Supplementary MaterialsSupplementary files 41598_2017_3681_MOESM1_ESM. uninjected contralateral and ipsilateral glands. Engrafted cells could be localized to the stroma surrounding acini and ducts. In summary, our data show that CD34+ derived SG-MSCs could be a promising cell source for adoptive cell-based SG therapies, and bioengineering of artificial SGs. Introduction Stem cells constitute an important facet of cell-based therapies in regenerative medicine. Mesenchymal stem/stromal cells (MSCs) are a group of specialized multipotent stem cells that have the ability to proliferate and differentiate into multiple mesodermal and non-mesodermal lineages and play key Lesinurad sodium roles after injury and in the homeostatic maintenance of tissue architecture1. Widespread availability in various organs, ease of isolation and propagation under conditions, lack of ethical and teratoma formation concerns, extensive proliferative and differentiation abilities, Lesinurad sodium paracrine secretory functions, and immunosuppressive behavior have rendered MSCs as the most sought after cell type in pre-clinical and clinical research fields2, 3. Additionally, MSCs play indispensable functions in the organogenesis and development of several epithelial organs including salivary glands (SGs)4, 5. It has been shown that MSCs isolated from various tissue sources and donors possess differences in phenotype and multilineage differentiation abilities6C8. Further, the phenotypic expression profile also differs among and cultured MSCs9. These limitations pose major hurdles in the widespread clinical power of MSCs. Therefore, investigation of unique phenotypic markers in various organs could lead to generation of more homogenous pools of MSCs with defined downstream functional implications in a tissue-specific manner. Herein, we report on the extensive phenotypic and functional characterization of MSCs in all three major SGs in humans: parotid (PAGs), sublingual (SLGs) and submandibular (SMGs) glands. SGs represent a group of exocrine organs with the primary function of synthesizing saliva. Saliva performs a plethora of crucial functions such as, mastication and digestion of food, protection of the oral cavity from bacterial infections, prevention of tooth decay, facilitating speech and induction of taste belief by stimulating taste buds10, 11. In humans, paired PAGs, SLGs and SMGs contribute to 90% of the total saliva secretion. SGs originate from the embryonic ectodermal epithelial progenitors around the 6th to 8th week of gestation. By the 28th week, a series of key physiological events lead to the IL-23A development of mature SGs that can secrete saliva at birth12, 13. Complex reciprocal Lesinurad sodium interactions between epithelial, mesenchymal, vascular and neuronal progenitors lead to the organogenesis and development of SGs. Embryonic mesenchyme, through secretion of growth and signaling factors, provides essential molecular cues to the developing epithelial progenitors at various stages of glandular development and histodifferentation5. Functional significance of MSCs has been further exhibited through recombination experiments between epithelial and mesenchymal progenitors, which have established that signals from mesenchyme regulate the branching pattern and type of saliva, either serous or mucus, secreted by the developing acinar cells in SGs14C16. Various groups have studied expression of stem/progenitor cell markers such as, aldehyde dehydrogenase (ALDH), c-kit (CD117), CD24, CD29, CD34, CD44, CD49f, CD90, CD133 and CD166 in SG cell aggregates known as salispheres and in cell monolayers produced under culture conditions, and have further exhibited regenerative and reparative functions of these cells in various mouse models of SG damage17C23. However, these cultured cells were derived from whole cell isolates of SGs made up of mixtures of cells of multiple lineages. The phenotypic markers identifying MSCs in freshly isolated SGs remain largely uncharacterized. Moreover, molecular nature of primitive embryonic mesenchyme from SMGs has been studied in mice, but little is known about the gene expression profile of the mesenchyme of human fetal SMGs14C16. The current study investigated human PAGs, SLGs and SMGs to understand (i) the phenotypic expression profile of MSCs in freshly collected SGs as compared to age-matched bone marrow (BM), (ii) localization and functional properties of a populace of SG-MSCs enriched for the adhesion molecule CD34, (iii) the gene expression profile of sorted CD34+ and CD34? cells derived from midgestation SMGs, (iv) growth and multilineage differentiation potential of cultured MSCs isolated from sorted CD34+ SG cells, and (v) functional ability of CD34+ cell-derived MSCs, when transplanted intraglandularly into immunodeficient NOD.Cg-localization and distribution of CD34+ cells was investigated in the context of other known lineage specific stem/progenitor and committed SG markers CD133 (prominin-1), K5 (cytokeratin 5) and c-Kit (CD117), as well as lineage markers for endothelial (CD31/PECAM-1), myoepithelial [-clean muscle actin (-SMA) and calponin], epithelial.