Identification And Functional Characterization Of Human Mesenchymal Stem Cells From Human Normal And Hyperplastic Gingiva

Gingiva surrounds the teeth and acts as mucosal barrier separating periodontium and outer space. Human gingiva plays an important role in the maintenance of oral health and shows not only unique fetal-like scarless healing process after wounding, but also tendency to undergo hyperplasia with usage of multiple drugs. To date, multiple types of cranial neural crest-derived mesenchymal stem cells (MSC) have been identified and proved not only to play essential role in local homeostasis but also to hold promise for regenerative therapy. Several lines of evidence have provided cues for imagination of presence of gingival mesenchymal stem cells, which in almost all the current studies are generally indistinguishable from gingival fibroblasts. This prompts us to corroborate and further find whether these mesenchymal stem cells exist within and differ between normal and hyperplastic gingival tissues. The main findings based on experimental results are described below. 1. Hyperplastic gingival tissues exhibited thickened layers and elongated rete ridges with abundant cellularity and extracellular matrix within lamina propria. Immunofluorescence assay showed that hyperplastic gingival tissues expressed more abundant COL-I, Ki-67and PCNA and demonstrated that the hyperplastic phenotype correlate with deregulation of cell proliferation and extracellular matrix production. Both normal and hyperplastic gingival propria displayed positive STRO-1 and SSEA-4 staining. These results indicated that despite the existence of differences between human normal and hyperplastic gingival tissues, they both showed evidence for harboring cells with features of mesenchymal stem cells.2. By using limiting dilution technique, single cell-derived colony cultures were obtained. The ability of gingival tissue derived cells to form adherent clonogenic cell clusters had no significant difference with those from bone marrow as shown by colony-forming efficiency analysis. N-GMSC and H-GMSC derived from single colonies showed more extensive repopulation capability than BMSC. For BMSC, the doubling time extended regarding the number of passages, while for H-GMSC and NGMSC group the doubling time maintained nearly at primary level. These findings highly suggested the GMSC was endowed with stemness property as highly self-renewable stem cells which can continue their growth for substantial doublings without evidence of senescence. Both types of GMSC showed the characteristic pattern of mesenchymal surface markers and negatively expressed hematopoietic markers after expansion.3. The multi-differentiation potential of both GMSC was determined. Under osteogenic induction conditions for 3 weeks, both GMSC could specifically differentiate and formed distinct nodules as stained by Alizarin Red S and express the osteogenic markers bone sialoprotein (BSP), osteocalcin (OCN) and alkaline phosphatase (ALP). After 3 weeks of culture in adipogenic induction medium, GMSC produced lipid droplets, the hallmark of functional adipogenesis. Adipogenic differentiation was further confirmed by the increased expression of specific adipogenic markers including peroxisome proliferator-activated receptorγ(PPARγ), CCAAT/enhancer-binding proteinα(C/EBPα) as determined by RT-PCR. Under chondrogenic induction as cell pellets, N- and H-GMSC underwent chondrogenic differentiation to exhibit hypertrophic chondrocyte-like morphology, which was densely stained with toluidine blue. The expression of COL2A1 and aggrecan mRNA in both types of GMSC under differentiation-inducing conditions appeared after induction. These results suggest that single colony-derived GMSC holds multi-potential properties.4. Immunostaining results demonstrated that matrix metalloproteinase-1 (MMP-1) expression within HGMSC was significantly lower than that in N-GMSC. There was no distinct variance of tissue inhibitors of metalloproteinase-1 (TIMP-1) expression between N- and HGMSC, suggesting role of MMP-1 expression within H-GMSC in mediating the pathological processes. N- and H-GMSC were subcutaneously transplanted respectively with fibrin gel as carrier in immunocompromised mice and regenerated connective tissue-like transplants. Histological examination showed that these regenerated transplants displayed collagenous connective tissue phenotype including presence of fibroblast-like cells. Immunofluorescence staining showed that there was distinct type I collagen expression in both GMSC transplants, while in H-GMSC it was more evident. We next performed serial transplantation with the same model. The results indicated that while after first transplantation the N-GMSC explants had lower frequency of derivation of single cell derived colony, some H-GMSC recovered from primary transplants maintained the in vivo ability to form connective-like tissues. These in vivo results demonstrated the definite regenerative capability of GMSC and indicated that H-GMSC may possess and maintain more robust activity to form large amount of tissues.5. We sought to determine this by using a highly immunogenic murine skin allograft model in which donor C57BL/6 mice (H-2d) was totally class II major histocompatibility complex-mismatched from BALB/c mice recipients (H-2b). The skin graft survival curves showed that compared to the control group injected with the same volume of PBS, the allograft rejection was delayed in GMSC or BMSC-treated group. Coculture experiments under contact condition showed that GMSC failed to exert inhibitory effect upon robust proliferative activity of splenocytes of recipient mice under alloantigen stimulation of splenocytes from C57BL/6 mice. This excluded the possibility that human GMSC relieved the active immune response of BALB/c mice against allogenic grafts through directly dampening the proliferative activity of immune cells. Flow cytometric analysis showed increased ratio of CD4+CD25+FOXP3+ Treg cells in response to coculture with both GMSC compared to control group. Pre-depletion of Tregs by anti-CD25 antibody treatment before H-GMSC injection not only abrogated the beneficial effect of H-GMSC on graft survival, but also accelerated rejection process in comparison with control group. These results demonstrated that human GMSC, irrespective from either normal or hyperplastic gingival, possessed strong immunosuppressive functions compared with their bone marrow counterpart under immune-activated environment, with Tregs probably being their in vivo mediators for immune regulation.