从小鼠胚胎植入前胚胎干细胞系的推导
Summary
这篇文章描述了一个协议，以有效地获得和囊胚阶段文化从小鼠胚胎多能干细胞系。
Abstract
小鼠胚胎干细胞 （卓） 推导是哪个多能干细胞系建立了从胚胎植入前胚胎的过程。这些行保留自我更新或区分在特定条件下的能力。由于这些特性，卓在再生医学、 疾病建模和组织工程研究中是一个有用的工具。这篇文章描述了一个简单的协议，以获得高推导效率 （60-80%) 的卓行通过培养从宽容小鼠品系对白血病抑制因子定义培养基中的饲养细胞的囊胚。议定书 》 还可有效地将卓行从非许可的小鼠品系，通过简单的加法到推导介质 （2i 中等） 的两种小分子抑制剂的鸡尾酒。提供了制备及文化的饲养层细胞，收集和小鼠胚胎和推导的文化和文化的卓行的详细的过程。该协议不需要专门的设备，可以在任何与基本的哺乳动物细胞培养的专门知识的实验室进行。
Introduction
胚胎干细胞 (ESC) 是多能干细胞来自胚胎植入前胚胎，保留自我更新或特定条件1下区分的能力。基于这些属性，按 esc 键已成为再生医学、 疾病建模和组织工程研究2有用工具。
按 ESC 取自于胚胎植入前胚胎，源自鼠标 ESC （卓） 线与低成功3，4第一次。多年来，推导效率仍然很低难以维持体外多向分化潜能。此外的进纸器细胞5，提高工作效率推导，存在促进殖民地附件和提高染色体稳定性6，全能维修改进协议导致的几种修改。最重要是加法白血病抑制因子 (LIF) 到卓培养基7，8，使用 129S2 宽松背景9从胚胎和卓与培养基的文化定义血清，免费的潜在分化因素10。最近，令人信服的证据表明，另外的两个小分子调节的信号传导通路在卓培养基 （称为 2i） 鸡尾酒是最好的保持多向分化潜能。2i 鸡尾酒由 MEK 抑制剂 PD0325901，减少分化信号抑制有丝分裂原活化蛋白激酶 (MAPK) 通路与都可诱发 gsk3 抑制剂 CHIR99021，增强了细胞存活在低密度的结合通过激活 Wnt 途径11。
在本文中，我们描述了一个简单的协议，有效地将卓行从小鼠囊胚。我们按照标准程序，培养从宽容菌株对 LIF 和血清更换12推导培养基中的饲养细胞的胚胎。按照此协议，卓行可以得到效率相当于那些获得 2i 介质中。尽管这样，可以添加 2i，以避免可能出现的问题，具有多向分化潜能维修或使用非许可株时。
Protocol
使用以下各节所述的动物已被批准，由动物和人类研究的大学 Autònoma de 巴塞罗那伦理委员会和部 d ' 农业，Ramaderia，以上我Alimentació 的加泰罗尼亚 （协议 #8741）。 1.馈线细胞失活和存储 注： 包皮成纤维细胞 (h f F-1) 以前被冰冻住的冷冻液组成的 90%胎牛血清 (FBS) 和 10%二甲基亚砜 （1 毫升的二甲基亚砜），直到使用存储在液态氮。 解冻 h f F-…
Representative Results
继本议定书中，超过 95%的产物形成应该达到后第一周的文化 (图 1A)。卓系后六通道的建立是变量实验复制，与平均成功的 60-80%之间。2i 的加法不能显著改善结果时使用许可小鼠品系，例如那些与 129S2 的背景，但它是必要的当工作与非许可的菌株。 鼠标按 ESC 殖民地应该与扁平的形状呈现形态规则和定义…
Discussion
虽然卓推导出来的线是广为人知的程序经常使用在许多实验室，其效率并不总是可以打扰全能维护的多因素预期那么高。在本文中我们表明，一步一步走，如何与高效率使用简单和可靠的协议建立卓行。这些效率相近的文献报道。
为了确保最高的效率，是重要的是控制细胞每日或每隔一天，从时间点表示只是说明性。至关重要的是，要避免过度的殖民地，因为这会促进细胞?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢 Jonatan 卢卡斯他与馈线细胞培养，Servei Estabulari de la UAB 为动物保健和 Domènec Martín 为录制视频的技术援助。这项工作得到了 Ministerio 德经济 y 与两性 AGL2014-52408-R 和自治区政府大楼和加泰罗尼亚 2014 SGR 524。MVC 是 PIF UAB 研究金的受益人。
Materials
| 1 mL sryringe | BD | 309628 | |
| 2-β-mercaptoethanol | Life Technologies | 31350-010 | |
| 4-well plate | Thermo Scientific Nunc | 176740 | |
| Acidic Tyrode's solution | Home-made | See recipe in Nagy et al, 2003. For short-term storage (up to 1 month) keep it at 4ºC. Alternatively, for long-term storage keep it at -20ºC. If the efficiency of the solution diminishes, it should be acidified by the adition of a small drop of HCl 1M. | |
| BSA | Sigma | A3311 | |
| Chicken anti-Mouse IgG, Alexa Fluor 488 | Molecular Probes – Invitrogen | A-21200 | 1:500 dilution. Secondary antibody for Oct4, Tuj1, αSMA and AFP mouse antibodies. |
| CHIR990212 | Axon Medchem | 1386 | Reconstitute with 2.15 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20ºC. Avoid freeze and thaw cycles. |
| CryoTube | Thermo Scientific Nunc | 3754518 | |
| DMSO | Sigma | 41640 | |
| DMEM | BioWest | L0107 | |
| FBS | BioWest | S1810 | Inactivate it prior to use by heating for 30 mins at 56ºC. Aliquot and store at -20ºC |
| Flushing needle | BD | 304000 | Cut the end of the needle and ground to a blunt tip on an abrasive stone |
| Gelatin from porcine skin | Fluka | 48724 | Dilute at 0,2% in destilled water and autoclave it. Each dilution can be used for a month. |
| Goat anti-Rabbit IgG, Alexa Fluor 594 | Molecular Probes – Invitrogen | A-11037 | 1:500 dilution. Secondary antibody for Sox2 rabbit antibody. |
| HBSS | BioWest | L0611 | |
| Hepes-buffered CZB | Home-made | See composition in Chatot et al, 1989 | |
| Human chorionic gonadotropin | Divasa-Farmavic | Veterin-Corion 3000 UI | Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20ºC for up to two months. Once thawed do not freeze again. |
| Human foreskin fibroblasts | ATCC | ATCCSCRC-1041 | For long term storage it is recommended to store them in liquid nitrogen. |
| KnockOut Serum Replacement | Life Technologies | 10828-028 | Photosensitive. It is recommended to aliquot it in small volumes such as 10 ml and store it at -20ºC (check the expiration date). Avoid freeze and thaw cycles. |
| KSOMaag Evolve | Zenith Biotech | ZEKS-050 | Supplement with 4 mg/ml BSA. Equilibrate at 37ºC and 5% CO2 a day prior to use. |
| Leukemia Inhibitory Factor | Merk Millipore | ESG1106 | |
| Mice | Charles River/Harlan | It is recommended to use mice from a permissive strain in terms of mESC derivation (such as 129S2 or C57BL). However, inbreed strains are less efficient producing embryos. Therefore, it is recomended to use a hybrid strain, such as 129S2 x C57BL or B6CBAF1 to take advantage of the hybrid vigor. | |
| Mineral oil | Sigma | M8410 | Embryo tested. Photosensitive. |
| Mitomycin C | Serva | 2980501 | Reconstitute the powder with MilliQ water at 0.5 mg/ml. Store at 4ºC protected from light. Attention, it is harmful and suspected of causing cancer. |
| Mouse anti-tubulin β III (Tuj1) | Biolegend | MMS-435P | 1:500 dilution |
| Mouse monoclonal anti-αSMA | Sigma | A5228 | 1:200 dilution |
| Mouse monoclonal anti-AFP | R&D Systems | MAB1368 | 1:50 dilution |
| Mouse monoclonal anti Oct3/4 | Santa cruz | Sc-5279 | 1:50 dilution |
| Non-Essential Amino Acids | Life Technologies | 11140-035 | |
| PD0325901 | Axon Medchem | 1408 | Reconstitute with 2.08 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20ºC. Avoid freeze and thaw cycles. |
| Petri dish (35mm) | Thermo Scientific Nunc | 153066 | |
| Petri dish (60mm) | Thermo Scientific Nunc | 150288 | |
| Pregnant mare's serum gonadotropin | Foligon | Foligon-1000 UI | Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20ºC for up to two months. Once thawed do not freeze again. |
| Rabbit policlonal anti-Sox2 | Merck Millipore | AB5603 | 1:200 dilution |
| T75 falcon | Thermo Scientific | 130190 | |
| Trypsin-EDTA 10x | BioWest | X0930 | Dilute 1:10 in HBSS to obtain a 1x solution |
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Citazione di questo articolo
Vila-Cejudo, M., Ibañez, E., Santalo, J. Derivation of Stem Cell Lines from Mouse Preimplantation Embryos. J. Vis. Exp. (126), e56171, doi:10.3791/56171 (2017).