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 Pdx...

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.

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			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
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		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
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			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
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		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
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			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;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 keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
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		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;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 micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
		</tr>
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</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 ini...

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 ...

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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 Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...