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

Tremblay, Jacob R. and LeBon, Jeanne M. and Luo, Angela and Quijano, Janine C. and Wedeken, Lena and Jou, Kevin and Riggs, Arthur D. and Tirrell, David A. and Ku, H. Teresa (2016) In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas. Journal of Visualized Experiments (112). ISSN 1940-087X . http://resolver.caltech.edu/CaltechAUTHORS:20160705-152413540

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Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.3791/54016DOIArticle
https://www.jove.com/video/54016/in-vitro-colony-assays-for-characterizing-tri-potent-progenitor-cellsPublisherArticle
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2016 JoVE. Date published 6/10/2016, Issue 112. 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. The authors have nothing to disclose.
Funders:
Funding AgencyGrant Number
NIHR01DK081587
NIHR01DK099734
NIHU01DK089533
NSFDMR-1206121
California Institute for Regenerative Medicine (CIRM)RB5-07398
Joseph J. Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Oxnard FoundationUNSPECIFIED
Ella Fitzgerald FoundationUNSPECIFIED
National Cancer InstituteP30CA33572
Subject Keywords:Developmental Biology, Issue 112, pancreatic colony-forming units, progenitor cells, fluorescence-activated cell sorting (FACS), single colony analyses, single cell analyses, extracellular matrix proteins, laminin hydrogel, methylcellulose, semi-solid medium, whole-mount immunostaining, microfluidic PCR, adult mice
Record Number:CaltechAUTHORS:20160705-152413540
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160705-152413540
Official Citation:Tremblay, J. R., LeBon, J. M., Luo, A., Quijano, J. C., Wedeken, L., Jou, K., et al. In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas. J. Vis. Exp. (112), e54016, doi:10.3791/54016 (2016).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:68838
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:07 Jul 2016 14:40
Last Modified:01 Sep 2016 17:39

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