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...

<ol>
	<li>Gu, G., Brown, J. R., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+lineage+tracing+reveals+the+ontogeny+of+pancreatic+cell+fates+during+mouse+embryogenesis.">Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis.</a> Mech Dev. 120, 35-43 (2003).</li><li>Kopp, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox9++ductal+cells+are+multipotent+progenitors+throughout+development+but+do+not+produce+new+endocrine+cells+in+the+normal+or+injured+adult+pancreas.">Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.</a> Development. 138, 653-665 (2011).</li><li>DiGiusto, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-based+gene+therapy+for+HIV+with+lentiviral+vector-modified+CD34(+)+cells+in+patients+undergoing+transplantation+for+AIDS-related+lymphoma.">RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma.</a> Science translational medicine. 2, 36-43 (2010).</li><li>Inada, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbonic+anhydrase+II-positive+pancreatic+cells+are+progenitors+for+both+endocrine+and+exocrine+pancreas+after+birth.">Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.</a> Proc Natl Acad Sci U S A. 105, 19915-19919 (2008).</li><li>Solar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+exocrine+duct+cells+give+rise+to+insulin-producing+beta+cells+during+embryogenesis+but+not+after+birth.">Pancreatic exocrine duct cells give rise to insulin-producing beta cells during embryogenesis but not after birth.</a> Dev Cell. 17, 849-860 (2009).</li><li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+pancreatic+progenitor+cells--recent+studies.">Minireview: pancreatic progenitor cells--recent studies.</a> Endocrinology. 149, 4312-4316 (2008).</li><li>Jin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony-forming+cells+in+the+adult+mouse+pancreas+are+expandable+in+Matrigel+and+form+endocrine/acinar+colonies+in+laminin+hydrogel.">Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.</a> Proc Natl Acad Sci U S A. 110, 3907-3912 (2013).</li><li>Fu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-26a+targets+ten+eleven+translocation+enzymes+and+is+regulated+during+pancreatic+cell+differentiation.">MicroRNA-26a targets ten eleven translocation enzymes and is regulated during pancreatic cell differentiation.</a> Proc Natl Acad Sci U S A. 110, 17892-17897 (2013).</li><li>Ghazalli, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+Pancreas+of+Mice+Contains+Tripotent+Progenitors+Capable+of+Giving+Rise+to+Duct,+Acinar,+and+Endocrine+Cells+In+Vitro.">Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro.</a> Stem Cells Dev. , (2015).</li><li>Jin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony-forming+progenitor+cells+in+the+postnatal+mouse+liver+and+pancreas+give+rise+to+morphologically+distinct+insulin-expressing+colonies+in+3D+cultures.">Colony-forming progenitor cells in the postnatal mouse liver and pancreas give rise to morphologically distinct insulin-expressing colonies in 3D cultures.</a> Rev Diabet Stud. 11, 35-50 (2014).</li><li>Jin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Multilineage+Differentiation+and+Self-Renewal+of+Single+Pancreatic+Colony-Forming+Cells+from+Adult+C57Bl/6+Mice.">In Vitro Multilineage Differentiation and Self-Renewal of Single Pancreatic Colony-Forming Cells from Adult C57Bl/6 Mice.</a> Stem Cells Dev. , (2014).</li><li>Huch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5/R-spondin+axis.">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> Embo J. 32, 2708-2721 (2013).</li><li>Greggio, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+and+conversion+of+human+pancreatic+ductal+cells+into+insulin-secreting+endocrine+cells.">Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.</a> Elife. 2, e00940 (2013).</li><li>Dorrell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organoid-initiating+cells+in+mouse+pancreas+and+liver+are+phenotypically+and+functionally+similar.">The organoid-initiating cells in mouse pancreas and liver are phenotypically and functionally similar.</a> Stem Cell Res. 13, 275-283 (2014).</li><li>Ku, H., Yonemura, Y., Kaushansky, K., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombopoietin,+the+ligand+for+the+Mpl+receptor,+synergizes+with+steel+factor+and+other+early+acting+cytokines+in+supporting+proliferation+of+primitive+hematopoietic+progenitors+of+mice.">Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice.</a> Blood. 87, 4544-4551 (1996).</li><li>Ku, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-expressing+colonies+developed+from+murine+embryonic+stem+cell-derived+progenitors.">Insulin-expressing colonies developed from murine embryonic stem cell-derived progenitors.</a> Diabetes. 56, 921-929 (2007).</li><li>Winkler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+insulin-expressing+colony-forming+progenitors.">A quantitative assay for insulin-expressing colony-forming progenitors.</a> J Vis Exp. , e3148 (2011).</li><li>Gong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425, 917-925 (2003).</li><li>Formeister, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+SOX9+levels+differentially+mark+stem/progenitor+populations+and+enteroendocrine+cells+of+the+small+intestine+epithelium.">Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium.</a> Am J Physiol Gastrointest Liver Physiol. 296, G1108-G1118 (2009).</li><li>Seymour, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dosage-dependent+requirement+for+Sox9+in+pancreatic+endocrine+cell+formation.">A dosage-dependent requirement for Sox9 in pancreatic endocrine cell formation.</a> Dev Biol. 323, 19-30 (2008).</li><li>Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+proliferation+of+hematopoietic+stem+cells.">Differentiation and proliferation of hematopoietic stem cells.</a> Blood. 81, 2844-2853 (1993).</li><li>Suzuki, A., Nakauchi, H., Taniguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+isolation+of+multipotent+pancreatic+progenitors+using+flow-cytometric+cell+sorting.">Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting.</a> Diabetes. 53, 2143-2152 (2004).</li><li>Seaberg, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nat Biotechnol. 22, 1115-1124 (2004).</li><li>Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+reprogramming+of+adult+pancreatic+exocrine+cells+to+beta-cells.">In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.</a> Nature. 455, 627-632 (2008).</li><li>Baeyens, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+cytokine+treatment+induces+acinar+cell+reprogramming+and+regenerates+functional+beta+cell+mass+in+diabetic+mice.">Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.</a> Nat Biotechnol. 32, 76-83 (2014).</li><li>Thorel, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+adult+pancreatic+alpha-cells+to+beta-cells+after+extreme+beta-cell+loss.">Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.</a> Nature. 464, 1149-1154 (2010).</li></ol>

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. ...

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...