Autologous mesenchymal stem cells offer a new paradigm for salivary gland regeneration

Abstract

Salivary gland (SG) dysfunction, due to radiotherapy, disease, or aging, is a clinical manifestation that has the potential to cause severe oral and/or systemic diseases and compromise quality of life. Currently, the standard-of-care for this condition remains palliative. A variety of approaches have been employed to restore saliva production, but they have largely failed due to damage to both secretory cells and the extracellular matrix (niche). Transplantation of allogeneic cells from healthy donors has been suggested as a potential solution, but no definitive population of SG stem cells, capable of regenerating the gland, has been identified. Alternatively, mesenchymal stem cells (MSCs) are abundant, well characterized, and during SG development/homeostasis engage in signaling crosstalk with the SG epithelium. Further, the trans-differentiation potential of these cells and their ability to regenerate SG tissues have been demonstrated. However, recent findings suggest that the "immuno-privileged" status of allogeneic adult MSCs may not reflect their status post-transplantation. In contrast, autologous MSCs can be recovered from healthy tissues and do not present a challenge to the recipient's immune system. With recent advances in our ability to expand MSCs in vitro on tissue-specific matrices, autologous MSCs may offer a new therapeutic paradigm for restoration of SG function.

Fig. 1

Epithelial-mesenchymal crosstalk during SG development. Studies of SG developmental morphogenesis have demonstrated a complex regulatory relationship between the maturing SG epithelium and the surrounding mesenchyme. The diagram shows signaling pathways that are common to both SG epithelium and mesenchyme and activated or inhibited during various phases of embryonic development. Adapted from previously published gene expression data by Jaskoll & Melnick and Suzuki et al. Created using BioRender.com

Fig. 2

Proposed biomarkers and locations of cells in the adult SG with regenerative capacity during homeostasis or in response to damage. Recent studies have demonstrated that multiple types of SG progenitors have the capacity to maintain homeostasis and regenerate SG function under various conditions. Clonal expansion of terminally differentiated Mist1⁺ and Pip⁺ acinar cells are involved in homeostatic maintenance and repair of the acinar compartment. The Mist1⁺ cells are also activated by ligation-induced damage to form acinar cells. In the intercalated duct, progenitors expressing Axin2⁺ or K5⁺ are responsible for homeostatic maintenance. However, in response to ligation-induced damage, cKit⁺ and K14⁺ cells self-renew to repair the intercalated duct or give rise to acinar cells. SMA-expressing myoepithelial cells also respond to ligation-induced damage by differentiating to form either acinar cells or ductal cells that express cKit and K14. Under IR-induced damage conditions, Axin2⁺ or K5⁺ ductal progenitors are capable of giving rise to functional secretory acinar cells. In addition, Sox2⁺ acinar cells have been shown to be capable of limited regeneration of the acinar compartment in response to IR-induced damage. Created using BioRender.com

Fig. 3

Ultrastructural characteristics of rat SMG tissue and SMG-ECM-treated aggregates were remarkably similar. a Transmission electron micrographs of rat SMG (positive control tissue) thin sections. Scale bar is shown in each panel. N: cell nucleus; SG: secretory granule (note the presence of two types of granules, electron dense and less electron dense); TJ: tight junction (identified with thin arrow pointers). b Transmission electron micrographs of cell aggregates that formed when BM-MSCs were incubated with SMG-ECM for 1 h and then cultured for 14 days. Note the presence of structures (e.g., electron dense secretory granules; formation of tight junctions; location of the nucleus near the cell membrane) found in rat SMG tissue that can also be seen in the cell aggregates. Scale bar is shown in each panel. c Transmission electron micrographs of BM-MSCs that formed a monolayer after treatment with SMG-ECM and culture for 14 days (Mono/SMG-ECM) and untreated BM-MSCs. Scale bar is shown in each panel. Images were selected from original work published by BioMed Central in Tran et al.

Fig. 4

Implantation of aggregates in a sub-renal capsule assay formed SG-like organoid structures in vivo that stained positively for markers of SG differentiation. SMG-ECM-treated aggregates were implanted under the kidney capsule in immunocompromised mice to study organoid development. Kidneys, containing the implants, were harvested after 30 days for histological analysis. After fixation, sectioning, and staining for immunofluorescence microscopy, the presence of amylase and Aqp5, both markers of SG differentiation, were identified in SMG tissue (positive control) and aggregates (formed during 14 days of culture) that had been implanted for 30 days. Staining with nonspecific isotype antibody was used as a negative control (not shown). Scale bar for SMG tissue = 50 µm; scale bar for aggregates implanted for 30 days = 20 µm. Images were selected from original work published by BioMed Central in Tran et al.

Fig. 5

Proposed clinical paradigm for use of autologous BM-MSCs for SG regeneration. MSCs harvested from the bone marrow of patients with SG hypofunction or dysfunction, would be cultured in an environment (i.e., ECM produced by BM cells from young donors [i.e., young BM-ECM]) which promotes the expansion of regenerative subpopulations of autologous MSCs. Once sufficient numbers of MSCs are obtained, they will be combined with a homogenate of decellularized SG-ECM (or, in the future, a mimetic of SG-ECM), which recapitulates components of the healthy SG microenvironment, and induces the trans-differentiation of the MSCs to the SG epithelial cell lineage (i.e., SG progenitors) during culture. After an initial period of culture with the decellularized SG-ECM, followed by the addition of differentiation media and additional time in culture, the cells begin to form SG salispheres, which are transplanted, along with the accompanying ECM, back into the damaged SG of the patient