体外胰腺器官从分散的小鼠胚胎祖细胞

Summary

在本协议中所述的三维培养方法概括胰腺发育从胚胎分散小鼠胰腺祖细胞，包括他们的大幅扩张，分化和形态发生成支器官。这种方法适合于成像，利基的功能的干扰和操纵。

Abstract

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells ¹. The whole embryonic organ can be cultured at multiple stages of development ^2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens.

We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas.

We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.

Introduction

器官培养提供了桥梁在体内研究和复杂，但具有很强的针对性之间的差距细胞系模型的方便，但近似模拟一个有用的模型。在胰腺的情况下，不存在细胞系完全等价于胰腺祖细胞虽然有转化细胞系模拟内分泌和外分泌细胞。成人全胰腺不能培养;分离的内分泌胰岛可维持数周而不细胞增殖和组织切片可以保持在体外对几个小时^5。胚胎胰腺培养已被广泛使用，不仅研究其发展，还探讨上皮-间充质相互作用^4,6,7，对图像进行处理⁸或化学上与它们⁹干扰。两个器官培养方法主要应用于：第一种是在纤连蛋白包被的板^2，它是型转换器中培养胰芽便于您进行成像的目的;第二个选项是文化在气-液界面^3,4的最佳形态保留在过滤器上的器官。虽然非常有用，这些方法会导致一定程度的平坦化的;相比于正常发育和起始群体是复杂的，包括所有类型的胰腺细胞和间充质细胞的前体细胞的扩张是非常有限的。

的能力，培养和扩大分散的初级细胞是有价值的研究谱系关系，并发现分离的细胞类型¹⁰的固有性质。杉山等 ¹¹能保持胰腺祖细胞和内分泌祖细胞是保留了一些功能特点3-5天培养的饲养层。 Pancreatospheres，类似于神经球¹²和¹³微球体，已经扩大，从成人胰岛细胞和导管细胞虽然祖细胞的性质/干细胞产生这些球体是不明确的。另外，在与生理发育相反，pancreatospheres含有一些神经元^14,15。领域也从最近胚胎胰腺祖细胞^16,17和再生胰腺¹⁸具有良好的祖扩张和随后的分化产生，但未能概括形态。

从分散，通常被定义细胞自组织成微型器官3D模型最近已蓬勃发展，并模拟多个器官如肠道^19,20，胃^21，肝脏^22，前列腺²³和气管的开发或成年营业额^24。在某些情况下，发育和形态发生分化已扼要重述在三维由ES细胞，因为是光学杯^25，小肠²⁶或脑²⁷的情况。

在这里，我们DEScribe一个扩大分离的多能胰腺祖细胞在三维基质胶支架在那里他们可以分化和自我组织的方法。

Protocol

该协议旨在扩大从鼠E10.5派生类器官胰腺上皮分离胰腺细胞。 该协议要求的伦理批准用于动物实验。 背胰芽1。解剖从E10.5小鼠胚胎牺牲定时妊娠小鼠胚胎一天（五）10.5，开有一对剪刀的腹部，取出两个子宫角，并放置在10厘米的培养皿中充满了冷磷酸盐缓冲液（PBS）或贝科改良基本培养基（DMEM）中保持在冰上。总的实验从祭祀细胞种植在60-90分钟…

Representative Results

分离并接种于三维基质胶E10.5胰背祖概括胰腺发育。祖细胞可以最容易遵循的荧光记者。在我们的例子中，我们使用了转基因小鼠，表达不激活Neurog3 4（ 图2），在没有他莫昔芬，从而通过的Pdx1启动子（ 的Pdx1，Ngn3的-ER TM-nGFP）（电影1）控制的核GFP蛋白。 与组织体介质中，小簇细胞的初始压实发生在第一个小时。膨胀然后可以由簇在…

Discussion

大规模生产的功能性β细胞在体外仍然无效^1。在这个充满挑战的背景下，发育生物学的研究可能有助于破译所必需的功能性β细胞分化的确切信号。此协议允许在体外胚胎胰腺祖细胞的维持，扩展和分化。这包括形成产生胰岛素的β细胞的不共表达其他内分泌激素，有高水平的Pdx1的，表达了亲convertases的成熟胰岛素和有胰岛素处理^28。系统内的重要的关键因素是FGF的活…

Materials

Penicillin-Streptomycin	Gibco	15070-063	Stock keept at -20°C
KnockOut Serum replacement (supplement)	Gibco	10828-028	Stock keept at -20°C
2-mercaptoethanol	Sigma Aldrich	3148-25ML	Stock keept at 4°C
Phorbol Myristate Acetate (PMA)	Calbiotech	524400-1MG	Stock keept at -20°C
Y-27632 (ROCK inhibitor)	Sigma Aldrich	ab120129	Stock keept at -20°C- Attention! Stability/source is a frequent source of problems
EGF	Sigma Aldrich	E9644-2MG	Stock keept at -80°C
Recombinant Human R-spondin 1	R&D	4645-RS-025/CF	Stock keept at -80°C
- or -
Recombinant Mouse R-spondin 1	R&D	3474-RS-050	Stock keept at -80°C
Recombinant Human FGF1 (aFGF)	R&D	232-FA-025	Stock keept at -80°C- do not include to increase beta cell production
Heparin (Liquemin)	Drossapharm		Stock keept at 4°C
Recombinant Human FGF10	R&D	345-FG-025	Stock keept at -80°C
DMEM/F-12	Gibco	21331-020
Penicillin-Streptomycin	Gibco	15070-063	Stock keept at -20°C
B27 x50 (supplement)	Gibco	17504-044	Stock keept at -20°C
Recombinant Human FGF2 (bFGF)	R&D	233-FB-025	Stock keept at -80°C
Y-27632 (ROCK inhibitor)	Sigma Aldrich	ab120129	Stock keept at -20°C- Attention! Stability/source is a frequent source of problems
DMEM/F-12	Gibco	21331-020
Matrigel	Corning	356231	Stock keept at -20°C
Trypsin 0.05%	Gibco	25300-054	Stock keept at 4°C
RNAlater – RNA stabilizing reagent	Qiagen	76104	Store at room temperature
Dispase	Sigma Aldrich	D4818-2MG	Stock keept at -20°C
BSA for reconstitution	Milipore	81-068	For reconstituition of cytokines  – Stock keept at -20°C
Fetal calf serum (FCS)	Gibco	16141079	Stock keept at -20°C
60 well MicroWell trays	Sigma Aldrich	M0815-100EA
4-well plates	Thermo Scientific	176740
95-well plates F bottom	Greiner Bio	6555180
Glas bottom plates	Ibidi	81158
Disposal micropittes	Blaubrand	708745