Can We Rescue Salivary Gland Function after Irradiation?

Hyposalivation induced by exposure of the salivary gland to radiation while treating head and neck cancer patients, can result in xerostomia (dry mouth syndrome), which burdens the patient with oral dryness or pain, dental caries, reduced taste and smell, increased risk for oral infections, hampered speech, and problems with food mastication. Stem cell therapy may be an option to reduce radiation-induced damage to the salivary glands permanently. This Directions in Science article reviews a recent study (Lombaert et al, 2008) using tissue stem cells to regenerate the salivary glands from cells that originate from putative stem cells residing in the ductal compartment. Lombaert et al showed restoration of function of irreversibly damaged mouse submandibular glands after intraglandular injection of an in vitro cultured c-Kit+ cell population containing salivary gland stem cells. The findings raise the prospect of clinical autologous salivary gland stem cell transplantation after radiotherapy.


ISOLATION OF SALIVARY GLAND STEM CELLS
With the fast development of techniques in the field of cell biology, many studies have revealed that adult stem cells are potentially able to regenerate organs or tissues. From such tissues as brain [23], mammary gland [24], pituitary gland [25], retina [26], skin [27], inner ear [28], and pancreas [29], stem cells can be isolated, characterized, and cultured as in vitro floating sphere cultures. With slight modification, Lombaert et al. isolated stem cells from salivary glands of mice using this in vitro floating sphere culture system for mouse salivary gland tissue [30]. A cell suspension of mice submandibular gland was obtained by enzymatic digestion. This was transferred to defined medium containing several growth factors. Within 3 days, from the initial 2-3 × 10 6 cells plated, ~9,000 spheres per digested submandibular gland were formed. Cells in these spheres were actively proliferating, indicating that the spheres did not originate from cell aggregation.

CHARACTERIZATION OF SALIVARY GLAND STEM CELLS
The salivary gland origin of the spheres was indicated by a set of (immuno-)histochemical analyses showing expression of a specific salivary gland protein, such as PAS (Periodic Acid Schiff's base), CK7, CK14, and amylase [30]. The potential presence of stem cells was shown by the expression of known stem cell markers, such as Sca-1 [31], c-Kit [32], and Musashi-1 [33]. Remarkably, the spheres could, when put into a 3D collagen culture system, form specific salivary gland tissue structures, such as elongated ducts and PAS + acinar cells.

SALIVARY GLAND STEM CELL TRANSPLANTATION
For transplantation studies [30], Lombaert et al. used male (Actbe-)GFP transgenic mice to track the progeny of the transplanted cells. Dissociated cells obtained from spheres cultured from male (Actbe-) GFP transgenic mice for 3 days were injected into submandibular glands of female mice, of which the salivary glands were irreversibly damaged by irradiation. Ninety days after transplantation, (immuno-) histochemical analyses indicated that in the transplanted glands, a high level of proliferation was ongoing. The morphology appeared as normal as in unirradiated glands and contained a large number of acinar cells. Quantification of the total surface area comprising acinar cells revealed a strong increase after salivary gland stem cell transplantation in most transplanted recipients. GFP expression and in situ hybridization for the donor Y-chromosome indicated that the newly formed acinar cells were donor derived. Furthermore, whereas nontransplanted irradiated animals hardly produce any saliva (0-5 µl/15 min), after transplantation, a clear improvement of salivary gland function was measured 90 days postirradiation, ranging from 23-70% of control flow rate (±200 µl/15 min, collected from restrained mice after pilocarpine injection).
To further characterize salivary stem cells, 300-1000 c-Kit + cells isolated by flow cytometry from 3day cultured spheres were transplanted in the same way, which resulted in a pronounced improvement in saliva secretion (30-150% of control) of nine out of 13 animals (69%) at 120 days postirradiation. This effect is extraordinary as only two of the six salivary glands were treated, whereas all were irradiated and normally contribute to the pilocarpine-induced saliva secretion. In contrast, transplantation of 10,000-90,000 of c-Kit-negative cells results in only one complete and two minor responders (out of nine). To test in vivo self-renewal and repopulating potential of engrafted cells, a secondary transplantation, with ckit + cells isolated from the primary recipients, was performed according to the same protocol [30]. Again, an amelioration of radiation-induced gland dysfunction in secondary recipients was evident.
In conclusion, we show restoration of function of irreversibly damaged mouse submandibular glands after intraglandular injection of an in vitro cultured c-Kit + cell population containing salivary gland stem cells. Our findings raise the prospect of clinical autologous salivary gland stem cell transplantation after radiotherapy.
Since surgical removal of salivary gland tissue during lymph node dissection prior to radiotherapy could provide stem cells to be transplanted postirradiation, transplantation of autologous salivary gland stem cells, compared to BMCs, may be a more adequate and elegant method of therapy. Indeed, we have been able to culture salispheres from both human parotid and submandibular gland, and show the presence of c-kit-expressing cells in the tissue, indicating the potential feasibility for translation to a clinical situation.
However, some questions still remain unanswered. To achieve more efficient transplantation, it is necessary to further characterize the exact cells with regenerative potential. In clinical therapy, more optimal techniques to inject the stem cells into the damaged salivary glands may increase the overall outcome. Injection into the excretory ducts of the glands seems to be optimal to get engrafted cells into the gland environment directly and with less clinical injury.
The presently described model appears readily translatable to the clinic and may lead, in the near future, to clinically applicable use of salivary gland tissue stem cells. Our data predict that transplantation of these cells will result in amelioration of the severely reduced quality of life of surviving cancer patients. Furthermore, our approach is the first proof for the potential use of stem cell transplantation to functionally rescue solid organ deficiency.