协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞
Summary
Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.
Abstract
滋养层干细胞（TSC的）出现在哺乳动物发育第一细胞命运决定的结果。它们可以在体外进行培养，保持能力自我更新和分化为滋养层谱系的所有子类型，等同于体内干细胞群从而引发胎盘的胎儿部。因此，的TSC提供了一个独特的模型来研究胎盘发育和胚胎与体外胚外细胞命运决定。从开始的囊胚阶段，包括DNA甲基化和组蛋白修饰明显的后生屏障分隔紧密谱系两者。在这里，我们描述了一个协议，通过瞬态过表达的滋养重点监管TFAP2C，GATA3，EOMES和ETS2在小鼠胚胎成纤维细胞完全克服这一障碍的血统。诱导滋养层干细胞是能自我更新和几乎相同的囊胚滋养衍生的干细胞我形态，标记基因的表达和甲基化模式的n项。功能的体外和体内测定确认这些细胞能够沿滋养层谱系产生多倍体滋养层巨细胞和当注入胚泡嵌合化胎盘区分。从体细胞组织滋养层干细胞的诱导打开了新的途径来研究这个胚外谱系遗传和表观遗传特性，并提供给产生滋养层干细胞系，而不破坏各自的胚胎的可能性。
Introduction
最近，比较小鼠胚胎干细胞的几种方法来滋养层干细胞转化的一项研究显示，在所有的分析系统，沿袭转换仍然是不完整的。代替诱导滋养层干细胞（iTSCs）所谓的滋养层干的已产生细胞样细胞保持原点1的细胞命运的存储器中。在这里，我们跟着ITSC一代不同的方法。类似于来自小鼠胚胎成纤维细胞（MEF中）2多能干细胞的直接感应，iTSCs已直接从分化的体细胞组织转化。首先，我们确定了MEF中过表达时诱导TSC命运12个候选的因素。稍后，因素TFAP2C，GATA3，EOMES和ETS2已经确定是必要的和足够的ITSC感应3。同时，另一组独立地发现TFAP2C，GATA3和EOMES足以MEF中转换成iTSCs。然而，在研究中，对转基因表达所需要的时间相当长相比，我们的研究中，表明不同的转化动力学，当ETS2从转鸡尾酒4不存在。
含有诱导和囊胚衍生滋养层干细胞的培养常规胎牛血清（FBS）依赖于由生长灭活的MEF 5,6-分泌的因子的存在。在ITSC感应过程中，由MEF中，缺乏转基因的充分结合，并且不接受转提供了这些因素。然而，一旦个人ITSC菌落亚培养，它们需要媒体，已预处理由生长灭活的MEF。从那里，iTSCs可以培养并根据标准协议，如胚泡衍生的TSC处理。值得注意的是，在对比田中等人的5，我们经常培养的TSC和iTSCs无凝胶化的细胞培养皿。
Protocol
Representative Results
Discussion
这里介绍的4因子（TFAP2C，GATA3，EOMES，ETS2）的转协议提供了一种可靠的方法来生成忠实地转换从小鼠胚胎成纤维细胞iTSCs。此外，该方法也适用于产后尾成纤维细胞，虽然在效率的降低相比胚胎成纤维细胞3。在一般情况下，初级成纤维细胞的质量是转结果和护理的一个关键因素，应采取使用早期传代细胞（传代两到三个）。
该协议的优点是使用强力霉素诱导的载体，…
Offenlegungen
The authors have nothing to disclose.
Materials
| Doxycycline hyclate | Sigma-Aldrich | D9891-10G | Dissolve in sterile PBS, prepare 2 mg/ml stock solution. Store aliquots at -20°C |
| Chloroquine diphosphate salt | Sigma-Aldrich | C6628-25G | Prepare 25 mM stock solution in sterile cell culture grade water. Store aliquots at -20°C |
| Polybrene (Hexadimethrine bromide) | Sigma-Aldrich | 107689-10G | Prepare 8 mg/ml stock solution in sterile cell culture grade water. Store aliquots at -20°C |
| human recombinant Fibroblast Growth Factor 4 | ReliaTech | 300-131L | Dissolve in sterile 0.1% BSA in PBS, prepare 25 µg/ml stock solution. Store aliquots at -80°C |
| Heparin sodium salt | Sigma-Aldrich | H3149-10KU | Prepare 1 mg/ml stock solution by dissolving in sterile PBS. Store aliquots at -80°C |
| ProFection Mammalian Transfection System | Promega | E1200 | |
| PBS, no calcium, no magnesium | ThermoFisher Scientific | 1419-094 | |
| DMEM (1x) + GlutaMAX | ThermoFisher Scientific | 31966-021 | |
| RPMI 1640, no glutamine | ThermoFisher Scientific | 31870-025 | |
| Advanced DMEM (1x) | ThermoFisher Scientific | 12491-015 | |
| Trypsin-EDTA (0.05%), phenol red | ThermoFisher Scientific | 25300-054 | |
| Poly-L-lysine | Sigma-Aldrich | P4707 | |
| Fetal bovine serum | Hyclone | SH30071.03IR | |
| Surfactant-free cellulose acetate-membrane filter | Corning | 431220 | |
| Non-essential amino acids | ThermoFisher Scientific | 11140-035 | |
| Penicillin Streptomycin | ThermoFisher Scientific | 15140-122 | |
| Sodium Pyruvate | ThermoFisher Scientific | 11360-039 | |
| L-glutamine | ThermoFisher Scientific | 25030-24 | |
| 2-Mercaptoethanol | ThermoFisher Scientific | 31350-010 | |
| Dimethyl sulfoxide (DMSO), cell culture grade | Panreac AppliChem GmbH | A3672,0100 | |
| pLV-tetO-Tfap2c | Addgene | 70269 | |
| pLV-tetO-Gata3 | Addgene | 70270 | |
| pLV-tetO-Eomes | Addgene | 70271 | |
| pLV-tetO-Ets2 | Addgene | 70272 | |
| pLV-tetO-mCherry | Addgene | 70273 | |
| psPAX2 | Addgene | 12260 | |
| pMD2.G | Addgene | 12259 | |
| FUdeltaGW-rtTA | Addgene | 19780 | |
| Mice B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J 7 | JAX Mice | 6965 | |
| 129S2SV | Charles River | 129 |
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