1Department of Cell Biology, University Medical Center Groningen, University of Groningen, 2Department of Radiation Oncology, University Medical Center Groningen, University of Groningen
Pringle, S., Nanduri, L. S. Y., Marianne, v. d. Z., Ronald, v. O., Coppes, R. P. Isolation of Mouse Salivary Gland Stem Cells. J. Vis. Exp. (48), e2484, doi:10.3791/2484 (2011).
Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sjögren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.
1. Regent Preparation
2. Mechanical and Enzymatic Tissue Digestion
3. Washing Steps
5. Plating and Medium
6. Representative Results:
After two to three days in culture, small aggregates of cells (salispheres) will be apparent in the cultures. Salispheres will continue to grow in size over a period of ten days in culture. Representative phase contrast microscopy images of salispheres are shown in Figure 1. Proliferating cells expressing stem cell-associated marker proteins can be isolated from these spheres, optimally between days 3-5 post isolation, and are capable of differentiation into functional, saliva producing acinar cells.
Figure 1. Salisphere formation in vitro. Following mechanical and enzymatic digestion using the present protocol, spheres of increasing size can be found in the floating cultures. Panels are representative phase contrast microscopy images of spheres from days 0 (A), 4 (B), 7 (C) and 10 (D). Scale bar = 50 μm.
The tissue culture method described here represents a reproducible protocol for the isolation of stem cell-containing salispheres from the salivary glands of mice. Studies using cells isolated in this manner have highlighted the regenerative capacity of salivary gland stem cells 9. Transplantation of one hundred of c-Kit+ cells derived from the salispheres induced functional recovery of irradiated mouse salivary glands. These data are exciting and provide a starting point for the investigation of stem-cell based therapy for xerostomia. Many avenues remain to be explored however, including the full marker protein expression profile of the stem cells, the ability of submandibular glands to rescue the function of irradiated parotid salivary glands and vice versa, and the characterisation of the putative in vivo stem cell niche of the cells. Ultimately, the translation of this protocol to human tissue samples and the subsequent potential for the therapy of xerostomia in human patients using the isolated cells is the most exciting application of the described method.
No conflicts of interest declared.
|Hyaluronidase||Sigma-Aldrich||H3506||Store at €“ 20 °C|
|Collagenase II||GIBCO, by Life Technologies||17101-015||Store at 4 °C|
|Epidermal Growth Factor-2||Sigma-Aldrich||E9644||Make a stock of 10 μg / mL in phosphate buffered saline (PBS). Store at €“ 20 °C in single use aliquots.|
|Fibroblast Growth Factor-2||Sigma-Aldrich||F0291||Make stock of 25 μg / mL in PBS. Store at €“ 20 °C in single use aliquots.|
|N2 supplement||GIBCO, by Life Technologies||17502-048||As manufacturer instructions.|
|Insulin||Sigma-Aldrich||I6634||Make stock of 2 mg / mL in tap water. Adjust water to pH 2-3 using glacial acetic acid prior to dissolving.|
|100 µM pore-size sterile cell strainers||BD Biosciences||352360|
|Polystyrene round-bottomed tubes with cell strainer caps (50 µM pore size)||BD Biosciences||352235|