用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成
Summary
该协议有效地引导小鼠胚胎干细胞衍生定形内胚层成熟呼吸道上皮细胞。这种分化技术使用的3维脱细胞肺支架直接肺谱系说明书中，在一个确定的，无血清培养的设置。
Abstract
肺谱系分化需要复杂的环境线索，包括生长因子信号传导，细胞 – 细胞相互作用和细胞 – 基质相互作用的集成。由于这种复杂性， 在体外肺发育的重演，促进干细胞肺上皮细胞的分化已经挑战。在这个协议中，脱细胞的肺支架被用来模仿肺的3维环境，并产生干细胞衍生的呼吸道上皮细胞。小鼠胚胎干细胞首先分化为使用具有激活素A.内胚层细胞的胚状体（EB）培养方法的内胚层谱系，然后接种于脱细胞的支架，并在长达21天的空气 – 液体界面中培养。这种技术促进无需额外生长因子的补充接种细胞功能的气道上皮细胞（纤毛细胞，俱乐部细胞和基底细胞）分化。这种文化建立的定义，塞鲁M-免费，价格低廉，重现性好。虽然有有限污染来自非肺胚层谱系中培养，该协议仅产生呼吸道上皮人群和不引起肺泡上皮细胞。与此协议产生的气道上皮细胞可用于在肺器官发生和疾病建模或气道相关的疾病，如囊性纤维化的药物发现平台来研究细胞 – 基质相互作用。
Introduction
多能细胞对肺谱系定向分化是依赖于微环境1,2-精确信号事件。由于此过程的动态本质，已经挑战模仿肺器官的准确事件体外 。最近的报道已经用于与二维培养物的可溶性生长因子补充分步进行谱系限制策略，实现肺分化3-8。在步分化方案，多能细胞，是否胚胎干细胞（ESC）或诱导的多能干细胞，首先分化为定形内胚层胚层。内胚层细胞随后推到前部内胚层的命运和其后肺祖细胞，所确定包含同源结构域转录因子NKX2-1的表达。这些肺祖细胞进一步分化近端（气）或远端（肺泡）肺上皮细胞的Wi日继续生长因子的补充。这种2维策略曾在产生肺上皮细胞取得了一些成功，但也有一些限制，包括不明效率，从其它内胚层谱系可能的污染，缺少一个3维（3D）结构，并且在某些情况下使用未定义培养物血清补充。上脱细胞肺支架多能或分化的细胞的培养日益用作测定，以评估在形成肺上皮结构3,5,6,8,9接种的细胞的再生潜能。这些报告接种文化与持续生长因子或血清补充支架的细胞。
龙开发涉及的划分，迁移，基因响应环境线索的表达和个体细胞的分化。细胞外基质（ECM）是糖蛋白的一个格子，除了提供结构支撑，引导Tissu酒店通过整合和调节这些过程10,11ê形态。通过使用肺ECM支架作为天然平台内胚层培养，以更好地模仿体内肺发育环境，我们已经产生干以限定三维培养细胞来源的呼吸道上皮细胞具有高效率和再现性设置。
由脱细胞以及小鼠ESC衍生的内胚层细胞产生的大鼠肺的ECM支架生成并随后接种到这些支架。 CXCR4和C-KIT蛋白的表达双重指示定型内胚层细胞的身份和阳性细胞都SOX2和NKX2-1的表达被确定为气道（近端肺）祖细胞。定形内胚层细胞在空气液体界面（ALI）长达三周生成官能呼吸道上皮细胞在体外进行培养。
该协议促进defini肺系分化略去内胚层早7天，用NKX2-1 + / + SOX2早期肺癌近祖的出现观察。第14天，文化成熟的气道上皮细胞群的21冒出包括纤毛（TUBB4A +），俱乐部（SCGB1A1 +），和基底（TRP63 +，KRT5 +）细胞的形态和功能相似本地鼠标气道。这个协议说明了3D-基质微环境的实现健壮分化到呼吸道上皮细胞的重要性。
Protocol
Representative Results
Discussion
这里描述的协议产生只使用天然肺支架直接，没有其他补充了分化成熟的ESC源性上皮气道。这种文化建立的定义，无血清，价格低廉，重现性好。没有基础分化培养基中生长因子的补充是必要的。用于产生干细胞衍生的肺上皮细胞中以前发表的方法已使用2维的策略与生长因子补充，以促进谱系限制3,4,8,18,19。此处所描述的技术是在这些方法中有几个原因，包括有利：定义培养条件下，分?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢Rossant博士和碧萝博士在图1-3中描述的实验中使用的Nkx2-1 mcherry ESC。 FACS是在病童医院，UHN流式细胞仪进行融资。这项工作是由操作从加拿大卫生研究院的研究，并从创新的加拿大基金会的资助基础设施（CSCCD）资助。
Materials
| Reagents | |||
| Perfusion solution | Sigma | H0777 | 10U/mL heparin |
| Perfusion solution | Gibco | 14170112 | dissolved in Hank's balanced salt solution (HBSS-) |
| Decellularization solution | BioShop | CHA003 | 8mM CHAPS |
| Decellularization solution | Sigma | E9884 | 25mM EDTA |
| Decellularization solution | BioShop | SOD002 | 1M NaCl |
| Decellularization solution | Gibco | 14190-144 | dissolved in PBS |
| Benzonase nuclease | Novagen | 70664-3 | 90U/mL Benzonase nuclease |
| Benzonase nuclease | Gibco | 14190-144 | diluted in PBS |
| Antimicrobial solution | Gibco | 15140 | 200U/mL penicillin streptomycin |
| Antimicrobial solution | Gibco | 15290 | 25μg/mL amphotericin B |
| Antimicrobial solution | Gibco | 14190-144 | diluted in PBS |
| Trypsinization | Gibco | 12605-028 | TrypLE |
| Serum free differentiation media (SFDM) | Gibco | IMDM 2440-053, F12 11765-054 | 3:1 ratio of IMDM and Ham’s modified F12 medium |
| Serum free differentiation media (SFDM) | Gibco | 12587-010 | B27 supplement (50x dilution) |
| Serum free differentiation media (SFDM) | Gibco | 17502-048 | N2 supplement (100x dilution) |
| Serum free differentiation media (SFDM) | Gibco | 15260-037 | 0.05% (Fraction V) bovine serum albumin |
| Serum free differentiation media (SFDM) | Gibco | 35050-061 | 200mM Glutamax |
| Serum free differentiation media (SFDM) | Sigma | M6145 | 4μM monothioglycerol |
| Serum free differentiation media (SFDM) | Sigma | A4403 | 0.05mg/mL ascobic acid |
| Endoderm induction | R&D | 338-AC/CF | Activin A |
| Antibodies | |||
| CDH1 | BD Biosciences | 610181 | Mouse, non-conjugated, 1:100 |
| C-KIT | BD Biosciences | 558163 | Rat, PE-Cy7, 1:100 |
| CXCR4 | BD Biosciences | 558644 | Rat, APC, 1:100 |
| KRT5 | Abcam | ab24647 | Rabbit, non-conjugated, 1:1000 |
| NKX2-1 | Abcam | ab76013 | Rabbit, non-conjugated, 1:200 |
| Laminin | Novus Biologicals | NB300-144 | Rabbit, non-conjugated, 1:200 |
| SCGB1A1 | Santa Cruz | sc-9772 | Goat, non-conjugated, 1:1000 |
| SOX2 | R&D Systems | AF2018 | Goat, non-conjugated, 1:400 |
| TRP63 | Santa Cruz | sc-8431 | Mouse, non-conjugated, 1:200 |
| TUBB4A | BioGenex | MU178-UC | Mouse, non-conjugated, 1:500 |
| Goat IgG | Invitrogen | A-11055 | Donkey, Alexa Fluor 488, 1:200 |
| Mouse IgG | Invitrogen | A-21202 | Donkey, Alexa Fluor 488, 1:200 |
| Mouse IgG | Invitrogen | A-31571 | Donkey, Alexa Fluor 647, 1:200 |
| Rabbit IgG | Invitrogen | A-21206 | Donkey, Alexa Fluor 488, 1:200 |
| Rabbit IgG | Invitrogen | A-31573 | Donkey, Alexa Fluor 647, 1:200 |
| Other Materials | |||
| Low adherent plates | Nunc | Z721050 | Low cell binding plates, 6 wells |
| Air-liquid interface membranes | Whatman | 110614 | Hydrophobic Nucleopore membrane, 8μm pore size |
| Vibratome | Leica | VT1200S | Leica Vibratome |
| Tissue Adhesive | Ted Pella | 10033 | Pelco tissue adhesive |
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