Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells

Abstract

In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPARγ2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration.

Fig. 1

Morphologies and population doubling time comparison of DFAT cells and PDLSCs. a, b DFAT cells from passage 1 (P1) mostly demonstrated in fibroblast-like cells but gradually flatten and provided polyhedral morphology when cultured to the passage 7 (P7). DFAT cells, which were isolated from 7 to 10 days of primary culture, were positive stained lipid droplets by Oil Red O (c, d). PDLSCs exhibited fibroblast-like cells homogenously (e). Population doubling time (PDT) comparing between DFAT cells and PDLSCs was analyzed every 2 days for 2 weeks. PDLSCs provided shorter PDT, which implied as higher proliferation potential (f). Scale bar without character indicated 100 μm

Fig. 2

Stem cells characterizations of PDLSCs. a Flow cytometric analysis was performed for detecting immunophenotypes, which displayed all mesenchymal stem cell markers: CD44, CD90, CD105 but negatively shown the vascular cell markers: CD34 and CD106. Immunoglobulin G was used as a negative control, which demonstrated in all grey filled. Positive cell expressions were present by red filled. b PDLSCs were successfully induced into osteogenic (Alizarin Red staining), adipogenic (Oil Red O staining), and chondrogenic (Toluidine Blue staining) differentiation, which were confirmed gene expression of each lineage by RT-PCR. GAPDH was used as endogenous control. UD means undifferentiated PDLSCs, (−) means negative control. O means osteogenic-differentiated PDLSCs. A means adipogenic-differentiated PDLSCs. C means chondrogenic-differentiated PDLSCs. bp means base pairs. Scale bar indicated 100 μm

Fig. 3

Cell morphology and distribution after co-cultured. Co-culture system was performed for 2 weeks followed by identifying cellular morphology. DFAT cells from co-culture group have dispersedly in cell distribution and shown more polyhedral shape comparing to non-co-culture group. Scale bar indicated 100 μm

Fig. 4

DNA methylation profiles of four adipogenic genes and RUNX2 gene by COBRA technique. (a, b) After co-cultured for 2 weeks, DNA methylation analysis of four adipogenic genes was analyzed by COBRA technique. Each PCR product was digested by restriction enzymes; HpyCH4IV (ACGT) for C/EBPα, LPL, PPARγ2 and Taq I (TCGA) for FABP4 and providing fragments as follows: C/EBPα gene (171, 249, and 420 bp), FABP4 gene (56, 85, 141, 272, 357, and 413 bp), LPL gene (121, 164, 172, 285, 336, and 457 bp), and PPARγ2 gene (62, 181, 237, 299, 418, and 480 bp). c Co-culture group demonstrated higher in methylation status in all adipogenic genes when compared with non-co-culture group, which implied for retardation in transcriptional activity. d RUNX2 gene has adversely demonstrated the methylation profile by showing lower methylation status after performed co-culture. NC means non-co-culture. C means co-culture, bp means base pairs. Scale bar indicated 100 μm. Astrerisk means that comparison was statistically significant difference at P < 0.05, double asterisk means that comparison was statistically significant difference at P < 0.01

Fig. 5

RUNX2 and PPARγ2 gene expression comparison by quantitative RT-PCR. After co-cultured for 2 weeks, osteogenic differentiation medium was replaced for induction the osteogenesis. a, b Real-time PCR was performed for analyzing the RUNX2 and PPARγ2 gene expression after osteogenic induction in co-culture DFAT cells comparing to non-co-culture and undifferentiated DFAT cells. There was statistically significant difference in RUNX2 gene expression level in co-culture group with in contrast of PPARγ2 gene expression level. c, d Semi-quantitative RT-PCR was analyzed in serial of RNA concentration. RUNX2 gene expression has up-regulated in all concentration of co-culture group. In contrast to PPARγ2, gene expression has down-regulated in co-culture group comparing to non-coculture group at 0.5 and 1.5 μg. NC means non-co-culture, C means co-culture. Astrerisk indicates that comparison was statistically significant difference at P < 0.05