Isolation of Mouse Salivary Gland Stem Cells

Biology
 

Summary

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

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Pringle, S., Nanduri, L. S., Marianne, v. d., Ronald, v. O., Coppes, R. P. Isolation of Mouse Salivary Gland Stem Cells. J. Vis. Exp. (48), e2484, doi:10.3791/2484 (2011).

Abstract

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.

Protocol

1. Regent Preparation

  1. Buffer: 1 % (w/v) bovine serum albumin in Hank's balanced salt solution
  2. Reconstitute enzymes. Hyaluronidase enzyme: 40mg / mL, dissolved in buffer. Collagenase II: 23mg / mL, dissolved in buffer. Use freshly prepared enzyme solutions fresh for each isolation. When dissolved, store at 4 °C until use for digestion.
  3. 50 mM calcium chloride in distilled water. Filter sterilize through a 0.2 uM pore size filter.
  4. Mouse salivary gland (MSG) culture medium: DMEM:F12 with penicillin (100 I.U. / mL), streptomycin (100 μg / mL), glutamax (2 mM), epidermal growth factor-2 (20 ng / mL), fibroblast growth factor-2 (20 ng / mL), N2 supplement (1 %), insulin (10 μg / mL) and dexamethasone (1 μM).

2. Mechanical and Enzymatic Tissue Digestion

  1. Weigh the dissected salivary glands.
  2. Chop glands into a homogenous pulp using sterile curved dissection scissors in a small petri dish.
  3. Collect minced tissue in 14 mL tubes, using 1mL of buffer per 80mg submandibular tissue. Rinse the petri dishes clean of tissue using some of the buffer.
  4. Add another 1 mL of buffer per 80 mg tissue, followed by 25 μL collagenase II enzyme solution, 25 μL hyaluronidase enzyme solution and 250 μL calcium chloride solution per 80 mg tissue. If working with large amounts of tissue, steps 2.4 - 2.9 can be performed in T25 tissue culture flasks for convenience.
  5. Incubate in a shaking waterbath set at 37 °C for 20 minutes. Remove tubes and triturate by pipette to mix enzyme thoroughly through tissue again.
  6. Replace in waterbath for another 20 mins.
  7. Collect tissue by centrifugation at 400 x g, for 8 minutes. Discard supernatant.
  8. Resuspend in 2 mL buffer for each 80 mg tissue, and repeat enzyme and calcium chloride addition as above. Incubate 20 minutes in shaking water bath. Remove tubes and triturate by pipette to mix enzymes thoroughly.
  9. Incubate for final 20 min in shaking water bath. Collect cells by centrifugation as above, discard supernatant.

3. Washing Steps

  1. Resuspend each 80 mg of tissue in 2 mL buffer and pipette to wash tissue free of enzymes.
  2. Centrifuge as previously to collect. Discard supernatant.
  3. Repeat wash using 1 mL buffer per 80 mg tissue. Centrifuge to collect, discard supernatant.

4. Filtering

  1. Resuspend tissue solution in 1 mL buffer per 80 mg tissue.
  2. Add solution to 100 μm pore-size filter placed over 50 mL Falcon tube. Do not apply more than 3 mLs of minced tissue solution per column, as filters may become blocked. Allow to seep through. Remove filtered material hanging on underside of filter by pipette, and add to filtrate.
  3. Use syringe with 26 gauge needle to take filtrate from 50mL tubes and apply to 50 μm pore size filters on 5 mL tubes. Allow to filter through, assisting by loosening lids if necessary.
  4. Centrifuge tubes as previously to collect. Discard supernatant.

5. Plating and Medium

  1. Combine all pellets into one volume. Count using automated cell counter or haemocytometer.
  2. Plate cells at density of 0.4 x 106 cells per well of 12-well plate, or 2.67 x 106 cells per T25 tissue culture flask. Add 1 mL MSG medium to each well or 6 mL to each T25 flask.
  3. Incubate at 37 °C. Spheres should be clearly visible by day 2.

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
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.

Discussion

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.

Disclosures

No conflicts of interest declared.

Materials

Name Company Catalog Number Comments
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.
Dexamethasone Sigma-Aldrich D4902
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

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References

  1. Vissink, A. Oral Sequelae of Head and Neck Radiotherapy. Crit Rev Oral Biol Med. 14, 199-212 (2003).
  2. Napeñ, as, J, J., Brennan, M. T., Fox, P. C. Diagnosis and treatment of xerostomia (dry mouth). Odontology. 97, 76-83 (2009).
  3. Denny, P. C. Parenchymal cell proliferation and mechanisms for maintenance of granular duct and acinar cell populations in adult male mouse submandibular gland. 235, 475-485 (2005).
  4. Man, Y. G. Persistence of a perinatal cellular phenotype in submandibular glands of adult rat. J. Histochem. Cytochem. 43, 1203-1215 (1995).
  5. Cotroneo, E., Proctor, G. B., Carpenter, G. H. Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation. Differentiation. 79, 120-130 (2010).
  6. Eirew, P. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nat Med. 14, 1384-1389 (2008).
  7. Gorjup, E. Glandular tissue from human pancreas and salivary gland yields similar stem cell populations. European Journal of Cell Biology. 88, 409-421 (2009).
  8. Alonso, L., Fuchs, E. Stem cells of the skin epithelium. Proc Natl Acad Sci U S A. 100, Suppl 1. 11830-11835 (2003).
  9. Lombaert, I. M. Rescue of salivary gland function after stem cell transplantation in irradiated glands. Plos One. 3, e2063-e2063 (2008).
  10. Coppes, R. P., Goot, A. vander, Lombaert, I. M. A. Stem Cell Therapy to Reduce Radiation-Induced Normal Tissue Damage. Seminars in Radiation Oncology. 19, 112-121 (2009).

Comments

8 Comments

  1. This is an urgent need for patients who had cancer radiation therapy.
    When are clinical trials on humans expected to commence?

    Reply
    Posted by: Anonymous
    April 25, 2011 - 8:43 AM
  2. We are working at the moment on the translation of our stem cell isolation technique to human salivary gland tissue, and are characterising the stem cell-like attributes of these isolated cells. We hope to achieve translation of the technique to the clinical setting for use for therapy of xerostomia in human patients by ²017.

    Reply
    Posted by: Sarah P.
    April 26, 2011 - 7:46 AM
  3. Why not earlier?

    Reply
    Posted by: Anonymous
    May 25, 2011 - 7:38 AM
  4. Mrs Pringle,How do you think ,Is it possible to be created or regenerated salivary glands if they were destroyed.
    I have atrophyc rhinopharyngitis.My salivary glands in the mouth are working ,but these on my throat were destroyed after incorect treatment over them and as a result of this
    I have lost my salivary glands in this area.Due to this I have a lot of health problems, and my hope is the scientists to discover a way of transplantation of salivary glands,using these glands ,that still are working.
    How do you think ,Is it possible?
    Thank you!

    Reply
    Posted by: Anonymous
    October 24, 2011 - 10:43 AM
  5. Dear Jana,

    Sorry to hear about your problems.
    We are focusing our research at the moment on providing at therapy for xerostomia in the major salivary glands of humans. This potential therapy is looking very promising. We work with these glands because they are the ones most commonly affected by radiation treatment. They are also large and easy to locate and work with.

    As far as we are aware, no-one is trying to develop a therapy for the minor salivary glands. This is mostly due to their high number and very small size - there are over 600 of them scattered throughout the oral cavity and throat. This would make a cell transplantation approach very challenging.

    So although I would love to be able to tell you that a stem cell-based therapy for xerostomia resulting from dysfunction of the minor salivary glands is in process, I think this is unlikely, I would not like to leave you with unrealistically high hopes!

    Regards,

    Sarah.

    Reply
    Posted by: Sarah P.
    October 31, 2011 - 4:31 AM
  6. Dear Sarah,
    thank you very much for your replay!

    The news are bad for me and maybe I couldn't see my small children grown up,but I believe that one day
    scientists like you will discover a way of solvetion of this serious problem.
    There are many people that suffer Seogren-sindrom and Empty nose(atrophic rhinitis),there is a web site "Empty nose sindrome association".I think ,if one day will be find a cure,many peopele would be saved!

    One more time,thank you for spared time!
    I wish you all the best and great success at your work!!!!
    regards:Jana

    Reply
    Posted by: Anonymous
    November 2, 2011 - 10:14 AM
  7. Dear Jana,

    My wife was diagnosed with a metastatic undifferentiated nasopharyngeal carcinoma The primary was found deep in her throat at the base of the tongue. Radiation therapy commenced on ²² January ²007 with the last treatment on 5 March ²007. She received a relatively high radiation dose of 6,000 cGy on the sides and 6,500 cGy in the centre. Consequently she had the usual side effects of a permanent dry mouth and throat, sore tongue, sensitive and painful teeth and gums, difficulty swallowing even soft food, etc. The oncologist said that the salivary glands will never recover if they have not recovered even partially over a ² year period.

    During October this year, 4 years and 7 months after her last radiation treatment, her saliva returned in one day; not gradually. The doctors don²17;t know why. One guess is her high protein diet of milk, yogurt, cheese, etc. but its just a guess not based on any science.

    Don²17;t loose hope
    Regards
    Paul http://www.ncbi.nlm.nih.gov/pubmed/²0698985 http://www.biomedcentral.com/content/pdf/1471-²407-10-417.pdf Restoration of radiation therapy-induced salivary gland dysfunction in mice by post therapy IGF-1 administration
    Abstract
    Background: Radiotherapy for head and neck cancer results in severe and chronic salivary gland dysfunction in most individuals. This results in significant side effects including xerostomia, dysphagia, and malnutrition which are linked to significant reductions in patients²17; quality of life. Currently there are few xerostomia treatment approaches that provide long-term results without significant side effects. To address this problem we investigated the potential for post-therapeutic IGF-1 to reverse radiation-induced salivary gland dysfunction.

    Methods: FVB mice were treated with targeted head and neck radiation and significant reductions in salivary function were confirmed 3 days after treatment. On days 4-8 after radiation, one group of mice was injected intravenously with IGF-1 while a second group served as a vehicle control. Stimulated salivary flow rates

    Conclusions: Post-therapeutic IGF-1 treatment restores salivary gland function potentially through normalization of
    cell proliferation and improved expression of amylase. These findings could aid in the rational design of therapy protocols or drugs for the treatment of radiation-induced salivary gland dysfunction in patients who have completed their anti-cancer therapies.

    Insulin-like growth factor 1 (IGF-1) also known as somatomedin C is a protein that in humans is encoded by the IGF1 gene.

    IGF-1 is produced primarily by the liver as an endocrine hormone

    In rat experiments the amount of IGF-1 mRNA in the liver was positively associated with dietary casein and negatively associated with a protein free diet.
    IGF-1 then stimulates systemic body growth, and has growth-promoting effects on almost every cell in the body, especially skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cell, and lungs. In addition to the insulin-like effects, IGF-1 can also regulate cell growth and development, especially in nerve cells, as well as cellular DNA synthesis.
    ---------
    Casein
    From Wikipedia, the free encyclopedia
    Jump to: navigation, search
    For information about casein usage in artistic painting, see Casein paint.
    Casein (pronunciation: /G²;keɪsiɪn/, from Latin caseus, "cheese") is the name for a family of related phosphoprotein proteins (αS1, αS², β, κ). These proteins are commonly found in mammalian milk, making up 80% of the proteins in cow milk and between 60% and 65% of the proteins in human milk. Casein has a wide variety of uses, from being a major component of cheese, to use as a food additive, to a binder for safety matches. As a food source, casein supplies amino acids; carbohydrates; and two inorganic elements, calcium and phosphorus
    --------------------------------------------

    Reply
    Posted by: Anonymous
    November 2, 2011 - 10:58 AM
  8. Dear Paul,

    thank you for your letter ,your support and advices!
    I fight everyday to make my health condition beter, becouse I have children and they need me!But now I have atrophic rhinitis due to atrophic pharingiitis and day after day it becomes harder and harder to cope with this really
    serious problem!Despite every medical data I don't lose hope,becouse when there aren't any others possibilities ,the faith is the most important thing that can help us and give us strength!

    I hope that your wife is fine now ,after partly recovering of her salivary glands!I wish your family whole the hapiness in the world and the most important thing-"Health",you deserve these thinngs!!!!

    Paul,if you learn something new at this area of medicine,I beg you to write me!Here is my e-mail: tisho²001@mail.bg.

    regards:Jana

    Reply
    Posted by: Anonymous
    November 3, 2011 - 10:18 AM

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