从小鼠羊水细胞多能干细胞使用转座子系统的生产
1Stem Cell and Regenerative Medicine Laboratory,Fondazione Istituto di Ricerca Pediatrica Citta della Speranza, 2Lunenfeld-Tanenbaum Research Institute,Mount Sinai Hospital, 3Stem Cells and Regenerative Medicine Section, Developmental Biology and Cancer Programme,UCL Institute of Child Health and Great Ormond Street Hospital
Summary
In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.
Abstract
Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.
Introduction
产前诊断是一个重要的临床工具，遗传性疾病（ 如染色体异常，单基因或多基因/多因素疾病）和先天性畸形（ 如先天性膈疝，囊性肺部病变，exomphalos，腹）评价。羊水（AF）细胞是简单怀孕的第二个三个月期间定期调度的程序，以获取（ 即羊膜穿刺术和amnioreduction）或剖腹产1，2。从产前或新生儿患者AF细胞的可用性提供了使用这种源再生医学的可能性，一些研究人员调查处理利用AF 3，4，5，6分离出来的干细胞群不同的组织损伤或疾病的可能性，7，8，9，10，11，12。容易地从患病的患者获得的AF细胞，其中的疾病是经常固定的时间窗口的可能性，开辟了道路使用用于重编程的目的本细胞来源的想法。的确，从AF细胞的诱导的多能干（iPS细胞）可以在用于体外药物测试或用于组织工程的方法感兴趣的细胞分化，为了分娩前准备适当的患者特异性治疗。许多研究已经证实的AF细胞的能力进行重新编程并分化成范围广泛的细胞类型13，14，15，16，17 <的/ SUP>，18，19，20，21，22，23，24，25，26，27。
由于通过四个转录因子（Oct4的，SOX2，cMYC的和Klf4）强制表达的发现高桥和重新编程体细胞的山中伸弥28日 ，取得了长足的重新编程领域取得。考虑到不同的方法，我们可以病毒和非病毒方法之间区别。所述第一预计的病毒载体（逆转录病毒和慢病毒），它们具有高效率，但在反转录病毒转基因的通常不完全沉默，具有部分重编程的细胞系的两种后果和风险的使用插入诱变29，30，31。的非病毒方法使用不同的策略： 即质粒，载体，基因，蛋白质，转座子。自由的转基因序列的iPS细胞的推导的目的，以规避渗漏的转基因表达和插入突变的潜在有害影响。在所有的上述的非病毒策略，所述piggyBac转（PB）转座子/转座系统只需要在反向末端重复侧翼的转基因和转座酶的瞬时表达来催化插入或切除事件32。在使用转座子上的其它方法iPS细胞产生的优点是获得免费矢量-iPS细胞与非病毒载体的方法，其中显示的逆转录病毒载体的相同的效率的可能性。这可以通过对reprogr集成座子编码的痕量少切除amming以下在iPS细胞33转座的新的瞬时表达的因素。鉴于PB为在不同的细胞类型34，35，36，37有效，是更适合于临床方法相对于病毒载体，并允许生产无异物-iPS细胞的违背使用的生物外源性电流病毒产生的协议条件下，该系统用于从鼠的AF获得iPS细胞。
在这里我们建议按照已经发表的作品展示从小鼠AF细胞（iPS-AF细胞）38生产多能干的iPS克隆的详细的协议。
Protocol
所有的程序都是按照意大利法律。鼠AF样品来自孕鼠13.5天交配后（DPC）从C57BL / 6-的Tg（UBC-GFP）30Scha / J小鼠被称为GFP收获。 1.转座子生产注意：使用标准克隆程序生成座子表达载体。使用商业试剂盒制备用于小鼠的AF细胞转染的质粒DNA。 混合的质粒DNA的10毫微克，在1.5ml微量离心管中加入50μl的DH5α细菌。在冰上孵育的微量离心管20分钟。 在42…
Representative Results
为了评估重新编程的能力，小鼠的AF细胞从GFP小鼠的胎儿收集。细胞用圆形座子的质粒PB-tetO2-IRES-OKMS，它表示连接到mCherry荧光蛋白在多西环素诱导的方式山因子（Oct4的，SOX2，cMYC的和Klf4）转染，和反向四环素式激活（PB- CAG-rtTA）与转座表达质粒（mPBase）一起质粒。小鼠的AF细胞1周超过MEF饲养层体外膨胀后的Oct4，Klf4的，cMYC的和Sox2染。对于外源因子的表达，强力霉?…
Discussion
选择以获得多能性的诱导的方法是相关的细胞的临床安全相对于长期的移植。如今，有适合的重编程的几种方法。间的非整合方法中，仙台病毒（SeV载体）载体是能产生大量的蛋白的未经结合进感染的细胞40的核，可以是获得iPS细胞的策略的RNA病毒。 SeV载体可能是平移级iPS细胞的产生有吸引力的候选者，但它提出了一些缺点。病毒复制酶是转基因序列的性质敏感。由于SeV载体组?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by CARIPARO Foundation Grant number 13/04 and Fondazione Istituto di Ricerca Pediatrica Città della Speranza Grant number 10/02. Martina Piccoli, Chiara Franzin and Michela Pozzobon are funded by Fondazione Istituto di Ricerca Pediatrica Città della Speranza. Enrica Bertin is funded by CARIPARO Foundation Grant number 13/04. Paolo De Coppi is funded by Great Ormond Street Hospital Children’s Charity.
Materials
| 100 mm Bacterial-grade Petri Dishes | BD Falcon | 351029 | For in vitro differentiation |
| 2-mercaptoethanol | Sigma | M6250 | For mouse AF, iPS-AF cells and differentiation medium |
| Alexa568-conjugated goat anti-mouse IgM | Thermo Fisher Scientific | A21043 | Secondary antibody (immunofluorescence) |
| Alexa594-conjugated chicken anti-goat IgG | Thermo Fisher Scientific | A21468 | Secondary antibody (immunofluorescence) |
| Alexa594-conjugated chicken anti-rabbit IgG | Thermo Fisher Scientific | A21442 | Secondary antibody (immunofluorescence) |
| Alexa594-conjugated goat anti-mouse IgG | Thermo Fisher Scientific | A11005 | Secondary antibody (immunofluorescence) |
| Alkaline Phosphatase kit | Sigma | 85L1 | Alkaline Phosphatase staining |
| Ampicillin | Sigma | A0166 | For bacterial selection |
| Bovine Serum Albumin | Sigma | A7906 | BSA, for blocking solution. Diluted in PBS 1X |
| Chloroform | Sigma | C2432 | For RNA extraction |
| DH5α cells | Thermo Fisher Scientific | 18265-017 | Bacteria for cloning procedure |
| Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific | 41965039 | For MEF, mouse AF, iPS-AF cells and differentiation medium |
| Doxycycline | Sigma | D9891 | For exogenous factors expression |
| Microcentrifuge tubes (1.5 mL) | Sarstedt | 72.706 | For PB production |
| ES FBS | Thermo Fisher Scientific | 10439024 | For mouse AF, iPS-AF cells and differentiation medium |
| FBS | Thermo Fisher Scientific | 10270106 | For MEF medium |
| Fine point forceps | F.S.T | Dumont #5 | AF isolation |
| Gelatin | J.T.Baker | 131 | Used 0.1%, diluted in PBS 1X |
| Glycine | Bio-Rad | 161-0718 | For blocking solution. Diluted in PBS 1X |
| Haematoxylin QS | Vector Laboratories | H3404 | Nuclei detection |
| HE | Bio-Optica | 04-061010 | Histological analysis of teratoma |
| Hoechst | Thermo Fisher Scientific | H3570 | Nuclei detection |
| Horse Serum | Thermo Fisher Scientific | 16050-122 | For blocking solution |
| HRP-conjugated goat anti-mouse IgG | SantaCruz | sc2005 | Secondary antibody (immunoperoxidase) |
| ImmPACT NovaRED | Vector Laboratories | SK4805 | Peroxidase substrate |
| Insulin syringe with needle (25G) | Terumo | SS+01H25161 | Amniocentesis procedure |
| Klf4 | SantaCruz | sc-20691 | Rabbit polyclonal IgG |
| L-glutamine | Thermo Fisher Scientific | 25030 | For mouse AF, iPS-AF cells and differentiation medium |
| LB broth (Lennox) | Sigma | L3022 | For bacterial growth |
| LIF | Sigma | L5158 | For mouse AF and iPS-AF cells medium |
| Matrigel | BD | 354234 | For in vitro differentiation. Diluted 1:10 in DMEM |
| Methanol | Sigma | 32213 | Peroxidase blocking |
| MULTIWELL 24 well plate | BD Falcon | 353047 | For in vitro differentiation |
| MULTIWELL 6 well plate | BD Falcon | 353046 | For MEF, mouse AF and iPS-AF cells culture |
| Nanog | ReproCELL | RCAB0002P-F | Rabbit polyclonal IgG |
| Non-essential amino acids | Sigma | M7145 | For mouse AF, iPS-AF cells and differentiation medium |
| Normal Goat Serum | Vector Laboratories | S2000 | For blocking solution. Diluted in PBS 1X |
| NP-40 | Sigma | 12087-87-0 | For cell permeabilization. Diluted in PBS 1X |
| Oct4 | SantaCruz | sc-5279 | Mouse monoclonal IgG2b |
| Oligo (dT) | Thermo Fisher Scientific | 18418012 | For RT-PCR |
| Paraformaldehyde (solution) | Sigma | 441244 | PFA, fixative, diluted in PBS |
| PBS 10X | Thermo Fisher Scientific | 14200-067 | D-PBS, free of Ca2+/Mg2+. Diluted with sterile water to obtain PBS 1X |
| Penicillin – Streptomycin | Thermo Fisher Scientific | 15070063 | For MEF, mouse AF, iPS-AF cells and differentiation medium |
| Petri Dish (150mm) | BD Falcon | 353025 | For MEF culture, tissue culture |
| PiggyBac transposase expression plasmid | Provided by professor Andras Nagy laboratory | – | mPBase |
| PiggyBac-tetO2-IRES-OKMS transposon plasmid | Provided by professor Andras Nagy laboratory | – | PB-tetO2-IRES-OKMS |
| QIAprep Spin Maxiprep Kit | Qiagen | 12663 | For plasmids purification |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 | For plasmids purification |
| Reverse tetracycline transactivator transposon plasmid | Provided by professor Andras Nagy laboratory | – | rtTA |
| RNeasy Mini Kit | Qiagen | 74134 | For RNA extraction |
| Sox2 | SantaCruz | sc-17320 | Goat polyclonal IgG |
| SSEA1 | Abcam | ab16285 | Mouse monoclonal IgM |
| SuperScript II Reverse Transcriptase | Thermo Fisher Scientific | 18064-014 | For RT-PCR |
| T | Abcam | ab20680 | Rabbit polyclonal IgG |
| Taq DNA Polymerase | Thermo Fisher Scientific | 10342020 | PCR |
| Trypsin | Thermo Fisher Scientific | 25300-054 | Cell culture passaging |
| Triton X-100 | Bio-Rad | 161-047 | For cell permeabilization, diluted in PBS 1X |
| TRIzol Reagent | Thermo Fisher Scientific | 15596-026 | For RNA extraction |
| Tubb3 | Promega | G712A | Mouse monoclonal IgG1 |
| TWEEN-20 | Sigma | P1379 | For cell permeabilization, diluted in PBS 1X |
| αfp | R&D Systems | MAB1368 | Mouse Monoclonal IgG1 |
| αSMA | Abcam | ab7817 | Mouse Monoclonal IgG2a |
| Transfection Reagent (FuGENE HD) | Promega | E2311 | For AF cells transfection |
| Stereomicroscope | Nikon | SM2645 | To perform amniocentesis |
| 200 ul tips | Sarstedt | 70.760012 | To pick bacteria colonies |
| Scissor | F.S.T | 14094-11 stainless 25U | To perform amniocentesis |
| Ethanol | Sigma | 2860 | To clean the abdominal wall of the pregnant dam |
| Tissue culture petri dish (150 mm) | BD Falcon | 353025 | For MEF expansion |
| Mitomycin C | Sigma | M4287-2MG | For MEF inactivation |
| MULTIWELL 96 well plate | BD Falcon | 353071 | For iPS-AF culture |
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Cite This Article
Bertin, E., Piccoli, M., Franzin, C., Nagy, A., Mileikovsky, M., De Coppi, P., Pozzobon, M. The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System. J. Vis. Exp. (120), e54598, doi:10.3791/54598 (2017).