Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.
The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.
Energy homeostasis and food digestion in mammals depend on proper pancreatic function. The adult pancreas is comprised of two main cellular compartments: the exocrine and the endocrine. Exocrine cells, including the acinar cells that produce and secrete digestive enzymes and the duct cells that transport these enzymes to the gut, encompass more than 80% of the total pancreatic mass1. Endocrine cells, which include insulin-producing beta cells and glucagon-producing alpha cells, are organized in the islets of Langerhans that are embedded in the exocrine tissue and secrete hormones to regulate blood glucose levels2.
Pancreatic cells acquire their differentiated fate through a highly-regulated, multistep process3. Evidence suggests that extrinsic cues provided by neuronal, endothelial, and mesenchymal cells guide pancreatic cell differentiation and proliferation in the embryo3,4,5. One example is the requirement of the aorta for the specification of early pancreatic precursors6. Later in development, endothelial cells were shown to play a central role in the development of both pancreatic endocrine and exocrine cells and to promote beta-cell differentiation4,6,7. Mesenchymal cells were shown to support the survival and expansion of common pancreatic progenitors, mainly through the secretion of the growth factor Fgf108,9. We have further shown that these cells support the proliferation of endocrine and exocrine precursors, as well as of differentiated cells (including acinar and beta cells) in the embryonic pancreas5. Recently, mesenchymal cells were further shown to regulate endocrine cells differentiation10.
In the adult, beta-cell function and mass were shown to depend on cells in their microenvironment, including neuronal, immune, and endothelial cells, as well as pericytes11,12,13. During injury, endothelial cells were shown to recruit immune cells to the pancreas to promote beta-cell replication13. Endothelial cells were further shown to produce extracellular matrix (ECM) components to support insulin expression and beta-cell function14. We recently demonstrated the requirement of islet pericytes for beta-cell function11. Lastly, cells of the pancreatic stroma were shown to regulate the progression of pancreatic ductal adenocarcinoma (PDAC)15,16. However, the identity of extrinsic cues that guide pancreas development, function, and tumorigenesis are largely unknown.
Identifying cues provided by cells of the pancreas microenvironment requires characterizing the genes and proteins expressed by these cells. This depends on isolating these cells from the pancreas in order to perform gene expression and proteomic analyses and/or on establishing cell lines. Here, we propose a method to isolate mesenchymal cells of the pancreas microenvironment by utilizing tissue enzymatic digestion and fluorescence-activated cell sorting (FACS) of either immunofluorescently-labeled cells or cells expressing fluorescent proteins. This protocol was successfully performed to isolate and analyze yellow fluorescent protein (YFP)-expressing mesenchymal cells of the embryonic, neonatal, and adult pancreas5,17.
Here, we describe a method to isolate and analyze cells of the pancreatic microenvironment. This method can be used to isolate mesenchymal cells from embryonic and adult pancreatic tissue. In addition, we successfully used this protocol to isolate endothelial cells from the adult and neonatal pancreas5,17. However, it may not be suitable for obtaining a reproducible single-cell suspension of pancreatic epithelial cells (alternative protocols are described in References 18, 23, and 24). Using this method, fluorescently-labeled cells, either expressing fluorescent proteins or immunostained for surface markers, can be purified by FACS or analyzed by flow cytometry. RNA can be extracted from purified cells to profile their gene expression pattern. Alternatively, purified cells can be cultured to establish a cell line for subsequent proteomic analysis. This method will enable the characterization of factors expressed by the pancreas microenvironment, which govern its organogenesis, physiology, and pathophysiology.
The pancreatic mesenchyme supports tissue organogenesis by promoting the proliferation of precursors and differentiated cells5,9. These cells were shown to support the expansion of human embryonic stem cell (hESC)-derived pancreatic progenitors17,25,26. Therefore, delineating the identity of embryonic mesenchymal factors would facilitate current efforts to generate insulin-producing beta cells from hESCs and induced pluripotent stem cells (iPSCs) as a potential cure to diabetes. Mouse genetic studies allowed the identification of growth factors, such as Fgf10, that are produced by the mesenchyme to promote pancreatic epithelium expansion during the early stages of pancreas development3,9. With the aim of identifying additional factors expressed in the embryonic mesenchyme, we isolated these cells using laser-captured microdissection, extracted their RNA, and performed gene expression analysis26. However, in addition to being labor-intense, this method relies on identifying cells based on their morphological features, which restricts its use to developmental stages prior to the branching of the epithelium into the surrounding mesenchyme (i.e., e12.5). To characterize mesenchymal cells at later developmental stages, we employed the method described here5,17.
We used this method to analyze surface marker expression by neonatal pancreatic mesenchyme5. In addition, mesenchymal cells were isolated from embryonic and neonatal pancreatic tissue of Nkx3.2-Cre;R26-EYFP mice, based on their fluorescent labeling in this mouse line, and were cultured to establish cell lines17. The proteomic analysis of these cells allowed for the identification of factors secreted by the pancreatic mesenchyme with the ability to promote hESC-derived pancreatic progenitors17. We further used this cell isolation method to purify mesenchymal cells from adult pancreatic tissues for RNA extraction and gene expression analysis17. Therefore, this method can be used to identify genes and proteins expressed by the pancreatic mesenchyme, with the ability to support pancreatic cell development.
Pancreatic mesenchymal cells were further shown to play a role in pancreas tumorigenesis. PDAC is characterized by the formation of a fibroblast-rich desmoplastic stroma comprised of fibroblasts, immune cells, and ECM27. While the stroma was thought to promote the development of many types of cancer, it was shown to restrain PDAC progression15,16,28. This suggests that components of the pancreatic stroma secrete factors that inhibit tumorigenesis. Furthermore, changes in stroma cellular composition as well as in cell phenotype can underlie their effect on epithelial cells15,16,28. The method described here can therefore assist in characterizing the different cell types that make up a PDAC stroma as compared to healthy pancreatic tissue. It would further allow the purification of the different stromal cell types to characterize potential changes in their gene expression profiles during PDAC progression. However, due to changes in pancreatic ECM composition during tumorigenesis27, adjustments of the tissue digestion parameters, such as the inclusion of additional collagenase types or increasing the incubation time, may be required.
The authors have nothing to disclose.
The authors thank Adi Sasson for the technical assistance and Helen Guez for the critical reading of the manuscript. This work was supported by European Research Council starting grant no. 336204.
Collagenase P | Roche | 11213865001 | |
DNase | Sigma-Aldrich | D5025-15KU | The effective units should be at least 2000 unitz/ml protein |
Hanks’ Balanced Salt solution (HBSS) | Sigma-Aldrich | H6648 | |
Fetal Bovine Serum (FBS) | Biological Industries | 04-127-1A | |
EDTA | Biological Industries | 01-862-1A | |
Sodium Azide | Sigma-Aldrich | S2002 | |
Goat IgG serum | Sigma-Aldrich | G9023 | |
DAPI | Sigma-Aldrich | D9542 | |
RNAse inhibitor | Invitrogen | N8080119 | |
Dulbecco's Modified Eagle Medium (DMEM) | Invitrogen | 11965092 | |
L-Glutamine | Biological Industries | 03-020-1B | |
Penicillin-streptomycin | Biological Industries | 03-031-1B | |
Dulbecco’s Phosphate Buffered Saline (PBS) | Sigma-Aldrich | D8537 | Without Calcium Chloride and Magnesium Chloride |
1.5 ml tubes | Sarstedt | 72 690 | |
15 ml conical tube | Corning | 430052 | |
Round Bottom Polystyrene 5 ml tube | Corning | 352008 | FACS tube'. Make sure tube is compatible with the flow cytomter to be used, as there are slight differences in required tubes between brands |
5ml tube with 35 μm cell strainer Snap Cap | Corning | 352235 | FACS tube' |
70 μm cell strainer | Miltenyi Biotec | 130-098-462 | |
Heating block with agitation | Eppendorf | ThermoMixer C | |
Centrifuge | ThermoFisher | Heraeus Megafuge 40R | |
Steromicroscope | Nikon | SMZ 745 | |
Cell sorter | BD Biosciences | FACSAria IIu | |
flow cytometer | Beckman Coulter | Gallios |