Characterization of Dental Pulp Pluripotent-like Stem Cells (DPPSC) and their mesodermal differentiation potential.

Dental pulp represents an easily accessible autologous source of adult stem cell populations. Among them, a population named dental pulp pluripotent-like stem cells (DPPSC) has been isolated from the dental pulp of human third molars and express pluripotency markers such as OCT3/4 and SOX2. DPPSC show pluripotent-like behaviour differentiating in vitro into tissues of the three embryonic layers and being able to form teratoma-like structures. This population also represents a source of adult stem cells without the ethical controversy or safety issues that are associated with the use of embryonic stem cells or induced pluripotent stem cells. In this project, we studied the growth rate and genetic stability of several populations of DPPSC from different donors, comparing them to other stem cell populations obtained from the dental pulp (DPSC). We observed that DPPSC divide faster than DPSC and present no chromosomal abnormalities, in contrast to DPSC. We also confirmed the pluripotency markers expression of these DPPSC populations at mRNA and protein level. We then analyzed how the expression of these pluripotency markers varies when the cells are cultured in vitro for several passages (at least until passage 15), when they are seeded at higher densities or split at higher confluence, and when they are cultured using a GMP-approved maintaining medium. Our results showed that DPPSC expression of pluripotency markers is higher at passage 10, and that the cells up-regulate some pluripotency markers and down-regulate some others when they are cultured at higher densities or using GMP-approved medium. In this project, we also evaluated the in vitro and in vivo DPPSC differentiation potential to different mesodermal-derived lineages. We demonstrated that DPPSC can differentiate in vitro into the endothelial lineage, as well as into the smooth and skeletal muscle lineage, using DPPSC from different donors and passages. Regarding their in vivo differentiation, we performed a wound healing assay in nude mice. We found that DPPSC are able to accelerate wound closure, revascularization and matrix organization in the regenerating wound area, as well as being able to differentiate in vivo to the smooth muscle lineage. In another experiment, we injected DPPSC in the skeletal muscle of two immunodeficient dystrophic mouse models, Scid/mdx and Sgcb-null Rag2-null γc-null. DPPSC engrafted in the skeletal muscle of both mouse models and differentiated into the endothelial, smooth muscle and skeletal muscle lineage, since the cells showed integration in muscular fibres and vessels. Taken together, our results showed that DPPSC are a genetically stable population that can be expanded in vitro in GMP conditions and that own mesodermal differentiation potential in vitro and in vivo. They represent a potential source of stem cells for translational therapies to enhance the wound healing process and slow down dystrophic muscle degeneration