Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Podsumowanie

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

Streszczenie

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells.

Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.

Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Wprowadzenie

Delineating the mechanisms of the normal development and the physiology is paramount to understand disease etiology and ultimately cultivate treatment methods. While culturing and differentiating stem cells enables quick and high-throughput analysis of development, it is limited by the existing body of knowledge regarding mechanisms regulating cell fate and artificially recapitulates development in a relatively homogenous, two-dimensional state^1,2. Not only is in vivo development affected by extrinsic influences, with different cell types in the niche and milieu providing paracrine signals and organizational support to guide organogenesis, but the function of these cells also relies on their surroundings for guidance^3,4,5. Given the importance of these external cues, the limitations of differentiation protocols, and the laborious nature of in vivo mouse models, new systems are needed to experimentally investigate basic developmental processes and physiology.

The emergence of protocols to generate three-dimensional, complex organoids provides a convenient and congruent system to study organogenesis, physiology, drug efficacy, and even pathogenesis. Establishing murine organoids for different systems such as the stomach⁶ and intestine⁷ have expanded our understanding of organogenesis, providing a tool to study developmental complexities with fewer restrictions than in vivo and in vitro models. Due to these advances in the murine organoid formation and the advent of human pluripotent stem cells, human intestinal⁸, retinal⁹, renal^10,11, and cerebral¹² organoids have been produced, and this repertoire is only limited by the existing knowledge regarding mechanisms of development.

Of particular interest is the generation of pancreatic organoids, as a myriad of diseases plagues different pancreatic cell types, including acinar cells and ducts in exocrine pancreatic insufficiency¹³, acinar cells in pancreatitis¹⁴, and beta cells in diabetes¹⁵. Gaining knowledge regarding the development of these different cell types could aid in understanding their pathology and can, also, act as a platform for personalized drug screening or transplantation. Previously, Greggio et al. developed a method to create murine pancreatic organoids that recapitulate in vivo morphogenesis and develop organized, three-dimensional, complex structures composed of all major pancreatic epithelial cell types^16,17. This is a major step forward in the pancreatic field, especially as making cells in vitro can enable biological investigation of beta-cell development. However, a scarcity of endocrine cells formed in this protocol unless the organoids were transplanted into tissue, where the niche could interact and provide instructional cues¹⁷. The mesenchyme constitutes the largest portion of the niche, heavily enveloping the developing epithelium from early stages of organogenesis to later stages including endocrine delamination and differentiation^3,4,18. The interaction of the mesenchyme with the developing pancreas is yet another example of extrinsic signaling and the importance of maintaining in vivo cellular complexity to study organogenesis.

Here, we describe how to generate three-dimensional pancreatic organoids, termed pancreatoids, from dissociated e10.5 murine pancreatic progenitors. These pancreatoids retain native mesenchyme, self-assemble in free-floating conditions, and generate all major pancreatic cell types, including a robust number of endocrine beta cells¹⁹. This approach is best suited for the analysis of endocrine development, as previous protocols lack robust endocrine differentiation. However, using the protocol for pancreatic organoids as described by Greggio et al. is better suited for analysis of pancreatic epithelial branching and morphogenesis, as branching is more limited in pancreatoids.

Protokół

All animal experiments described in this method were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine.

1. Preparation of Mouse Embryonic Day 10.5 Pancreatic Progenitors

Note: This protocol does not need to be followed under sterile conditions until step 2, however it is optimal to sterilize dissection tools and spray with 70% ethanol prior to use.

1. To set up for dissection, fill an ice bucket and place a container of phosphate buffered saline (PBS) in the ice. Clean two fine-tipped forceps and dissection scissors with 70% ethanol. Set near the dissection area, a container with at least three borosilicate capillary tubes, a mouth pipette, a lighter, and filtered 1,000 µL pipette tips.
   1. Prepare at least 1 mL of 1.25 mg/mL cold dispase diluted in sterilized water and place in the second well of a 12 well plate on ice. Put PBS in the first, third, and fourth wells of the 12 well plate. Place 1 mL of 0.05% Trypsin in a 1.5 mL tube on ice. Pre-warm a shaking incubator to 37 ^oC.
2. Euthanize e10.5 timed-pregnant mice in accordance with IACUC guidelines. Spray the abdomen with 70% ethanol to clean the region before making a V-shaped incision at the genital area of the mouse using scissors and forceps and continue it up to the diaphragm.
   ​NOTE: Appearance of a vaginal plug in this protocol indicates day 0.5. Using 5 - 6-week-old mice, a weight gain of more than 2 g acts as a secondary measure of successful impregnation.
   1. Carefully excise the uterus by making an incision at the uppermost lateral portions of the uterus, pulling the organ upwards away from the mouse, and following this incision down to the genital region before repeating on the opposite side. Place it in a 10 cm Petri dish with the cold PBS.
      NOTE: It is possible to obtain organoids from e11.5 dissociated cells, however unlike the e10.5 pancreatoids, these organoids are not yet characterized and thus may not retain the capacity to differentiate into all three major pancreatic lineages.
3. Under a light dissection microscope, place the Petri dish and carefully open the uterine tissue by placing two forceps between each embryo and peeling tissue away from the yolk sac. Transfer embryos by grasping the yolk sac gently with forceps and place in a 10 cm Petri dish with fresh, cold PBS. Place the Petri dish on ice.
   NOTE: It is important to heedfully remove embryos, preferably maintained within the yolk sac, as forceful removal can pull the umbilical cord, ripping tissue of the embryo and compromising the structural integrity.
4. Place multiple PBS drops on a 10 cm Petri dish lid. Use these droplets to transfer the embryo during dissection as extraembryonic tissues are removed to ensure visibility. Transfer an embryo to a PBS drop and gently remove from the yolk sac using forceps, while the remaining embryos stay in PBS on ice (Figure 1A). Remove the head before moving the embryo to a new PBS drop using forceps to lightly scoop the embryo up, to not damage the tissue (Figure 1B).
   ​NOTE: The tissue might need to be transferred more frequently to fresh PBS droplets than described here, depending on the opacity of the liquid.
   1. In a new PBS drop, remove the forelimb buds using forceps (Figure 1B). Place forceps into the opening where limb bud was present and gently tear only the most external tissue anteriorly (Figure 1B). Rotate embryo and repeat in the posterior direction, stopping at the hindlimb bud. At this point, the gastrointestinal tract should be visible (Figure 1B).
   2. The posterior region of the gastrointestinal tract has a slight bend (Figure 1F). Insert forceps behind this bend in the opening between the gastrointestinal tract and the spinal region of the body wall. Detach the gastrointestinal tract, working slowly upwards until the most anterior region is reached where the cardiac region connects (Figure 1B-E).
      OPTIONAL: Remove the primordial heart and the liver buds from the ventral regions of the gastrointestinal tract, leaving only the continuous gut tube. The first few times this protocol is performed it might be easier to leave the heart and liver as ventral landmarks until the morphology of the gastrointestinal tract and the dorsal pancreatic bud are more familiar (Figure 1C and D).
   3. Transfer the gastrointestinal tract to the fresh PBS droplet.
   4. Take forceps and gently pinch the tissue beneath the protruding bud and the intestine and slightly lift the outer tissue off (Figure 1D-F).
      NOTE: The dorsal pancreatic bud is located between the stomach and intestine (Figure 1C-G). The pancreatic bud beneath should look like a round, knobby structure (Figure 1G).
   5. Place forceps where the bud connects to the intestine and pinch upward to loosen the bud (Figure 1G). Wash one time in a new PBS bubble before transferring the bud into the first well of the 12 well plate in cold PBS on ice. Repeat until all buds are collected from embryos and placed in the first well of the 12 well plate.
5. Place the 12 well dish under the light dissection microscope. Count the number of pancreatic buds successfully dissected to later calculate the split ratio.
   1. Place a filtered 1,000 µL pipette tip into the mouth pipette with a capillary tube attached. Flame sterilize the capillary tube and create a bend in the tube for the ease of use. Use the capillary to transfer buds to the second well containing cold dispase solution for 2 min before transferring to the third well with the clean PBS (Figure 1G).
      NOTE: While transferring buds from dispase to PBS, avoid touching the tissue to the tip of the capillary tube. If the tissue gets stuck at the tip of the tube, pipette up and down or shake in the clean PBS solution until loosened.
   2. Pipette buds up and down, and transfer to the fourth well with clean PBS. Finally, using the capillary device transfer buds to the 1.5 mL tube with 0.05% Trypsin. Place the tube in the pre-warmed 37 ^oC shaker and shake at 1,500 rpm for 4 min.
   3. Vortex for approximately 10 s then immediately place the tube in a centrifuge and spin for 5 min at 200 g. Remove all but approximately 50 µL of solution cautiously as to not disturb the centrifuged cells (Figure 1G).

2. Culturing Dissociated Progenitors to Form Pancreatoids

Note: The following should be performed in a sterile atmosphere in a standard tissue culture hood, using standard sterile procedures.

1. For every bud collected, add 400 µL organogenesis media^16,17 (Table 1) to the centrifuged cells. Pulse vortex three times and plate 100 µL per well of a low attachment 96 well plate, splitting buds at a 1:4 ratio (Figure 1G).
2. Check under the microscope to visualize dissociated cells freely floating (Figure 1H).Place the culture dish in a 37 ^oC incubator with 5% CO₂ on a rocker at medium speed.
3. Monitor the progress of pancreatoids daily. Replace with 100 µL of fresh media every 3 days, carefully pipetting media under microscopic control in the hood to remove while leaving pancreatoid in the well. Alternatively, fresh 100 µL of media can be added to a new well and pancreatoid transferred using the borosilicate capillary tube attached to mouth pipette and 1000 µL filtered pipette tip.

3. Processing Pancreatoids for Immunofluorescent Imaging

1. To process pancreatoids for immunofluorescent images, first prepare 4% paraformaldehyde/PBS, pH7.4 (PFA) solution. Place 50 µL of 4% PFA in wells of a 96 well plate.
   1. Using the borosilicate capillary tubes and mouth pipette with the 1,000 µL filtered pipette tip, transfer pancreatoids from culture medium taking as little media as possible into the fresh well with 4% PFA. Set on the rocker at room temperature for 15 min.
   2. Transfer pancreatoids from 4% PFA into a well with 100 µL of fresh PBS, rock at room temperature for 5 - 10 min. Repeat for a total of three fresh PBS washes.
      NOTE: The protocol can be paused here, with pancreatoids stored in 100 µL of PBS at 4 ^oC for up to one week.
2. For wholemount imaging (recommended if antibodies are suitable, alternative approach in section 3.3. and microscopy in 3.4.), transfer from PBS into 50 µL 5% donkey serum in 1x PBS with 0.1% nonionic detergent (PBST) to block. Rock overnight at 4 ^oC or alternatively for 4 h at room temperature.
   1. Transfer pancreatoids to a new well containing primary antibodies diluted in 50 µL of 5% donkey serum in 1x PBST. Optimally, rock for 24 h at 4 ^oC (at least 12 h but up to 36 h).
      NOTE: Antibodies listed in the Table of Materials against Chga (1:100), DBA (1:400), Vim (1:400), and Pdx1 (1:100) are suitable for wholemount staining; other antibodies may require optimization.
   2. Transfer pancreatoids to a well containing 100 µL of fresh PBST to wash and rock for 30 min at room temperature. Repeat this step for a total of three fresh PBST washes.
   3. Transfer pancreatoids to a new well containing secondary antibodies diluted in 50 µL of 5% donkey serum in 1x PBST. Protect the plate from light and place in 4 ^oC, rocking for optimally 24 h (at least 12 h but up to 36 h).
      NOTE: All secondary antibodies listed in the Table of Materials and DAPI nuclear counterstain are suitable for wholemount staining.
   4. Transfer pancreatoids to a new well containing 100 µL of 1x PBST and rock at room temperature for 30 min. Repeat for a total of three washes in 1x PBST. In the third and final wash, add 300 nM DAPI.
   5. Finally, place into 100 µL of clean PBS and store at 4 ^oC protected from light for up to a week before imaging by confocal microscopy, although best results come from imaging immediately after staining. To prevent movement of pancreatoids during imaging but prevent drying out, place each pancreatoid into a 20 µL droplet of PBS. If pancreatoids do not remain still, reduce PBS volume.
3. For frozen sections, transfer pancreatoids from PBS into 30% sucrose solution overnight at 4 ^oC.
   1. Add embedding media in a bubble under a dissection microscope. Using forceps under microscopic control, soak pancreatoids by gently pushing through the embedding media several times to remove residual sucrose before transferring to a tissue block with frozen embedding media.
   2. Place the tissue block on dry ice until frozen and section on the cryostat at 8 µm.
      NOTE: Sections can be stored at -80 ^oC or immunostained immediately.
   3. Allow sections from -80 ^oC to thaw at room temperature for approximately 5 min until dry. Draw a hydrophobic barrier using the hydrophobic pap pen around the tissue and the block using 5% donkey serum diluted in 1x PBST for 30 min at room temperature.
   4. Remove media and replace immediately with primary antibodies diluted in 5% donkey serum diluted in 1x PBST overnight at 4 ^oC.
   5. Remove the primary solution and wash tissues three times with 1x PBST for 10 min each. Following this, add secondary antibody solution diluted in 5% donkey serum diluted in 1x PBST for 1 h at room temperature and protect slides from light.
   6. Remove the secondary solution and wash slides three times in 1x PBST for 10 min each. In the final wash, add 300 nM DAPI.
   7. Remove media and add mounting media and a coverslip. Store slides away from light in 4 ^oC until ready to image.

4. Isolation of RNA from Pancreatoids for Transcript Analysis

1. Collect pancreatoids using borosilicate capillary attached to a mouth pipette with a filtered 1,000 µL pipette tip for sterility and place into 500 µL of acid guanidinium thiocyanate-phenol-chloroform solution in a 1.5 mL tube. Store at -80 ^oC or proceed immediately to RNA isolation.
2. For RNA isolation, thaw samples on ice if necessary and add 100 µL of chloroform. Vortex well and place in 4 ^oC for 15 min. Pre-cool a centrifuge to 4 ^oC.
   1. Place tubes into centrifuge and spin for 20 min at 12,000 g at 4 ^oC.
   2. Carefully remove tubes to not disturb the separation of layers. Using a 200 µL pipette, collect the clear aqueous solution and place into a new 1.5 mL tube, leaving behind a small amount to buffer from the white protein layer and pink DNA layer. Do not touch the tip of the pipette to anything in this process and do not touch the walls of the 1.5 mL tube.
   3. Add 500 µL of 100% isopropanol to the new tube containing the aqueous layer and vortex. Let sit at room temperature for 20 min, longer on the ice, or for maximum precipitation leave overnight at -20 ^oC.
   4. Centrifuge at 12,000 g for 15 min at 4 ^oC to pellet the RNA. Remove the isopropanol without disturbing the pellet.
      NOTE: As pancreatoids are small, it is likely the pellet will not be visible.
   5. Add cold, 75% ethanol and invert the tube several times. Place in the centrifuge and spin at 9,000 x g for 10 min at 4 ^oC. Remove the ethanol and spin for 2 min at 9,000 x g.
   6. Use a 200 µL pipette to remove any remaining ethanol in the tube, without contacting the region the pellet resides in. Leave the cap open on the tube for 5 - 10 min at room temperature to ensure evaporation of residual ethanol.
   7. Resuspend pellet in by vortexing in 20 µL of clean, nuclease-free water.
      Optional: Heat the RNA at 65 ^oC for 3 min and immediately return to the ice. RNA can be stored at -80 ^oC or immediately used for reverse transcription and qPCR.

Wyniki

Careful dissection of mouse embryos at e10.5 from the uterine horn should yield undamaged embryos in PBS for further dissection (Figure 1A). The gastrointestinal tract can be efficiently removed from the embryo (Figure 1B), permitting discernment of the dorsal pancreatic bud at the junction of the intestine and stomach (Figure 1C-F). The e10.5 pancre...

Dyskusje

The progression of cell culture models is critical to properly model development, produce clinically relevant cell types, test drug efficacy, or even transplant to patients. However, artificially recapitulating development in a dish is challenging as we are still far from understanding the mechanisms of organogenesis and physiology in vivo. Thus in vitro cells are inefficiently generated, not fully functional, unable to be maintained for long periods of time, or harbor other abnormalities from comparabl...

Materiały

Name	Company	Catalog Number	Comments
2-Mercaptoethanol	Sigma-Aldrich	M6250
Aspirator Tube Assemblies for Calibrated Microcapillary Pipettes	Sigma-Aldrich	A5177
BarnStead NanoPure Nuclease-free water	ThermoFisher	D119
Borosilicate Capillary Tubes	Sutter Instruments	GB1007515	O.D. 1mm, I.D. 0.75mm, 1.5cm length
CaCl2	Sigma-Aldrich	C5080
Cell-Repellent 96-Well Microplate	Greiner Bio-One	650970	U-bottom
Centrifuge 5424 R	Eppendorf	5401000013
Chloroform	Sigma-Aldrich	233306
Chromogranin-A antibody	Abcam	ab15160
Compact, Modular Stereo Microscope M60	Leica
Countess Automated Cell Counter	Invitrogen	C10310
Countess Cell Counter Slides	Invitrogen	C10312
CryoStar NX70	ThermoFisher	957000L
D-(+)-Glucose	Sigma-Aldrich	G7528
DAPI (4',6-Diamidine-2'-phenylindole-dihydrochloride)	Roche	10 236 276 001	Powder
DBA antibody	Vector Lab	RL-1032
Dispase II, Powder	Gibco	17105041
DMEM/F-12, HEPES	Gibco	11330032
Dnase I	Invitrogen	18068-015
Dumont #5 Forceps	Fine Science Tools	11251-10	0.05 x 0.02 mm; Titanium; Biology tip
EGF (Epidermal growth factor)	Sigma-Aldrich	E9644
Ethanol, 200 Proof	Decon Laboratories	2716
Forma Steri Cycle CO2 Incubators	ThermoFisher	370
Fluoromount-G	Southern Biotech	OB10001
Heparin sodium salt from porcine intestinal mucosa	Sigma-Aldrich	H3149-10KU
INSM1 Antibody	Santa Cruz BioTechnology	sc-271408	Polyclonal Mouse IgG
Isopropanol	Fisher	a4164
Isothesia Isoflurane, USP	Henry Schein	11695-6776-2
Insulin Antibody	Dako	A056401	Polyclonal Guinea Pig
KAPA SYBR FAST Universal	KAPA Biosystems	KK4618
KCl	KaryoMax	10575090
KnockOut Serum Replacement	Invitrogen	10828028
Leica TCS SPE High-Resolution Spectral Confocal	Leica
MgCl2	Sigma-Aldrich	442615
Mouse C-Peptide ELISA	ALPCO	80-CPTMS-E01
Mouse Ultrasensitive Insulin ELISA	ALPCO	80-INSMSU-E01
MX35 Microtome Blades	ThermoFisher	3052835
NaCl	Sigma-Aldrich	S7653
NaHCO3	Sigma-Aldrich	S3817
NaH2PO4	Sigma-Aldrich
Normal Donkey Serum	Jackson Immuno Research	017-000-121
Paraformaldehyde	Sigma-Aldrich	158127
PBS 1X	Corning	21-040-CV
Pdx1 antibody	DSHB	F6A11	Monoclonal Mouse MIgG1
Peel-A-Way Disposable Embedding Molds	VWR	15160-157
Penicillin-Streptomycin Solution	Corning	MT30002CI
PMA (Phorbol 12-Myristate 13-Acetate)	Sigma-Aldrich	P1585
Protein LoBind Microcentrifuge Tubes	Eppendorf	22431081	1.5mL Capacity
Recombinant Human FGF-10 Protein	R&D Systems	345-FG
Recombinant Human FGF-Acidic	Peprotech	100-17A
Recombinant Human R-Spondin I Protein	R&D Systems	4546-RS
BenchRocker 2D	Benchmark	BR2000
Sucrose 500g	Sigma-Aldrich	S0389
SuperFrost Plus Microscope Slides	Fisher Scientific	12-550-15
Super Pap Pen	Electron Microscopy Sciences	71310
Thermomixer R	Eppendorf	05-412-401
Tissue Tek O.C.T. Compound	Sakura	4583
Triton X-100	Sigma-Aldrich	T8787
TRIzol Reagent	Invitrogen	15596018
TrypLE Express	Invitrogen	12604039	(1x), no Phenol Red
Trypan Blue Stain	Invitrogen	15250061	For cell counting slides
Trypsin-EDTA (0.05%)	Corning	25-052-CI
Trypsin-EDTA (0.25%)	Gibco	25200072	Phenol Red
Ultra-Low Attachment 24-Well Plate	Corning	3473
Ultra-Low Attachment Spheroid Plate 96-Well	Corning	4520
Vimentin Antibody	EMD Millipore	AB5733	Polyclonal Chicken IgY
Vortex Genie	BioExpres	S-7350-1
Y-27632 Dihydrochloride	R&D Systems	1254	Also known as ROCK inhibitor
Zeiss 710 Confocal Microscope	Zeiss