Dental pulp pluripotent-like stem cells (DPPSC), a new stem cell population with chromosomal stability and osteogenic capacity for biomaterials evaluation.

BACKGROUND: Biomaterials are widely used to regenerate or substitute bone tissue. In order to evaluate their potential use for clinical applications, these need to be tested and evaluated in vitro with cell culture models. Frequently, immortalized osteoblastic cell lines are used in these studies. However, their uncontrolled proliferation rate, phenotypic changes or aberrations in mitotic processes limits their use in long-term investigations. Recently, we described a new pluripotent-like subpopulation of dental pulp stem cells derived from the third molars (DPPSC) that shows genetic stability and shares some pluripotent characteristics with embryonic stem cells. In this study we aim to describe the use of DPPSC to test biomaterials, since we believe that the biomaterial cues will be more critical in order to enhance the differentiation of pluripotent stem cells. METHODS: The capacity of DPPSC to differentiate into osteogenic lineage was compared with human sarcoma osteogenic cell line (SAOS-2). Collagen and titanium were used to assess the cell behavior in commonly used biomaterials. The analyses were performed by flow cytometry, alkaline phosphatase and mineralization stains, RT-PCR, immunohistochemistry, scanning electron microscopy, Western blot and enzymatic activity. Moreover, the genetic stability was evaluated and compared before and after differentiation by short-comparative genomic hybridization (sCGH). RESULTS: DPPSC showed excellent differentiation into osteogenic lineages expressing bone-related markers similar to SAOS-2. When cells were cultured on biomaterials, DPPSC showed higher initial adhesion levels. Nevertheless, their osteogenic differentiation showed similar trend among both cell types. Interestingly, only DPPSC maintained a normal chromosomal dosage before and after differentiation on 2D monolayer and on biomaterials. CONCLUSIONS: Taken together, these results promote the use of DPPSC as a new pluripotent-like cell model to evaluate the biocompatibility and the differentiation capacity of biomaterials used in bone regeneration.

Dental pulp of the third molar: a new source of pluripotent-like stem cells.

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However, no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work, we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF, EGF and PDGF. These cells are SSEA4(+), OCT3/4(+), NANOG(+), SOX2(+), LIN28(+), CD13(+), CD105(+), CD34(-), CD45(-), CD90(+), CD29(+), CD73(+), STRO1(+) and CD146(-), and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly, DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm, endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4, GATA6, MIXL1, NANOG, OCT3/4, SOX1 and SOX2 to determine the degree of similarity between DPPSCs, EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs, hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages, they represent an easily accessible source of stem cells, which opens a range of new possibilities for regenerative medicine.

Chemically Defined Conditions Mediate an Efficient Induction of Dental Pulp Pluripotent-Like Stem Cells into Hepatocyte-Like Cells.

Liver diseases are major causes of morbidity and mortality. Dental pulp pluripotent-like stem cells (DPPSCs) are of a considerable promise in tissue engineering and regenerative medicine as a new source of tissue-specific cells; therefore, this study is aimed at demonstrating their ability to generate functional hepatocyte-like cells in vitro. Cells were differentiated on a collagen scaffold in serum-free media supplemented with growth factors and cytokines to recapitulate liver development. At day 5, the differentiated DPPSC cells expressed the endodermal markers FOXA1 and FOXA2. Then, the cells were derived into the hepatic lineage generating hepatocyte-like cells. In addition to the associated morphological changes, the cells expressed the hepatic genes HNF6 and AFP. The terminally differentiated hepatocyte-like cells expressed the liver functional proteins albumin and CYP3A4. In this study, we report an efficient serum-free protocol to differentiate DPPSCs into functional hepatocyte-like cells. Our approach promotes the use of DPPSCs as a new source of adult stem cells for prospective use in liver regenerative medicine.

Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration.

BACKGROUND: Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance. METHODS: DPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy (Scid/mdx and Sgcb-null Rag2-null γc-null). RESULTS: DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206+ macrophages. CONCLUSIONS: Overall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration.