bioinformatic assessment of pluripotency, accepts microarray data – *.idat files from HT-12 v4 platform, soon to support U133, U219 microarray and RNA sequencing data | |
| CellNet | Johns Hopkins University | cellnet.hms.harvard.edu, free service for bioinformatic identification of cell type, including plutipotent stem cells, based on U133 microarray data – *.cel files, soon to support RNA sequencing data |
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Tags
Keyword Extraction:Amniotic FluidMembrane CellsPluripotencyXeno-free ConditionsInduced Pluripotent Stem CellsEpisomal ReprogrammingFetal Stem CellsDisease ModelingBasic ResearchLongitudinal StudiesChemically Defined ConditionsPediatric Cell-based TherapiesPlacentasAmnionRPMI-1640 MediumAFMC MediumAmniotic Fluid Stem CellsMembrane Stem CellsCell Detachment EnzymePBS
Cite This Article
Slamecka, J., Laurini, J., Shirley, T., Hoerstrup, S. P., Weber, B., Owen, L., McClellan, S. Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions. J. Vis. Exp. (129), e56003, doi:10.3791/56003 (2017).