Isolation and characterization of human periodontal ligament stem cells

by Gay, Isabel C.

The periodontal ligament (PDL) is thought to include progenitor cells capable of forming fibroblasts, osteoblasts and cementoblasts. However, characterization of PDL stem cell populations (SC) remains undetermined due in part, to the diversity of cell types that conform to the periodontal apparatus. Objective: To isolate and characterize PDLSC and assess their pluripotent capabilities to differentiate into bone, cartilage and adipose cell types. Methods: PDL was scraped from human teeth, enzymatically digested, and cells stained for STRO- 1 (SC marker) using fluorescent immunohistochemistry. Positive cells were FACS sorted and expanded in culture. Human bone marrow SC (BMSC) served as a positive control. PDLSC and BMSC were cultured using conditions conducive for osteogenic, chondrogenic and adipogenic differentiation. Osteogenic induction was assayed using alizarine red staining, and expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) by RT-PCR and immunohistochemistry. Adipogenic induction was assayed using Oil Red O staining and expression of PPAR? 2 and LPL (early/late specific markers) by RT-PCR. Chondrogenic induction was immunoassayed by collagen type II expression (cartilage marker) and toluidine blue staining. Results: Of the PDL cells isolated, 27% were positive for STRO-1, with 3% staining strongly. ALP expression was initially observed in PDLSC osteogenic cultures by day-14, where BMSC showed expression by day-7. BSP expression was detectable by day-7; with more intense staining found in PDLSC cultures. Under adipogenic conditions both population showed positive Oil Red O staining by dayii 35 with expression of PPAR? 2 and LPL. By day-21 both BMSC and PDLSC chondrogenic cultures expressed collagen type II. Control cultures showed no differentiation. Conclusions: Human PDL tissue contains a high percentage of STRO-1 positive stem cells. These PDLSC remain undifferentiated until challenged with various differentiating conditions having the potential to differentiate into osteoblasts, chondrocytes and adipocytes, comparable to BMSC. Our method for obtaining stem cell populations can be utilized for potential therapy procedures and possibly formation of a periodontal ligament around dental implants. - iii -