In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Summary

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

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

Organ culture provides a useful model that bridges the gaps between the complex but highly relevant in vivo investigations and the convenient but approximate simulation of cell line models. In the case of the pancreas, there is no cell line perfectly equivalent to pancreas progenitors although there are transformed cell lines simulating endocrine and exocrine cells. The adult whole pancreas cannot be cultured; isolated endocrine islets can be maintained for few weeks without cell proliferation and tissue slices can be kept in vitro for few hours ⁵. Embryonic pancreas culture has been widely used not only to study its development, but also ....

Protocol

This protocol aims to grow pancreatic organoids derived from murine E10.5 dissociated epithelial pancreatic cells.

The protocol requires ethical approval for animal experimentation.

1. Dissection of Dorsal Pancreatic Bud from E10.5 Mouse Embryos

1. Sacrifice timed-pregnant mice at embryonic day (E) 10.5, open the abdomen with a pair of scissors, remove the two uterine horns and place them in a 10 cm Petri dish filled with cold phosphate buffer saline (PBS) or Dulbecco modified essential medium (DMEM) kept on ice. The total experiment from the sacrifice to cell seeding is done in 60-90 min to prevent cell....

Results

E10.5 dorsal pancreatic progenitors dissociated and seeded in 3D Matrigel recapitulate pancreas development. Progenitors can be most easily followed with fluorescent reporters. In our case we used a transgenic mouse that expresses a nuclear GFP protein controlled by Pdx1 promoter (Pdx1-Ngn3-ER^TM-nGFP) (Movie 1) in the absence of tamoxifen and thus without activating Neurog3 ⁴ (Figure 2).

With the organoid medium, an ini.......

Discussion

Large-scale production of functional beta cells in vitro is still ineffective ¹. In this challenging context, developmental biology studies may help deciphering the exact signals that are required for the differentiation of functional beta cells. This protocol allows for the maintenance, expansion and differentiation of embryonic pancreatic progenitors in vitro. This includes the formation of insulin-producing beta cells that do not co-express other endocrine hormones, have high levels of Pdx.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Penicillin-Streptomycin	Gibco	15070-063	Stock kept at -20 °C
KnockOut Serum replacement (supplement)	Gibco	10828-028	Stock kept at -20 °C
2-mercaptoethanol	Sigma Aldrich	3148-25ML	Stock kept at 4 °C
Phorbol Myristate Acetate (PMA)	Calbiotech	524400-1MG	Stock kept at -20 °C
Y-27632 (ROCK inhibitor)	Sigma Aldrich	ab120129	Stock kept at -20 °C. Attention! Stability/source is a frequent source of problems.
EGF	Sigma Aldrich	E9644-2MG	Stock kept at -80 °C
Recombinant Human R-spondin 1	R&D	4645-RS-025/CF	Stock kept at -80 °C
Recombinant Mouse R-spondin 1	R&D	3474-RS-050	Stock kept at -80 °C
Recombinant Human FGF1 (aFGF)	R&D	232-FA-025	Stock kept at -80 °C - do not include to increase beta cell production
Heparin (Liquemin)	Drossapharm		Stock kept at 4 °C
Recombinant Human FGF10	R&D	345-FG-025	Stock kept at -80 °C
DMEM/F-12	Gibco	21331-020
Penicillin-Streptomycin	Gibco	15070-063	Stock kept at -20 °C
B27 x50 (supplement)	Gibco	17504-044	Stock kept at -20 °C
Recombinant Human FGF2 (bFGF)	R&D	233-FB-025	Stock kept at -80 °C
Matrigel	Corning	356231	Stock kept at -20 °C
Trypsin 0.05%	Gibco	25300-054	Stock kept at 4 °C
RNAlater - RNA stabilizing reagent	Qiagen	76104	Store at RT
Dispase	Sigma Aldrich	D4818-2MG	Working concentration: 1.25 mg/ml. Stock kept at -20 °C
BSA for reconstitution	Milipore	81-068	For reconstituition of cytokines  - stock kept at -20 °C
Fetal calf serum (FCS)	Gibco	16141079	Stock kept 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 glass micropipettes	Blaubrand	708745
Microscope			Cell® imaging station (motorized inverted Olympus IX81 stand) equipped with a Hamamatsu ORCA ER B7W camera and the Ludin Cube and Box. Leica DMI6000 B stand surrounded with a Ludin Cube and Box, equipped with a Leica DFC365 FX camera and the AF6000 Expert/Matrix software command interface.
Objective			Olympus UPLAN FL NA 0.30 air 9.50 mm 10X long distance; Leica HC PL FLUOTAR NA 0.30 air 11.0 mm 10X long distance