A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

Abstract

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 μL of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin βB, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level. Video LinkThe video component of this article can be found at https://www.jove.com/video/3148/ Protocol Differentiation of murine ES cells to pancreatic-like cells in vitro to generate conditioned media (CM) for colony assayProcedures for murine ES...

Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel

“…A cell capable of forming a colony is termed a "colony-forming unit" (CFU). To test whether the adult pancreas contains CFUs, we replaced hematopoietic growth factors with factors that we thought would be helpful for growing pancreatic cells, including various growth factors (18)(19)(20)(21) and ECM proteins, such as the commercially available Matrigel or an artificial ECM protein (22) containing an α1 laminin sequence (termed "laminin hydrogel").…”

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

“…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 .…”

“…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 2.…”

“…For troubleshooting, ensure that cells from dissociated primary colonies are kept in RT before plating into secondary cultures. For culturing in murine ECM proteins, add the cells last to the tube containing cold culture medium before mixing 18 , thereby minimizing the exposure of the cells to cold temperature, which may kill the dissociated cells obtained from primary colonies. However, the re-plating of cells into laminin hydrogel does not need to be performed on ice before 37 °C incubation (see step 7).…”

“…Therefore, it will be important to verify progenitor cells in follow-up studies using in vivo assays. Second, the culture components contain undefined reagents, such as murine ECM proteins, FCS, and conditioned media 18 . These undefined components may affect the function of specific growth factors on the progenitor cells indirectly and more work is needed to create a fully defined set of culture conditions.…”