Name: in vitro colony assays for characterizing tri-potent progenitor cells isolated from the adult murine pancreas
Uploaded: 2016-06-10T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas at JoVE.com

We thank Lucy Brown and Alexander Spalla from the Analytical Cytometry Core at City of Hope for assistance in sorting. This work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K., and U01DK089533 to A.D.R., and by National Science Foundation grant NSF-DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T. Supports from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine at Caltech to D.A.T., and those from Oxnard Foundation and Ella Fitzgerald Foundation to H.T.K. are also gratefully acknowledged.
Funding: This work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K., and U01DK089533 to A.D.R., and by National Science Foundation grant NSF-DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T. Supports from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine at Caltech to D.A.T., and those from Oxnard Foundation and Ella Fitzgerald Foundation to H.T.K. are also gratefully acknowledged. Research reported in this publication included work performed in the Analytical Cytometry Core and Light Microscopy Digital Imaging Core supported by the National Cancer Institute of the National Institutes of Health under award number P30CA33572.
Study sponsor: The sponsor did not participate in the study design, collection, analysis, or interpretation of data.

Ethical Statement: We adhere to the widely accepted ethical standards in conducting research to ensure the quality and the integrity of the results. Animal experimentation is conducted according to protocols approved by the Institutional Animal Care and Use Committee at City of Hope.
1. Prepare Single-cell Suspension from Adult Murine Pancreata
NOTE: In prior publications 7,11, CD-1 or B6 background mice were used; both backgrounds yielded similar results. A transgenic mouse line (designated Sox9/E...

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The pancreatic colony assays and single colony analyses described here were inspired by the methylcellulose-containing hematopoietic colony assays that have played major roles in deciphering the biology of hematopoietic progenitor cells in the past decades 23. In these assays (Figure 5), dissociated pancreatic cells are plated into methylcellulose-containing semi-solid media with appropriate growth factors and ECM proteins that support the formation of Ring, Dense or Endocrine/Acinar colonies ...

The pancreas is composed of three major cell lineages; acinar cells secrete digestive enzymes, ducts secrete mucin to fend off pathogens and transport digestive enzymes to the gut, and endocrine cells secrete hormones, including insulin and glucagon, that maintain glucose homeostasis. During the embryonic development of the pancreas, the early ductal cells are the source of the tri-potent progenitor cells capable of giving rise to the three lineages in the pancreata of adult animals 1,2. Because adult stem and progenitor cells, such as bone marrow stem cells, are already successfully used to treat various diseases 3, there is intense interest in finding the stem and progenitor cells in the adult pancreas. If isolation and manipulation of adult pancreatic stem and progenitor cells were possible, these cells could be used to treat diseases such as type 1 diabetes, in which the insulin-secreting cells are destroyed by autoimmunity.
Whether tri-potent progenitor cells still exist in adult pancreatic ducts after the completion of embryonic development is a question that is heavily debated in the scientific community. In this debate, and using in vivo cre-lox lineage-tracing techniques, Inada and coworkers showed that adult murine ductal cells labeled with a marker, carbonic anhydrase II, could give rise to all three pancreatic lineages 4. However, using other ductal markers, such as HNF1b 5 and Sox9 2, it was concluded that ductal cells are not the major source of beta cells in adult mice.
Several years ago, we proposed that the cause of the aforementioned debate may be due to the lack, in the field 6,7, of appropriate analytical tools that can be used to measure self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. The in vivo cre-lox lineage-tracing technique mentioned above can provide evidence for the progenitor-progeny relationship on a population level. However, this lineage tracing technique is limited in its power to discern whether single progenitor cells can self-renew and differentiate into multiple lineages. Single-cell analysis is important because if several mono-potent progenitors, each with a different lineage potential, were analyzed together, they may collectively appear to have multi-lineage differentiation abilities. In addition, stem cells are usually a minor population of an adult organ. The activities of a minor cell population could be masked by the major population. Therefore, a negative result from a population study does not necessarily indicate the absence of stem cells. Finally, cre-lox lineage tracing does not currently allow the measurement of self-renewal.
To begin addressing the technical gap in the field of pancreatic progenitor cell biology, colony 7-11 or organoid 12-15 assays using 3D culture systems were devised. Two colony assays for pancreatic progenitors were developed in our laboratory: one contains a commercial preparation of murine extracellular matrix proteins (ECM) (see Methods and Equipment Table), and the other contains laminin hydrogel, a defined artificial ECM protein 7-11. Progenitor cells are mixed in semi-solid medium containing methylcellulose. Methylcellulose is a biologically inert and viscous material prepared from wood fibers, and has been routinely used in hematopoietic colony assays 16. The methylcellulose-containing semi-solid medium restricts the movement of single progenitor cells so that they cannot re-aggregate. Yet, the medium is soft enough to allow a progenitor cell to grow and differentiate into a colony of cells in the 3D space. Following the tradition of the hematologists, a pancreatic progenitor cell that was capable of giving rise to a colony of cells was named a pancreatic colony-forming unit (PCFU). PCFUs, when grown in the murine ECM-containing colony assay, give rise to cystic colonies that are named &#34;Ring&#34; colonies 7. Upon addition of a Wnt agonist, R-spondin1, into the murine ECM-containing culture, some Ring colonies turn into &#34;Dense&#34; colonies 7. In this article, these two types of colonies grown in murine ECM culture are collectively referred to as &#34;Ring/Dense&#34; colonies. When Ring/Dense colonies are dissociated into single cell suspension and re-plated into cultures that contain laminin hydrogel, &#34;Endocrine/Acinar&#34; colonies are formed 7.
Using single colony analyses, it was found that the majority of Ring/Dense and Endocrine/Acinar colonies, either from adult (2-4 month-old) 7,11 or young (1 week-old) 9 murine pancreas, express all three lineage markers. This suggests that most of the originating PCFUs are tri-potent. In the murine ECM-containing colony assay, adult murine PCFUs robustly self-renew and expand approximately 500,000 times over 11 weeks in culture 7. Murine ECM preferentially supports the differentiation of ductal cells over endocrine and acinar lineages, whereas in the presence of laminin hydrogel, murine PCFUs are encouraged to differentiate preferentially into endocrine and acinar cells and less so to the ductal lineage 7,9,11. Importantly, Insulin+Glucagon- mono-hormonal cells are generated in the laminin hydrogel culture and secrete insulin in response to glucose stimulation in vitro 7,9, suggesting functional maturity. The tri-lineage differentiation potential 7,9 and self-renewal 11 of individual PCFUs are confirmed by single-cell micromanipulation, i.e., culturing one cell per well for colony formation. Together, these results provide evidence that there are self-renewing, tri-potent, progenitor-like cells in the postnatal murine pancreas that show activities in 3D culture.
The murine PCFU assays described in this article are derived from a prior colony assay designed for progenitor cells differentiated from murine embryonic stem cells (mESCs) 17. That protocol is documented in detail in another JoVE publication 18. The culture components and techniques required to perform the murine ECM-containing colony assay for adult PCFUs are the same as for mESC-derived progenitors 17,18. Therefore, these aspects of the assay will not be repeated here; instead the following procedures will be addressed: 1) dissociation of the adult pancreas and sorting CD133+Sox9/EGFP+ ductal cells, which enrich PCFUs from adult mice 7, 2) single-cell manipulation of the sorted cells, 3) single-colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of colonies into single-cell suspension and re-plating into murine ECM or laminin hydrogel colony assays.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Murine ECM proteins (Matrigel)</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>354230</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Laminin Hydrogel</td>
			<td>Provided by David Tirrell (Pasadena, CA USA)</td>
			<td></td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Shinetsu Chemical (Tokyo, Japan)</td>
			<td></td>
			<td>1500 centipoise (dynamic viscosity unit equal to 15g/cm/s) (high viscosity)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Mediatech (Manassas, VA, USA)</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Flacon Conical vial</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>100mmx20mm Suspension culture dish</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>A8412</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase1</td>
			<td>Calbiochem (Darmstadt, Germany)</td>
			<td>260913</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase B</td>
			<td>Roche (CH-4070, Basel, Schweiz, Switzerland)</td>
			<td>11088831001</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/32</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>101310</td>
			<td>low endotoxin, azide free</td>
		</tr>
		<tr>
			<td>PE-Cy7 Rat IgG2a &#954; Isotype Control</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>400522</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG1 &#954; Isotype Control</td>
			<td>eBioscience (San Diego, CA, USA)</td>
			<td>13-4301-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD133-Biotin</td>
			<td>eBioscience (San Diego, CA, USA)</td>
			<td>13-1331-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD71-PE-Cy7</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>113812</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-Allophycocyanin</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>405207</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-phenylindole</td>
			<td>Invitrogen (Waltham, MA,&#160; USA)</td>
			<td>3571</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Anti-mucin 1</td>
			<td>Thermo Fisher Scientific (Waltham, MA USA)</td>
			<td>HM-1630-P1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 649 Goat anti-Armenian Hamster</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>127-495-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Mediatech(Manassas, VA, USA)</td>
			<td>10-092-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Tissure Culture Biologicals (Long Beach, CA, USA)</td>
			<td>101</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Tris Ethylenediaminetetraacetic acid</td>
			<td>TEKnova (Hollister, CA, USA)</td>
			<td>T0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy Micro Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTec Reverse Transcription Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>205310</td>
			<td></td>
		</tr>
		<tr>
			<td>CellsDirect One-Step qRT-PCR Kit</td>
			<td>Ambion/Invitrogen(Grand Island, NY, USA)</td>
			<td>11753-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Santa Cruz Bio (Santa Cruz, CA, USA)</td>
			<td>SC-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>005-000-121</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>0017-000-121</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>T-4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Invitrogen (Waltham, MA,&#160; USA)</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies (Waltham, MA, USA)</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Water</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15230-147</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Pasteur Pipette</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>13-678-8B</td>
			<td></td>
		</tr>
		<tr>
			<td>40um Filter Mesh</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um filter mesh</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 96 Well Suspension</td>
			<td>Sarstedt</td>
			<td>83.3924 (Previously 83.1835)</td>
			<td></td>
		</tr>
		<tr>
			<td>1cc Syringe</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10cc Syringe</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr>
			<td>48.48 Dyanmic Array Chip</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td>BMK-M-48.48</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluidigm GE 48.48 Dynamic Array Sample &#38; Assay Loading Reagent Kit</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td>85000800</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems (Grand Island, NY, USA)</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol sorbitan monolaurate</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dish</td>
			<td>MatTek (Ashland, MA, USA)</td>
			<td>P35G-1.5-14-C</td>
			<td>35 mm petri dish with glass bottom</td>
		</tr>
		<tr>
			<td>Mouth Piece/ Rubber Tubing</td>
			<td>Renova Life Inc. (College Park, MD, USA)</td>
			<td>MP-SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>N0636</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Vascular Endothelial Growth Factor</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>293-VE</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Activin B</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>659-AB</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Extendin 4</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>E7144</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Rspondin-1</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>3474-RS</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Falcon 5mL Polystyrene Round-Bottom Tube</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 18Gx1 1/2</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 16Gx1 1/2</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>305198</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Ultra-Low Attachment Surface 24 well flat bottom plate&#160;</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 96 Black Well Plate</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>3603</td>
			<td>Flat, clear bottom with lid.&#160; Black polystyrene TC-treated microplates</td>
		</tr>
		<tr>
			<td>Zeiss LSM510 META NLO Axiovert 200M Inverted Microscope</td>
			<td>Carl Zeiss AG (Oberkochen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biomark HD&#160;</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aria Special Order Research Product Cell Sorter</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Adult pancreatic progenitor cells can be enriched by fluorescence-activated cell sorting (Figure 1). The Sox9/EGFP transgenic mouse line used here was first generated as a result of the GENSAT Brain Atlas Project 19, and the EGFP reporter is under the control of a bacterial artificial chromosome containing ~75 kb upstream and ~150 kb downstream sequences of Sox9 20. In these mice, EGFP labels pancreatic ducts efficiently and specifically 22</sup...

Watch this Scientific Journal Video about In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas at JoVE.com

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.

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. ...

The overall goal of these protocols is to characterize the cell colonies grown from pancreatic progenitor cells isolated from adult mice using cultures assays that employ methylcellulose containing semi-solid medium. Our methods can help answer key questions in the pancreatic progenitor cell biology field, such as, what are the self renewal and differentiation capacities of progenitor cells in the adult pancreas? The main advantage of these techniques are that they allow the study of individual progenitor cells which is important for discerning their true biological potentials.We first had the idea for these methods when we realized that the colony assays were studying hematopoietic stem cells could also be applied to pancreatic cell populations. Demonstrating the procedures will be Jacob Tremblay, a graduate student, Dr.Janine Quijano, a post doc, and Miss Angela Luo, a technician, all from my laboratory. As soon as possible after sacrifice, use scissors to make a vertical incision on the midline of the abdominal wall, and open the abdominal cavity.Using forceps, gently lift the spleen, cutting the connective tissue between the spleen and the splenic lobe of the pancreas with scissors. Sever the connective tissues for the duodenal and gastric lobes in the same way, and place the pancreatic tissues in a Petri dish containing DPBS supplemented with 0.1%BSA and antibiotics on ice. Then, under a dissecting microscope, use fine tip forceps to remove the fat tissue.Now transfer the pancreata to a tissue culture hood and rinse the tissue in three sequential 100 millimeter Petri dishes containing 10 milliliters of cold PBS plus BSA each. After the final wash, place the pancreata into a sterile dry Petri dish to remove as much PBS plus BSA as possible, and transfer the tissues to another dry Petri dish on ice. Next, use spring scissors to mince the pancreata for two to three minutes until fine pieces of tissue are generated, and suspend the tissue fragments in two to three milliliters of freshly prepared cold PBS BSA DNase I solution.Using a 10 milliliter pipette, transfer the pieces to a 50 milliliter conical tube and add enough PBS BSA DNase I to bring the total volume up to 10 milliliters. Then add 350 to 450 microliters of collagenase B to the tissues. Place the tube in a 37 degree Celsius water bath for eight minutes, with swirling every three to four minutes.At the end of the incubation, use a 10 milliliter syringe with a 16 and a half gauge needle to draw up and subsequently spray the tissue solution down the wall of the 50 milliliter tube at room temperature seven times. Then return the tissues to the water bath for another eight minutes with occasional swirling, followed by seven passes through the syringe as just demonstrated. After the last mechanical disruption, transfer a 10 microliter aliquot of the cell suspension onto a dry Petri dish and confirm the presence of single cells and small sized cell clusters under an inverted phase contrast light microscope with a 10x objective lens.Next, place the tube on ice and bring the total volume up to 50 milliliters with PBS BSA DNase I.Centrifuge the cells, and use a P1000 pipette to re-suspend the pellet in five milliliters of cold PBS BSA DNase I.Then filter the cells through a 70 micron nylon mesh filter followed by a 40 micron nylon mesh filter into a new 50 milliliter polypropylene conical tube. Adjust the volume to 5 milliliters of PBS BSA DNase I, and count the cells. Then dilute the single cell suspension to a final concentration of two times 10 to the seven cells per milliliter in cold PBS BSA DNase I on ice.To whole mount immunostain pancreatic colonies grown in methylcellulose containing semi-solid medium, first prepare a glass Pasteur pipette with a fine opening using a Bunsen burner. Next, attach the small end of a one milliliter pipette tip to one end of a thin walled rubber tube and a mouthpiece to the other end. Attach the glass Pasteur pipette to the large end of a pipette tip.Then draw up 10 to 50 microliters of DMEM/F12 medium with one percent methylcellulose into the mouth pipette. Now place the dish of colonies on an inverted phase contrast microscope stage and use the 10x objective to find suitable colonies for picking. Then place the opening of the pipette next to the colony of interest, and slowly apply suction to the mouth to pick a single colony.For an effective collection, it is important to make sure the colony of interest is in focus before placing the opening of the glass pipette next to the cell cluster. Once the colony has been harvested, place the pipette tip into one well of a 96 well plate containing 4%paraformaldehyde and deposit the colony into the well. After all of the colonies have been collected, fix the colonies overnight at four degrees Celsius with gently shaking.The next morning, wash the colonies two times in PBS for 10 to 30 minutes at room temperature. From this point onward, pick up colonies using a plastic P10 pipette with a bent tip. After the second wash, store the fixed colonies overnight in 1.5 milliliter tubes filled with paraffin at four degrees Celsius.Then transfer the colonies to individual wells of a 96 well black plate with a clear bottom containing 200 microliters of blocking buffer per well. Incubate the plate overnight at four degrees Celsius with gentle shaking. The next morning, transfer the colonies to new wells containing 200 microliters of the primary antibody of interest for another overnight culture at four degrees Celsius with gentle shaking.The next morning, transfer the colonies into three consecutive wells containing 200 microliters of PBS plus 0.1%Tween 20 for 10 minute washes at room temperature. After the third wash, transfer the colonies to clean wells containing 200 microliters of the appropriate secondary antibody, and incubate the plate at room temperature for two hours in the dark. At the end of the incubation, wash the colonies three times in PBS Tween as just demonstrated, and transfer the colonies into new wells of DAPI for five minutes at room temperature.Then pool the immunostained colonies in a 35 millimeter glass bottom Petri dish and cover them with a cover slip. Finally, image the colonies on a confocal microscope. Adult pancreatic progenitor cells can be enriched by flourescence activated cell sorting using the appropriate gating parameters, as the colony forming capability is found only within CD133 Sox9/EGFP positive ductal cells.The pancreatic colonies can then be classified according to their morphologies by inverted phase contrast light microscopy. After hand picking, the colonies can also be analyzed by microfluidic qRT-PCR analysis for gene expression, or by whole mount immunostaining for protein expression. Indeed, many individual colonies express lineage markers for duct, endocrine, and acinar cells, for example, indicating that the majority of the initiating pancreatic colony forming progenitors for these colonies are tri-potent.Once mastered, the dissection, dissocation, cell staining and sorting, and cell plating into methylcellulose containing semi-solid medium procedures can be completed in six to seven hours if they are done properly. Following the colony formation, other methods like quantitative RT-PCR, western blot, or insulin secretion assays can be performed to answer additional questions about the gene expression or functional abilities of the colonies. After watching this video, you should have a good understanding of how to dissociate an adult murine pancreas and how to characterize pancreatic progenitor cell generated colonies in methylcellulose containing semi-solid cultures.

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