在人多能的体外干细胞向滋养层细胞
Summary
Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.
Abstract
胎盘是胚胎发育过程中，开发的第一个器官，并需要对发育中的胚胎的生存。胎盘包括从植入前胚泡的胚外滋养外胚层细胞分化的各种滋养细胞。因此，我们的人胎盘的早期分化事件的认识，是因为对人胚胎的分离和操作的伦理和法律限制的限制。人多能干细胞（hPSCs）是用于调查人类发展的稳健的模型系统，也可以在体外分化成表达各滋养层细胞类型的标志物滋养层细胞。在这里，我们提出了一个详细的协议，用于区分hPSCs到使用骨形态发生蛋白4和激活素/ Nodal信号通路抑制剂的滋养层细胞。该协议生成可以用的siRNA转染各种滋养层细胞类型调查失功能表型或可感染病原体。此外，hPSCs可以进行遗传修饰，然后分化成滋养层祖换的功能增益分析。用于产生人类滋养细胞从hPSCs开始本体外分化的方法克服了与早期的人类胚胎工作的伦理和法律的限制，并且该系统可用于多种应用，包括药物发现和干细胞研究。
Introduction
怀孕期间胎盘需要胎儿的生长和存活，并促进气体，营养物，废物和激素的母体和胎儿循环之间的交换。哺乳动物胚胎发育过程中形成的第一器官是胎盘，它开始显影6-7天在人类受孕后和在小鼠中1，2，3，4 3.5-4.5天。滋养层细胞是胎盘的最重要的细胞，这些细胞所代表的哺乳动物胚胎的最早谱系分化的事件之一。他们从植入前胚泡的外胚外滋养层细胞产生。我们的胎盘发育的早期阶段的知识是通过模拟早期人类发展的伦理和后勤限制的限制。
在胚胎移植，滋养细胞侵入母体上皮细胞和分化成专门的祖细胞5。细胞滋养细胞（CTB介）的单核，即融合和分化成合体（SYN包）和绒毛外滋养层细胞侵入（EVTs）未分化的祖细胞，它的锚胎盘在子宫。 SYN包的多核，即合成所需的持续受孕激素影响终末分化细胞。产生EVTs和SYN包的早期分化事件对于胎盘形成所必需的，如在滋养层细胞的损伤导致流产，先兆子痫，宫内生长受限1。已经开发人类滋养细胞系的类型包括永生个CTB和绒毛膜癌，其产生的胎盘激素和显示侵入属性6。从人类早期妊娠胎盘的主要滋养层细胞可以分离，但很快细胞DIFferentiate并停止在体外增殖。重要的是，转化和原代细胞系具有不同的基因表达图谱，表明致瘤性和永生滋养层细胞系可能不准确地表示主滋养层7。此外，这些线是不可能像胎盘滋养层干细胞祖细胞，因为它们是从后级通过第三孕期导出的第一。
有为了研究胎盘的形成和功能的早期事件需要在早期人类滋养细胞体外培养体系稳健。人类胚胎干细胞（胚胎干细胞），它们共享与早期胚胎的内细胞团性质，常用于早期人类的发展模式，包括早期胎盘的形成。人类诱导多能干细胞（iPS细胞）和人类胚胎干细胞可以分化成骨用在铁道部滋养细胞体外phogenic蛋白4（BMP4）8，9，10，11，12，13，14，15。多能细胞用BMP4的滋养层细胞这种转换是特异于人细胞，并广泛用于研究早期人类胎盘的发展，因为它不要求访问人类早期胚胎9,16。最近，人们发现，在加入抑制剂A83-01（A）和PD173074（P），其阻断SMAD2 / 3和MEK1 / 2信号途径，增加HPSC分化的效率为滋养外胚层样祖细胞，主要的SYN和EVTs，没有广泛代中胚层，内胚层或外胚层细胞9，17的</suP>。使用这些介质的条件下，人类胚胎干细胞分化12天具有相似的基因表达谱从人胚泡期胚胎中分离滋养层细胞和分泌多种胎盘特异性生长激素，支撑该体外模型系统9，11的有效性。在这里，我们提出了hPSCs 体外分化为使用BMP4 / A / p培养基中的人类祖先滋养了详细的方案。这些条件产生细胞的数量丰富为各种各样的应用，包括RNA测序，基因破坏用的siRNAs，病原体感染，遗传修饰使用脂转染介导的转染。
Protocol
Representative Results
Discussion
我们提出了分化的hESC入滋养层祖细胞的基本步骤。这个协议最近被优化以迅速分化的hESC与除了激活素/ Nodal信号抑制剂，增加分化为滋养层细胞和避免胚层祖细胞，其通常单独用BMP4治疗观察到的产生。该模型BMP4系统允许滋养层沿袭规格和扩展的最早阶段的审查。此外，这种BMP4模型系统也调查滋养层细胞向特定子谱系，使用绒毛膜细胞系和绒毛外滋养细胞系，这是不可能的分化是有用的。 体?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by a Pennsylvania Health Research Formula Fund.
Materials
| DMEM/F12 | Invitrogen | 11330-057 | |
| Knock Out Serum Replacement | Invitrogen | 10828-028 | This is referred to as "serum replacement" in this protocol. |
| NEAA | Invitrogen | 11140-050 | |
| FBS | Invitrogen | 16000-044 | |
| L-Glutamine | Invitrogen | 10828-028 | |
| Penicillin/Streptomycin | Invitrogen | 15140-155 | |
| 2-Mercaptoethanol | Sigma | M-7522 | |
| B-FGF | Millipore | GF-003 | |
| DMEM | Invitrogen | 11965-118 | |
| Dispase | Invitrogen | 17105-041 | |
| Collagenase Type IV | Invitrogen | 17104-019 | |
| Rock inhibitor Y27632 | Calbiochem | 688000 | |
| Irradiated CF1 MEFs | GlobalStem | 6001G | MEFs can be generated from embryonic day 13.5 embyos and irradiated. |
| 0.22 um syringe filter | Millipore | SLGS033SS | |
| Heracell 150i low oxygen incubator | Heracell/VWR | 89187-192 | Any tissue culture incubator with capacity to regulate oxygen concentrations is sufficient. |
| BMP4 | R&D Systems | 314-BP-01M | |
| A 83-01 | R&D Systems | 2939/10 | |
| PD173074 | R&D Systems | 3044/10 | |
| RNAiMax | Invitrogen | 13778150 | |
| Trizol | ThermoFisher | 15596026 | Trizol is used to isolate total RNA. |
| X-tremeGENE 9 | Roche | 6365779001 | |
| Matrigel | Corning | 356231 | This is referred to as "extracellular matrix" in this protocol. |
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