This work was funded sequentially by a NCCR Frontiers in Genetics pilot award, Juvenile Diabetes Research Foundation Grant 41-2009-775 and Grant 12-126875 from Det Frie Forskningsr&#229;d/Sundhed og Sygdom. The authors thank the Spagnoli lab for hosting the video shooting.

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
<ol>
	<li>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 damage.</li>
	<li>Separate the uterus into individual embryo segments using small scissors. Transfer one embryo to a 35 mm Petri dish with cold PBS and visualize it under a dissecting microscope with illumination from above. With dissection forceps remove the surrounding muscle, decidua and yolk sac and expose the embryo. Place the embryo back into cold DMEM medium. Embryos can easily be transferred using a 3 ml plastic transfer pipette/dropper. Isolate every individual embryo in a similar manner before continuing.</li>
	<li>Place an embryo into a clean 35 mm Petri dish in PBS.
	<ol>
		<li>Use thin forceps (0.05 mm width) to remove the forelimb. Gently insert the forceps in the opening to detach the digestive tract from the spinal cord region (Figure 1). OPTIONAL: To more conveniently see the stomach, remove the upper body of the embryo, down to the heart and the tail region below the yolk stalk.</li>
		<li>Locate the stomach, liver and intestine. Using the forceps, isolate the gastrointestinal tract from the stomach to the intestine and place it in cold DMEM on ice (Figure 1). The dorsal pancreatic bud is attached dorsally, posterior to the stomach (Figure 1).</li>
		<li>Isolate every individual gastrointestinal tract in a similar manner before continuing. If the embryos need to be genotyped, collect the tail at the time of dissection and keep every individual gastrointestinal tract in a different well in a 24-well plate with cold DMEM on ice.</li>
	</ol>
	</li>
	<li>Place one gastrointestinal tract in a clean 35 mm Petri dish in cold PBS. Now use illumination from below in bright field to visualize and dissect the dorsal pancreatic bud under the dissection microscope. These conditions will optimize the visualization of volumes. Using electrolytically-sharpened tungsten needles or 20 G syringe needles, isolate the pancreatic bud with as little mesenchyme as possible around the epithelium (Figure 1).
	<ol>
		<li>Transfer the isolated bud into a petri dish containing a cold dispase solution (1.25 mg/ml) for 2-3 min. From this point, transfer can most conveniently be done with flame-pulled 50 &#181;l glass capillaries attached to a mouth-controlled flexible tube. Alternatively, but with more risk of losing the bud, use a 10 &#181;l automatic pipette (Pipetman) with appropriate plastic tips.</li>
		<li>Perform pancreatic bud aspiration and ejection under microscopic control. Put the pancreatic bud back in PBS. Further clean the isolated pancreatic bud from the mesenchyme with the needles and gentle aspiration using the glass capillary (Figure 1).</li>
		<li>When the entire mesenchyme is removed, rinse the pancreatic bud in cold PBS; transfer each bud to cold DMEM in individual wells (60-well mini-trays filled with 10 &#181;l of cold DMEM). It is important not to remove the mesenchyme in dispase, which makes the tissue very sticky.</li>
	</ol>
	</li>
</ol>
2. Plating and Culture of Dispersed Cells
<ol>
	<li>Transfer the dissected epithelia from all embryos with a flame-pulled glass capillary into conical wells of 60-well mini-trays filled with 10 &#181;l PBS for rinsing.
	<ol>
		<li>Transfer the bud into 10 &#181;l of Trypsin 0.05 % and let it incubate at 37 &#176;C for 4 min. Inactivate the trypsin by transferring the bud into a well with 10 &#181;l DMEM + 10% fetal calf serum (FCS).</li>
		<li>Dissociate the cell suspension by aspiration through a thin capillary pulled with a pipette puller. It is important to avoid bubbles at this stage while pipetting up and down to dissociate the cells. Pancreas organoids optimally start from small groups of 5-15 cells and therefore partial dissociation is recommended (Figure 1).</li>
	</ol>
	</li>
	<li>Pool the cells from several embryos into an Eppendorf tube in order to minimize differences due to individual processing. Dilute the cell suspension in chilled Matrigel at a 1:3 ratio. Aliquot this mixture to a 96-well plate, 8 &#181;l/well or in a plate optimized for imaging (see below).</li>
	<li>Incubate the plate at 37 &#176;C for 5 min, allowing the Matrigel to thicken. Fill the wells with 70 &#181;l of medium of choice (organoid or sphere, see Tables 1 and 2) and leave in a humidified environment containing 5% CO2 and 95% air at 37 &#176;C.</li>
	<li>Replace the medium every 4th day. Monitor the growing pancreatic colonies daily and document the process by imaging.</li>
	<li>Small molecules or proteins of interest can be added to the medium at this stage for interference experiments, as reported previously 28.</li>
</ol>
Table 1: Organoid medium.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material</td>
			<td>Stock Concentration</td>
			<td>Concentration in final medium</td>
			<td>Volume of stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>100%</td>
			<td>1%</td>
			<td>50 &#181;l</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>100%</td>
			<td>10%</td>
			<td>500 &#181;l</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>14.3 M</td>
			<td>0.1 mM</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>16 &#181;M</td>
			<td>16 nM</td>
			<td>5 &#181;l</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>50 mM</td>
			<td>10 &#181;M</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>50 &#181;g/ml</td>
			<td>25 ng/ml</td>
			<td>2.5 &#181;l</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>250 &#181;g/ml</td>
			<td>500 ng/ml</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>250 &#181;g/ml</td>
			<td>500 ng/ml</td>
			<td>10 &#181;l</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>100 &#181;g/ml</td>
			<td>25 &#181;g/ml</td>
			<td>1.25 &#181;l</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>2500 U/ml</td>
			<td>2.5 U/ml</td>
			<td>2 &#181;l</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>100 &#181;g/ml</td>
			<td>100 ng/ml</td>
			<td>5 &#181;l</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td></td>
			<td></td>
			<td>4,412.25 &#181;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>5,000 &#181;l</td>
		</tr>
	</tbody>
</table>
Table 2: Sphere medium.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material</td>
			<td>Stock Concentration</td>
			<td>Concentration in final medium</td>
			<td>Volume of stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>100%</td>
			<td>1%</td>
			<td>50 &#181;l</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>100%</td>
			<td>10%</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>100 &#181;g/ml</td>
			<td>64 ng/ml</td>
			<td>3.2 &#181;l</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>50 mM</td>
			<td>10 &#181;M</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td></td>
			<td></td>
			<td>4845.8 &#181;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td></td>
			<td>5000 &#181;l</td>
		</tr>
	</tbody>
</table>
3. Imaging of the Progression of Organoid Development
<ol>
	<li>Image organoids either daily or by time lapse microscopy using a fluorescent time-lapse microscope. For time lapse imaging, use an XY(Z) automated inverted fluorescent microscope.</li>
	<li>Deposit small droplets of 3 &#181;l in 4-well plates or glass-bottom plates filled with 2-5 ml medium. Image with a 10x long distance objective. Note: Transgenic mice expressing fluorescent tracers can be used. Movie 1 shows for example the initial expansion of organoids from Pdx1-Ngn3-ERTM-ires-nGFP+ mice4. The nuclear GFP enables the user to track cells as individual objects but similar principles can be applied to track cells with membrane fluorescence such as mT/mG mice29.</li>
	<li>Start time-lapse imaging 3 hr after seeding the cells to avoid focus drifts and set the software controlling automation to take 1 picture/hr for 3 or more days at manually defined positions. For every position, acquire a differential interference contrast (DIC) image as well as the GFP signal, reporting fluorescent marker expression.</li>
</ol>
4. Recovery of Organoids for Histology
<ol>
	<li>Place the 96-well plate on ice and remove the medium, replacing it with ice cold PBS. This partially depolymerizes Matrigel.</li>
	<li>Gently aspirate each individual organoid, removing the surrounding Matrigel using a 1,000 &#181;l tip in order to not disrupt the overall architecture. Transfer each organoid to a well with ice cold PBS. Keep the plate on ice. Direct fixation in Matrigel is also possible.</li>
	<li>Fix the organoid for 15 min in 4% paraformaldehyde (PFA), cryopreserve it in sucrose and embed it in gelatin. Process each organoid for cryosectioning and histology as previously described (Johansson et al., 2007) 4.</li>
</ol>
5. Recovery of Organoids for PCR and Biochemistry
<ol>
	<li>Place the 96-well plate on ice and remove the medium. Add 60 &#181;l of RNAlater per well in order to stabilize and protect cellular RNA.</li>
	<li>Disrupt the gel in each well mechanically by partially depolymerizing it on ice. Either recover individual organoids using a 1,000 &#181;l tip in order to not disrupt the overall architecture or recover the entire well (with Matrigel) by disrupting the gel mechanically with a 200 &#181;l tip. Use a 1,000 &#181;l tip to transfer the well content into an RNAse free-non-sticky Eppendorf tube kept on ice.</li>
	<li>Wash the wells with 60 &#181;l RNAlater and add the remaining content to the same Eppendorf tube.</li>
	<li>Spin the tubes for 5 min at 500-1,000 x g at 4 &#176;C.</li>
	<li>Remove the supernatant, only leaving 20-30 &#181;l of RNAlater in the tube together with the pellet. For biochemistry, store the samples as dry as possible.</li>
	<li>Do not freeze samples in RNAlater immediately; store at 4 &#176;C O/N (to allow RNAlater to thoroughly penetrate the tissue). The tissue can be stored at -20 &#176;C for long term storage and can later be processed for tissue disruption and extraction of small quantities of RNA.</li>
</ol>

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The authors have nothing to disclose.

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 Pdx1, express the pro-convertases that mature insulin and have the processed insulin 28. Important key factors within the system are the activity of FGF (exogenously stimulated by added FGF potentiated by heparin) and Notch (endogenous) signaling pathways, as well as ROCK-inhibition by Y-27632: in the absence of those factors, no or very limited numbers of organoids and spheres were generated 28. The requirement for FGF and Notch activity is easily understood based on their importance for pancreas development in vivo 28. ROCK inhibitor can be substituted by blebbistatin, thus revealing that hyperactivation of microfilament dynamics upon dissociation leads to both increased cell death, a strong inhibition of the progenitor transcription factor Pdx1 and a lack of expansion. Interestingly, many additional components of the organoid medium were proven to be individually unnecessary, but their combined absence resulted in the loss of epithelial branching 28. In addition to certain essential components of the medium, it is important to control the level of dissociation of progenitors. Indeed, progenitor proliferation and Pdx1 maintenance are significantly promoted in groups of more than 4 cells. A compaction can be observed within the first 12 hr and failure to compact results in failure to maintain Pdx1 and expand. The ROCK inhibitor is essential for this process.
At the moment organoids do not form after FACS sorting but the efficiency of the system could potentially be improved by reaggregating a controlled number of progenitors. Another critical component of this culture system is the 3D matrix. Cells put on Matrigel or in the Matrigel too close to the bottom of the plate spread and lose Pdx1 expression. Matrigel most likely provides biochemical components, notably laminin as well as mechanical cues 28. Indeed, the stiffness of the matrix plays a pivotal role. Stiff hydrogels are not permissive for pancreatic progenitors maintenance and expansion 28 and diluted Matrigel is not either. When Matrigel is diluted 1:10 pancreas progenitor cannot be cultured.
The organoid system can be used to test the effects of small molecules and recombinant proteins on pancreatic progenitors in terms of survival, proliferation, differentiation, polarization and branching 28. It can also be used to test the cooperation of different cell types during pancreas development 28. We are confident that the accessibility of pancreatic progenitor cells will also allow genetic manipulations, such as viral targeting, as seen in other organoid systems 17,27. This could be used for screening 17 with a system that enables morphogenesis in contrast to the spheres described previously as well as here 16,17,28. The culture conditions we developed also present the advantage of being serum-free, feeder-free and devoid of mesenchyme and blood vessels thereby reducing the cellular and biochemical complexity. However, there is a limitation in the ability to passage the organoids and thus to obtain large quantities of progenitors. This could be circumvented in the future by adaptations of the protocol to later stages of development where progenitors are more abundant, to sources of pancreatic progenitors produced from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. This paves the way for a 3D model of human pancreas development.
This system can also potentially be used for the production of pancreatic cells in the future perspective of therapy. In this context, the production of functional beta cells for transplantation would potentially help in diabetes therapy. The adaptations of the system to human ES or iPS cells would be important for this purpose. It is still unclear whether the organoid or sphere conditions should be used. The organoid conditions allow for the production of cells that have several characteristics of mature beta cells but their function remains to be tested. However, these cells are currently not numerous and are mixed among other cells. It is also likely that the early appearance of heterogeneity in the organoid system leads to uncontrolled signaling between cells and is therefore detrimental to production.
The sphere system that maintains progenitors is in principle preferable for controlled expansion and passaging of progenitors but their subsequent differentiation remains to be controlled. Others have recently produced pancreatospheres that can be efficiently differentiated. It will be important to compare the nature of the spheres obtained in the current protocol devoid of feeders and serum to spheres obtained with the other protocols 16,17. From a therapeutic point of view, the complexity and biological origin of Matrigel may constitute an issue of reproducibility, health and scalability. Preliminary results have shown that soft hydrogels functionalized with laminin are permissive for pancreatic progenitor expansion in vitro. Further optimization is required as these gels are not yet as efficient as Matrigel 28.
The production of beta cells in their natural context could also potentially be useful to test drugs that boost beta cell activity or increase their survival or proliferation but for this purpose it will be important to first test the degree of maturity of the beta cells, to increase the efficiency of their differentiation and to test whether the culture conditions presented here better maintain islets than the current suspension cultures. Producing the exocrine pancreas could also be useful to develop drugs to target pancreatic cancers and pancreatitis. Here again, the degree of maturity of the exocrine cells produced needs to be thoroughly investigated.

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 to investigate epithelial-mesenchymal interactions 4,6,7, to image processes 8 or to chemically interfere with them 9. Two organ culture methods are mainly used: the first consists in culturing pancreatic buds on fibronectin coated plates 2, which is convenient for imaging purposes; the second option is to culture the organs on filters at the air-liquid interface 3,4 which best preserves morphogenesis. Although very useful, these methods lead to a certain degree of flattening; the expansion of progenitors is very limited as compared to the normal development and the starting population is complex comprising all types of pancreatic cells and mesenchymal cells.
The ability to culture and expand dispersed primary cells is valuable to study lineage relationships and uncover the intrinsic properties of isolated cell types 10. Sugiyama et al. 11 could maintain pancreas progenitors and endocrine progenitors that retained some functional characters for 3-5 days in culture on feeder layers. Pancreatospheres, akin to neurospheres 12 and mammospheres 13, have been expanded from adult islets and ductal cells although the nature of the progenitors/stem cells that generate these spheres is not clear. In addition, in contrast with physiological development, the pancreatospheres contained some neurons 14,15. Spheres were also recently produced from embryonic pancreas progenitors 16,17 and regenerating pancreata18 with good progenitor expansion and subsequent differentiation but failed to recapitulate morphogenesis.
3D models from dispersed and often defined cells that self-organize into miniaturized organs have recently flourished and simulate the development or adult turnover of multiple organs such as the intestine 19,20, the stomach 21, the liver 22, the prostate 23 and the trachea 24. In some instances, developmental morphogenesis and differentiation have been recapitulated in 3D from ES cells, as is the case of optic cups 25, intestine 26 or brain 27.
Here, we describe a method to expand dissociated multipotent pancreatic progenitors in a 3D Matrigel scaffold where they can differentiate and self-organize.

<table><tbody><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>KnockOut Serum replacement (supplement)</td><td>Gibco</td><td>10828-028</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma Aldrich</td><td>3148-25ML</td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>Phorbol Myristate Acetate (PMA)</td><td>Calbiotech</td><td>524400-1MG</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Y-27632 (ROCK inhibitor)</td><td>Sigma Aldrich</td><td>ab120129</td><td>Stock kept at -20 &amp;deg;C. Attention! Stability/source is a frequent source of problems.</td></tr><tr><td>EGF</td><td>Sigma Aldrich</td><td>E9644-2MG</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Human R-spondin 1</td><td>R&amp;amp;D</td><td>4645-RS-025/CF</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Mouse R-spondin 1</td><td>R&amp;amp;D</td><td>3474-RS-050</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF1 (aFGF)</td><td>R&amp;amp;D</td><td>232-FA-025</td><td>Stock kept at -80 &amp;deg;C - do not include to increase beta cell production</td></tr><tr><td>Heparin (Liquemin)</td><td>Drossapharm</td><td></td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF10</td><td>R&amp;amp;D</td><td>345-FG-025</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>21331-020</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>B27 x50 (supplement)</td><td>Gibco</td><td>17504-044</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF2 (bFGF)</td><td>R&amp;amp;D</td><td>233-FB-025</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Trypsin 0.05%</td><td>Gibco</td><td>25300-054</td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>RNAlater - RNA stabilizing reagent</td><td>Qiagen</td><td>76104</td><td>Store at RT</td></tr><tr><td>Dispase&amp;nbsp;</td><td>Sigma Aldrich</td><td>D4818-2MG</td><td>Working concentration: 1.25 mg/ml. Stock kept at -20 &amp;deg;C</td></tr><tr><td>BSA for reconstitution</td><td>Milipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/item/81-068-3&quot;&gt;81-068&lt;/a&gt;</td><td>For reconstituition of cytokines&amp;nbsp; - stock kept at -20 &amp;deg;C</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Gibco</td><td>16141079</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>60-well MicroWell trays</td><td>Sigma Aldrich</td><td>M0815-100EA</td><td></td></tr><tr><td>4-well plates</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>95-well plates F bottom</td><td>Greiner Bio</td><td>6555180</td><td></td></tr><tr><td>Glas bottom plates</td><td>Ibidi</td><td>81158</td><td></td></tr><tr><td>Disposal glass micropipettes</td><td>Blaubrand</td><td>708745</td><td></td></tr><tr><td>Microscope</td><td></td><td></td><td>Cell&amp;#174; imaging station (motorized inverted Olympus IX81 stand) equipped with a Hamamatsu ORCA ER B7W camera and the Ludin Cube and Box.&lt;br /&gt;
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.</td></tr><tr><td>Objective</td><td></td><td></td><td>Olympus UPLAN FL NA 0.30 air 9.50 mm 10X long distance;&lt;br /&gt;
Leica HC PL FLUOTAR NA 0.30 air 11.0 mm 10X long distance</td></tr></tbody></table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

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&#180;s tensegrity.

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-ERTM-nGFP) (Movie 1) in the absence of tamoxifen and thus without activating Neurog3 4 (Figure 2).
With the organoid medium, an initial compaction of small clusters of cells occurs in the first hours. Expansion can then be detected by the enlargement of the clusters in the first 4 days (Figure 2A). From day 5, branches form in the 20% largest organoids. Single cells do not expand and lose Pdx1 expression while the large clusters retain Pdx1 expression 28.
In these conditions, progenitors undergo a spectacular morphogenesis with the emergence of branched epithelial structures. This process takes place only when the progenitors are seeded in &#8805;4-cells clusters, indicating a strong requirement for community signals. Histological analysis reveals that after day 7 of culture, the resulting mini-organs are composed of pancreatic progenitors (SOX9+/HNF1B+/Pdx1+ cells: Figure 3B) and differentiated cells expressing either exocrine (Amylase+) or endocrine (Insulin+ or Glucagon+) markers (Figure 3A,C). The differentiation into endocrine cells is lower than in the endogenous pancreas (around 0.1%) but is increased to 1% when FGF1 is not added to the culture medium 28. Remarkably, not only do the seeded progenitors differentiate into the expected pancreatic lineages, but they also spontaneously adopt the normal pancreatic architecture. Although E10.5 multipotent pancreas progenitors are not polarized, the cells in culture polarize as demonstrated by the segregation of Mucin1 and aPKC in the membrane facing the central lumen and they organize into a branched tubular network. Regionalized &#8220;tip and trunk&#8221; domains emerge: HNF1B+ progenitors and endocrine cells are localized in the central region, while acinar cells are located at the periphery as a partial or complete crown of cells. The organoids can be maintained in culture for 10 days; after this period, they generally lose their architectural organization and become cystic (not shown). Passaging can be done after partial dissociation but quickly leads to cyst formation, a phenomenon that is reduced by the addition of the BMP inhibitor Noggin 28.
With the sphere medium, expansion is more frequent and is seen from 2% of single cells; nevertheless the efficiency of sphere formation correlates with the size of the seeded clusters 28. At day 2/3, a lumen is detected in the small clusters and expands thereafter, leading to largely mono-layered hollow spheres with occasional local multilayered areas (Figure 2B). These spheres collapse when retrieved from Matrigel (Figure 3D-H). Under these conditions, the resulting structures are mainly composed of pancreatic progenitors, with a small percentage of differentiated exocrine and endocrine cells at day 7 (Figure 3D-H). Progenitors in the spheres also become apically polarized, as demonstrated by the segregation of aPKC at the membrane facing the central lumen of all cells (Figure 3F). Pancreatospheres can be passaged at least twice (not shown).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51725/51725fig1highres.jpg" width="500" /> 
Figure 1. Schematic representation of the procedure. The gastro-intestinal tract is initially dissected from the embryo and subsequently the dorsal pancreatic bud is isolated. The mesenchyme is removed and the pancreatic progenitors are dissociated using trypsin. The resulting partially-dispersed cells are then seeded at low density in growth factor-depleted Matrigel. Scale bars: 1.00 mm except for the resulting organoid picture where the scale bar is 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51725/51725fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51725/51725fig2highres.jpg" width="500" /> 
Figure 2. Progress of culture over time. (A) A small cluster of pancreatic progenitors grown with the organoid medium and followed with time-lapse microscopy from 3 hr after plating, for 60 hr. In the bottom panels, an organoid after 7 days of culture. Scale bar: 200 &#181;m; applies to all panels in A. (B) Example of a sphere followed in a 60 hr time-lapse and captured at day 7 of culture. Scale bar: 200 &#956;m; applies to all panels in B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51725/51725fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51725/51725fig3highres.jpg" width="500" /> 
Figure 3. Histology. (A-C) Serial sections of a 7-day organoid stained for progenitor (B &#8211; HNF1B) and differentiation (A &#8211; amylase, C &#8211; insulin) markers. The organoid is composed of epithelial (A &#8211; E-cadherin) and apically polarized (B &#8211; mucin1) cells. The dashed line corresponds to the non-acinar central region (A), where HNF1B (B) and endocrine (C) cells are detected. (D-H) Sections of 7-day spheres stained for progenitor (G &#8211; HNF1B; H &#8211; SOX9) and endocrine (D &#8211; insulin and glucagon) markers. The spheres are composed of apically polarized (D, F &#8211; aPKC) epithelial (E &#8211; E-cadherin) cells. Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51725/51725fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

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.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

in-vitro-pancreas-organogenesis-from-dispersed-mouse-embryonic

Nanyang Technological University

Research

JoVE Journal

Biology

13.7K Views.  Swiss Institute for Experimental Cancer Research. 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.

Video: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Developmental Biology

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Medicine

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

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.

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

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

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Summary

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Chapters in this video

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1⁺ Pancreatic Progenitors

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

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

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1⁺ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

A System for ex vivo Culturing of Embryonic Pancreas