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Developmental Biology

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

Published: June 10th, 2016

DOI:

10.3791/54016

1Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope, 2Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 3Division of Chemistry and Chemical Engineering, California Institute of Technology
* These authors contributed equally

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

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

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 ....

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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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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

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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 .......

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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....

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Name Company Catalog Number Comments
Murine ECM proteins (Matrigel) Becton Dickson (Franklin Lakes, NJ, USA) 354230 Stock kept at -20oC
Laminin Hydrogel Provided by David Tirrell (Pasadena, CA USA) Stock kept at -20oC
Methylcellulose Shinetsu Chemical (Tokyo, Japan) 1500 centipoise (dynamic viscosity unit equal to 15g/cm/s) (high viscosity)
Dulbecco's Phosphate-Buffered Saline Mediatech (Manassas, VA, USA) 21-031-CV
Phosphate Buffered Saline Gibco (Grand Island, NY, USA) 15070-063
50mL Flacon Conical vial Corning Inc. (Corning, NY, USA) 352070
100mmx20mm Suspension culture dish Corning Inc. (Corning, NY, USA) 430591
Bovine Serum Albumin Sigma (St. Louis, MO, USA) A8412
Penicillin/Streptomycin Gibco (Grand Island, NY, USA) 15070-063
DNase1 Calbiochem (Darmstadt, Germany) 260913
Collagenase B Roche (CH-4070, Basel, Schweiz, Switzerland) 11088831001 Stock kept at -20oC
Anti-mouse CD16/32 Biolegend (San Diego, CA, USA) 101310 low endotoxin, azide free
PE-Cy7 Rat IgG2a κ Isotype Control Biolegend (San Diego, CA, USA) 400522
Rat IgG1 κ Isotype Control eBioscience (San Diego, CA, USA) 13-4301-82
Anti-CD133-Biotin eBioscience (San Diego, CA, USA) 13-1331-82
Anti-CD71-PE-Cy7 Biolegend (San Diego, CA, USA) 113812
Streptavidin-Allophycocyanin Biolegend (San Diego, CA, USA) 405207
4',6-Diamidino-2-phenylindole Invitrogen (Waltham, MA,  USA) 3571 Stock kept at -20oC
Anti-mucin 1 Thermo Fisher Scientific (Waltham, MA USA) HM-1630-P1
Dylight 649 Goat anti-Armenian Hamster Jackson Immuno (West Grove, PA, USA) 127-495-160
Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 Mediatech(Manassas, VA, USA) 10-092-CV
Fetal Bovine Serum Tissure Culture Biologicals (Long Beach, CA, USA) 101 Stock kept at -20oC
Tris Ethylenediaminetetraacetic acid TEKnova (Hollister, CA, USA) T0221
Rneasy Micro Kit Qiagen (Venlo, Netherlands) 74004
QuantiTec Reverse Transcription Kit Qiagen (Venlo, Netherlands) 205310
CellsDirect One-Step qRT-PCR Kit Ambion/Invitrogen(Grand Island, NY, USA) 11753-100
Paraformaldehyde Santa Cruz Bio (Santa Cruz, CA, USA) SC-281692
Goat serum Jackson Immuno (West Grove, PA, USA) 005-000-121 Stock kept at -20oC
Donkey serum Jackson Immuno (West Grove, PA, USA) 0017-000-121 Stock kept at -20oC
Triton X-100 Sigma (St. Louis, MO, USA) T9284
Trypsin Sigma (St. Louis, MO, USA) T-4799
Ethylenediaminetetraacetic acid Invitrogen (Waltham, MA,  USA) 15575-020
Trypsin-EDTA Life Technologies (Waltham, MA, USA) 25200-056
Sterile Water Gibco (Grand Island, NY, USA) 15230-147 Molecular biology grade
Pasteur Pipette Fisher Scientific (Pittsburgh, PA , USA) 13-678-8B
40um Filter Mesh Fisher Scientific (Pittsburgh, PA , USA) 08-771-1
70 um filter mesh Fisher Scientific (Pittsburgh, PA , USA) 08-771-2
TC Plate 96 Well Suspension Sarstedt 83.3924 (Previously 83.1835)
1cc Syringe Becton Dickson (Franklin Lakes, NJ, USA) 309659
10cc Syringe Becton Dickson (Franklin Lakes, NJ, USA) 301604
48.48 Dyanmic Array Chip Fluidigm (San Francisco, CA, USA) BMK-M-48.48
Fluidigm GE 48.48 Dynamic Array Sample & Assay Loading Reagent Kit Fluidigm (San Francisco, CA, USA) 85000800
TaqMan Universal PCR Master Mix Applied Biosystems (Grand Island, NY, USA) 4304437
Polyethylene glycol sorbitan monolaurate Sigma (St. Louis, MO, USA) P7949
Glass Bottom Dish MatTek (Ashland, MA, USA) P35G-1.5-14-C 35 mm petri dish with glass bottom
Mouth Piece/ Rubber Tubing Renova Life Inc. (College Park, MD, USA) MP-SET
Nicotinamide Sigma (St. Louis, MO, USA) N0636 Stock kept at -20oC
Vascular Endothelial Growth Factor R&D Systems (Minneapolis, MN, USA) 293-VE Stock kept at -80oC
Activin B R&D Systems (Minneapolis, MN, USA) 659-AB Stock kept at -80oC
Extendin 4 Sigma (St. Louis, MO, USA) E7144 Stock kept at -20oC
Rspondin-1 R&D Systems (Minneapolis, MN, USA) 3474-RS Stock kept at -80oC
Falcon 5mL Polystyrene Round-Bottom Tube Corning Inc. (Corning, NY, USA) 352054
PrecisionGlide Needle 18Gx1 1/2 Becton Dickson (Franklin Lakes, NJ, USA) 305196
PrecisionGlide Needle 16Gx1 1/2 Becton Dickson (Franklin Lakes, NJ, USA) 305198
Costar Ultra-Low Attachment Surface 24 well flat bottom plate  Corning Inc. (Corning, NY, USA) 3473
Costar 96 Black Well Plate Corning Inc. (Corning, NY, USA) 3603 Flat, clear bottom with lid.  Black polystyrene TC-treated microplates
Zeiss LSM510 META NLO Axiovert 200M Inverted Microscope Carl Zeiss AG (Oberkochen, Germany)
Biomark HD  Fluidigm (San Francisco, CA, USA)
Aria Special Order Research Product Cell Sorter Becton Dickson (Franklin Lakes, NJ, USA)

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