Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers

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Abstract

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile.

Highlights

► Novel monoclonal antibodies marking murine pancreatic cell types are described. ► Live cell isolation of alpha, beta and exocrine cells by FACS is demonstrated. ► DGKB, GPM6A, TTR, DPPIV show alpha or beta cell selective expression. ► Labeling changes from E14.5−E18.5 show early or late developmental marking.

Introduction

The development of strategies for the de novo generation of beta cells, the enhancement of in situ beta cell proliferation, and/or the reprogramming of other adult cells to serve as beta surrogates requires a sufficient understanding of the regulation of beta cell identity. Experimental tools that allow the convenient isolation of specific endocrine cell subsets from mice – whether wild type or compound transgenics – and the selective comparison of their gene expression profile to that of other defined cell types are in short supply. A transcriptional profile of beta cells from different genetic environments, for comparison with candidate progenitors and reference populations of mature pancreatic cells, will be particularly useful.

Markers of adult pancreatic cell types have been comprehensively identified, but detection of the expression of these genes nearly always requires cell fixation. In certain cases, however, the consistent physical properties of the cell type(s) have been used to facilitate viable isolation. For beta cells, a naturally high intracellular zinc ion concentration has been exploited using the low-toxicity membrane permeable fluorescent dye Newport Green (NG) in combination with orthogonal scatter gating (Lukowiak et al., 2001). Enrichment of insulin expression among NG+ progeny of differentiated embryonic stem (ES) cells has also been observed (Narushima et al., 2005). In addition, certain transgenic mice incorporating marker genes driven by promoters associated with known cell types have proven quite useful. Mouse insulin promoter-green fluorescence protein (MIP-GFP) transgenic animals (Hara et al., 2003) have aided the identification and isolation of pancreatic islets and beta cells, and the more recently derived GluCre-ROSA26EYFP mice (Quoix et al., 2007) may facilitate the convenient isolation of alpha cells. However, a comprehensive collection of transgenic animals with pancreatic cell lineage-restricted marker gene expression has not yet been assembled – and the costs of maintaining or back-crossing additional mouse lines are significant.

Viable cell isolation by antibody labeling has been instrumental in the characterization of functional cell subsets of hematopoietic, neural, and other cell types (Lawson et al., 2007, Maric and Barker, 2004, Swart et al., 2005). Excluding the well-studied hematopoietic field, however, the introduction of new cell lineage markers has been disappointingly elusive. Recently, we reported the development of a collection of antibodies marking human endocrine and exocrine pancreatic cell populations (Dorrell et al., 2008b). Although these have proven useful for the isolation and study of important human cell types, these reagents do not work on mouse cells.

In this report we describe the development and application of novel tools for the study of murine pancreatic biology. These antibodies allow the isolation of duct and acinar cells (and subsets thereof) from “bulk” pancreatic tissue. When applied to mouse islet samples, alpha and beta cells can be selectively marked and purified. Expression analysis of these populations reveals striking differences between alpha and beta cells including the alpha cell specificity of transthyretin (TTR) and dipeptidyl peptidase 4 (DPPIV) and the selective expression of diacylglycerol kinase beta (DGKB) and glycoprotein M6A (GPM6A) in beta cells. The ability to conveniently isolate viable exocrine and endocrine populations should facilitate the study of these important cell populations.

Section snippets

Tissue sources and pancreatic cell isolation

Animal care and immunization procedures were performed in accordance with the institutional review committee at Oregon Health & Science University. BALB/cBy, 129/S3, and NOD.Cg-Prkdcscid Tg(Ins1-EGFP/GH1)14Hara/Sz (“MIP-GFP”) mice were obtained from the Jackson Laboratory. F344 rats were acquired from Charles River Laboratories. Adult pancreatic tissue was collected from c129/S3 mice aged 2–4 months. For the optimal preparation of a single cell suspension of whole pancreas tissue, a modified

Generation of monoclonal antibodies by negative selection

To favor the production of antibodies with selective reactivity against cells of interest, a subtractive immunization strategy was employed (Williams et al., 1992). A F344 rat was pre-immunized with murine hematopoietic cells and FCS followed by cyclophosphamide treatment to ablate reactive lymphoid cells. Subsequent immunizations of this rat and a negative selection-free control animal used enriched preparations of murine islet cells (500 islets per animal per immunization). The resulting

Discussion

The investigation of pancreatic endocrinology and stem cell biology has not been adequately matched by reagents and tools from the field of mouse genetics. Transgenic animals with useful marker properties (e.g., MIP-GFP (Hara et al., 2003)) have proven useful, but investigators of pancreatic endocrine and exocrine biology still lack a comprehensive collection of transgenic animals with useful cell-lineage restricted marker expression. In this report we describe the development and

Duality of interest

OHSU has commercially licensed part of the technology disclosed herein (MPdi1); authors C.D., P.R.S. and M.G. are inventors of this reagent. This potential conflict of interest is reviewed and managed by OHSU. The other authors disclose no conflicts.

Acknowledgements

We gratefully acknowledge the hard work of Jean Leif, Michael Bates, and Elaine Norowski who prepared the mouse islet samples used in this study. The advice of Dr. Soren Impey regarding qRT-PCR primer design was also very useful.

This work was supported by the Beta Cell Biology Consortium with grant U01 DK072477.

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