Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells

Abstract

Although mesenchymal progenitor cells can be isolated from periodontal ligament (PDL) tissues using stem cell markers STRO-1 and CD146, the proportion of these cells that have the capacity to differentiate into multiple cell lineages remains to be determined. This study was designed to quantify the proportions of primary human PDL cells that can undergo multilineage differentiation and to compare the magnitude of these capabilities relative to bone marrow-derived mesenchymal stem cells (MSCs) and parental PDL (PPDL) cells. PDL mesenchymal progenitor (PMP) cells were isolated from PPDL cells using the markers STRO-1 and CD146. The colony-forming efficiency and multilineage differentiation potential of PMP, PPDL, and MSCs under chondrogenic, osteogenic, and adipogenic conditions were determined. Flow cytometry revealed that on average 2.6% of PPDL cells were STRO-1(+)/CD146(+), whereas more than 63% were STRO-1(-)/CD146(-). Colony-forming efficiency of STRO-1(+)/CD146(+) PMP cells (19.3%) and MSCs (16.7%) was significantly higher than that of PPDL cells (6.8%). Cartilage-specific genes, early markers of osteoblastic differentiation, and adipogenic markers were significantly upregulated under appropriate conditions in PMP cells and MSCs compared to either their noninduced counterparts or induced PPDL cells. Consistent with these findings, immunohistochemistry revealed substantial accumulation of cartilaginous macromolecules, mineralized calcium nodules, and lipid vacuoles under chondrogenic, osteogenic, or adipogenic conditions in PMP and MSC cultures, respectively, compared to noninduced controls or induced PPDL cells. Thus STRO-1(+)/CD146(+) PMP cells demonstrate multilineage differentiation capacity comparable in magnitude to MSCs and could potentially be utilized for regeneration of the periodontium and other tissues.

FIG. 1.

Isolation and characterization of PDL mesenchymal progenitor (PMP) cells and evaluation of their stem cell-like self-renewal ability. (A) The proportions of STRO-1 and CD146 double positive, single positive, and double negative PDL cells from six individuals was determined by examining the surface expression profiles of these stem cell surface markers using flow cytometry. Female (F) and male (M) subjects ranged from 16 to 25 years of age. The proportions of STRO-1 and CD146 double positive, single positive, and double negative mesenchymal stem cells (MSCs) was determined by examining the surface expression profiles of these stem cell surface markers using flow cytometry. (B) The mean percent of STRO-1 and CD146 double positive, single positive, and double negative cells was calculated and depicted graphically. (C) The stability of STRO-1 and CD146 double positive PMP cells prior to commencing the differentiation experiments was determined from a subset of three individuals. The STRO-1 single positive cells were isolated, expanded for 1 week before the CD146⁺ cells were secondarily isolated, and expanded for further 3 weeks before being assayed by flow cytometry. (D) Assays performed to assess stem cell-like adherent clonogenic cell clusters formed in colony-forming assay plates by the presumptive PMP cells, the counterpart PPDL cells, and bone marrow-derived MSCs showed a high number of colonies for PMP cells and MSCs but not for PPDL cells. Newly formed colonies were stained with toluidine blue. Histogram depicting the colony-forming efficiency for cells were quantified from colony-forming assays. The colony-forming efficiency was determined as the number of colonies standardized by the total number of seeded cells in each plate. Aggregates of more than 50 cells were scored as colonies. Data, expressed as mean ± SD, were obtained from experiments performed in triplicate for three independent cultures (*P ≤ 0.01 for PMP or MSC compared to PPDL).

FIG. 2.

Osteoblastic gene expression and alkaline phosphatase activity is induced in PMP cells and MSCs by osteogenic media (Ost Media). (A) Mean ± SD relative expression levels of osterix (A) and alkaline phosphatase (B) mRNA measured by qRT-PCR in noninduced (−) and induced (+) PPDL cells, PMP cells, and MSCs on Day 5 of culture. (C) Histograms of mean ± SD alkaline phosphatase activity in noninduced and osteoblastically induced PPDL cells, PMP cells, and MSCs on Day 7 of culture. Alkaline phosphatase activity was quantified spectrophotometrically and standardized by the total amount of protein in the cell lysate. Data were obtained from experiments performed in triplicate on three independent cultures (*P ≤ 0.01 for osteoblastically induced PMP cells or MSCs compared to noninduced PMP cells, MSCs, or PPDL cells, or osteoblastically induced PPDL cells).

FIG. 3.

PMP cells and MSCs but not PPDL cells cultured in osteogenic media show increased deposition of mineralized ECM. (A) Images of monolayer cultures of PPDL cells, PMP cells, and MSCs incubated in control or osteogenic media (Ost Media) for 14 days stained with Alizarin red S staining shows substantial staining in the induced PMP cells and MSCs but not PPDL cells. (B) The amount of calcium deposition in the mineralized ECM was quantitated by releasing the calcium-bound alizarin red into solution, measured spectrophotometrically, and standardized by the total amount of protein in each culture. The mean ± SD for experiments performed in triplicate on three independent cultures is presented as a histogram (*P ≤ 0.01 for osteoblastically-induced PMP cells or MSCs vs. noninduced PMP cells, MSCs, or PPDL cells, or osteoblastically induced PPDL cultures).

FIG. 4.

Chondrogenic media enhances the expression of cartilage-specific genes in PMP cells and MSCs but not in PPDL cells. Cells were cultured in pellet, exposed to control or chondrogenic media (Chondro Media) for 7 days, and the RNA retrieved and assayed for collagen type II (A) and COMP (B) by qRT-PCR. Data, expressed as mean ± SD, were obtained from experiments performed in triplicate on three independent cultures (*P ≤ 0.01 for chondrogenically-induced PMP cells or MSCs vs. noninduced PMP, MSC, or PPDL cultures, or chondrogenically induced PPDL cultures).

FIG. 5.

Chondrogenic conditions increase the levels of cartilaginous ECM in PMP cell and MSC pellets. Cell pellets were cultured as described in Figure 4 for 21 days, the pellets fixed, sectioned, and stained. (A) Representative photomicrographs of sections stained with Safranin-O show substantial amounts of staining for newly synthesized glycosaminoglycans in induced PMP and MSC pellets but not in noninduced PMP, MSC, or PPDL pellets, or chondrogenically-induced PPDL pellets. Sections of the pellets immunostained for aggrecan (B) and collagen type II (C) also demonstrate high levels of staining for these macromolecules in chondrogenically-induced PMP and MSC pellets but not in any noninduced pellets. Images were captured under 40× magnification (Bar represents 50 μm).

FIG. 6.

Adipogenic-specific genes are upregulated and lipid vacuoles accumulate in adipogenically-induced PMP cells and MSCs but not in PPDL cells. (A) Cells were cultured in control media or adipogenic media (Adipo Media) for 7 days, the RNA extracted and subjected to qRT-PCR for LPL (A) and PPARγ2 (B), and the relative mean ± SD expression of these genes for experiments performed in triplicate on three samples was plotted as histograms. (C) Cells from similar experiments cultured for 18 days were stained with Oil Red O staining and photomicrographed to show accumulated lipid vacuoles. Images were captured under 10× magnification (*P ≤ 0.01 for adipogenically-induced PMP cells or MSCs vs. noninduced PMP cells, MSCs, or PPDL cells, or adipogenically induced PPDL cultures; Bar represents 100 μm).