Multi-lineage Differentiation Potential of CD۱۴۶+ Dental Pulp Derived Stem Cells

Background: Routinely, cultures of dental pulp stem cells (DPSCs)are achieved by direct culturing of tissue derived singlecell suspensions which are very heterogeneous and contaminatedby fibroblasts. Recently we have shown that CD۱۴۶ positiveselection can discriminate between dental pulp fibroblasts(DPFs) and DPSCs. Here, we aimed to assess tri-lineage differentiationpotential of CD۱۴۶+ DPSCs.Materials and Methods: Based on the CD۱۴۶ expression,magnetic cell sorting was done to purify DPSCs from culturesof pulp derived cells. For adipogenic differentiation was doneby culturing the cells in MesenCult medium supplemented with۱۰% Adipogenic Stimulatory Supplements (both from StemCell Technologies. Differentiation was assessed using oil redstaining and PCR. For osteogenic differentiation, the cells werecultured in NH-osteoDiff Medium (Miltenyi Biotec) accordingto the manufacturer’s guidelines. To approve the differentiation,the cells were stained with Alizarin red and underwent RT-PCR.For neural differentiation, the media was replaced by neurogenicmedia (neurobasal medium, ۲% B۲۷, ۲۰ ng/ml bFGF, ۱۰μM retinoic acid) at passage ۳-۴. The media was changed every۲ days till ۱۴ days. Differentiation was confirmed by Immunofluroscentstaining for ß-tubulin III. For hepatocyte differentiation,cells were serum deprived for ۲ days and then were culturedin DMEM supplemented with ۱۰ ng/mL basic fibroblastgrowth factor (bFGF) and ۲۰ ng/mL epidermal growth factor(EGF). Then a two-step differentiation protocol was performedas follows: Step-۱ was accomplished by adding DMEM supplementedwith ۱۰ ng/mL bFGF, ۴.۹ mmol/L nicotinamide and۲۰ ng/mL hepatocyte growth factor (HGF) for ۷ days. In step۲, DMEM supplemented with ۱ μmol/L dexamethasone ۲۰ ng/mL oncostatin M (OSM), ۱.۲۵ mg/mL bovine serum albumin(BSA), ۱۰ μL/mL ITS (insulin, transferrin, selenious acid), and۱۹۰ μmol/L linoleic acid was added to the cells in order to completecell maturation up to day ۲۱ and hepatic differentiationwas assessed morphologically and using RT-PCR.Results: After induction of differentiation cells could changeinto appropriate morphological appearance and staining andPCRs confirmed successful differentiation into desired fates.Conclusion: Our results reveal that the CD۱۴۶ positive portionsof the dental pulp derived cells were able to differentiateinto all tri-lineages (mesoderm, ectoderm and endoderm) andare multipotent and to some-how pluripotent.

کلیدواژه ها:

Pluripotency ، Dental Pulp Stem Cell ، CD۱۴۶