To date, specific markers for the localization of NCSCs in human tissues have not been identified. Previously, we reported that nestin+/CD44+ cells are localized in the lamina propria of the oral mucosa.23 Nestin is a widely used NSC and stem cell marker in the dental pulp (including apical papilla)3,5,17−19, periodontal ligament,22 oral mucosa/gingiva,9,11,23,24 skin,12,13,15,16 heart,15 and bone marrow.16 Previous reports have suggested that nestin-positive bone marrow cells are enriched multipotent MSCs with sphere-forming and self-renewing abilities.29–31 Although nestin is expressed in NSCs, Liu et al. reported that human embryonic stem cell-derived NCSCs are nestin+/CD44+, whereas NSCs are nestin+/CD44−.32 Therefore, both nestin and CD44 were utilized to identify NCSCs; a nestin+/CD44+ population was observed among the cells derived from each tissue. Surprisingly, most cells were nestin+/CD44+ (approximately 80%−87%), although they may have been derived from NCs.
In this study, the cells did not differ in proliferative capacity, although OMSDCs had significantly higher colony-forming ability than APDCs (Fig. 2b-d). Previously, several reports suggested a lack of difference in the colony-forming abilities of DPSCs, PDSCs, and gingival mesenchymal stem cells (GMSCs), whereas others have shown that GMSCs form more colonies than DPSCs and PDLSCs.33–36 The colony-forming abilities of these cells correlate with their proliferative capability, although the colony details have not been assessed.33–36 Measurement of the colony area in this study showed that APDCs tended to have a larger distribution area in the histogram than OMSDCs (Fig. 2e). This observation was consistent with the low colony-forming ability and high proliferation rate of APDCs.
The sphere culture system has been widely used for the enrichment of NSCs and other types of stem cells.37,38 Many reports have suggested that NCSCs, MSCs, and cancer stem cells possess sphere-forming abilities.13–24,30,31,38 Furthermore, sphere-forming APDCs, PDLDCs, and OMSDCs express NES (NSC and NCSC markers), CD44 (a possible maker of NCSC-like cells from neuroblastoma), SNAI1 (a marker for the appearance of NC or their precursors in the neural plate border), SNAI2 (a marker of epithelial-mesenchymal transition in the NC), MSX1 (a neural plate border induction marker), and HES1 (the transcription factor for Notch effectors; Notch signaling is activated during NC differentiation).23,39 These data suggest that NC-derived cells or NCSCs are present in sphere-forming APDCs, PDLDCs, and OMSDCs.
Human stem/progenitor cells derived from APDCs, PDLDCs, and OMSDCs can differentiate into NC lineage cells in vitro.1–9,17−24 However, whether the multipotency of each tissue-derived stem/progenitor cell type is equivalent was unclear. In this study, we evaluated these cells from the same individual and observed that the in vitro differentiation abilities of APDCs and PDLDCs were similar, and that the mineralized-cell differentiation abilities of these cells were high, although their adipogenic ability was low (Fig. 4a, c). OMSDCs showed the opposite tendency (Fig. 4a, c). Lei et al. reported that the expression pattern of differentiation markers in mineralized cells, adipocytes, and chondrocytes differentiated from the DPSCs and PDLSCs does not differ.34 Yang et al. compared the multipotency of PDLSCs and gingiva-derived MSCs (GMSCs) and observed that PDLSCs have a higher ability to differentiate into mineralized cells and chondrocytes than GMSCs and that there is no significant difference in their abilities to differentiation into adipocytes.33 Monterubbianes et al. reported that GMSCs have a higher adipogenic differentiation ability than DPSCs and that DPSCs exhibit a higher osteogenic potential than GMSCs.40 A comparative study of abilities for differentiation into smooth muscle cells and neuronal cells in these cells is lacking. Our results regarding mineralized-cell and adipocyte differentiation abilities were similar to those reported by Monterubbianes et al. However, we did not observe any difference in chondrocyte differentiation between tissues. Previous studies isolated these stem/progenitor cells through monolayer culture, whereas we used enriched stem/progenitor cells isolated using the sphere culture technique in this study. We believe that differentiation abilities may differ with the stem/progenitor cell isolation technique.
The in vivo hard tissue regenerative capacity of APDCs, PDLDCs, and OMSDCs has been reported previously.1–9,17−23 Previously, we reported that not only APDCs but also OMSDCs regenerate into hard tissue when transplanted subcutaneously into immunodeficient mice following culture in BMP-2-supplemented mineralization-inducing cell differentiation medium.5,6,17,18,23 Although human APDCs retained osteodentin-like hard tissue-forming abilities, PDLDCs and OMSDCs had negligible hard tissue-forming ability (Fig. 6). Surprisingly, PDLDCs showed different hard tissue-forming abilities in in vitro and in vivo transplantation experiments. Grzesik et al. reported that hard tissue is not formed upon transplantation of PDLDCs, although these cells form calcified nodules and express mineralized-cell differentiation markers when cultured in mineralized-cell differentiation medium in vitro.41 Seo et al. reported that, unlike DPSCs, PDLSCs have the potential to generate cementum/PDL-like tissue, in which hard tissue is thinly formed around HA in vivo.7 Grzesik et al. suggested that differentiated mineralized cells are lost in the early stage of differentiation and that the remaining cells do not form hard tissue in vivo; alternatively, the differentiated mineralized cells may be negatively regulated by other fibroblastic populations that exert a suppressive influence in vivo.41,42 In the present study, APDCs, PDLDCs, and OMSDCs were cultured in BMP-2-supplemented osteogenic differentiation medium. Hence, the mineralized cells differentiated from PDLDCs may require BMP-2 and might have formed hard tissue only when it was supplied as a stimulus.41 The difference in the ability of each tissue-derived cell to form hard tissue in vivo is closely associated with biological roles. That is, APDCs have a high ability to form hard tissues, as they form tooth roots. In contrast, in the periodontal ligament, the ability to form cementum remains unaltered, despite the application of occlusal force. In addition, ectopic bone formation in the oral mucosa is extremely rare.
We detected genes with different expression levels among distinct tissue-derived cells. CD24 has been reported as a marker of several normal tissue-derived stem/progenitor cells and cancer stem cells.20 These stem/progenitor cells show higher CD24 expression than terminally differentiated cells, revealing the close relationship between CD24 expression and cellular pluripotency and self-renewal capability.20 This phenomenon has been reported in previous studies on tooth development. For example, Sonoyama et al. reported that CD24 is a specific marker of the apical papilla, whereas it is not detected in DPSCs and bone marrow MSCs, and that mineralized cells differentiated from APDCs do not express CD24.4 Recently, Chen et al. reported that CD24 expression increases by 72.9% during sphere formation in mouse APDCs.20 However, in our study, the expression of CD24 in sphere-forming human APDCs was only 2.9% (Fig. 6). Similar to our results, Sonoyama et al. reported an average of 7.56% for their CD24-positive cells.4 Hence, further studies regarding the expression of CD24 in APDCs are required. CD56 (NCAM1) is a marker of natural killer cells, neural cells, muscles, and MSCs generated from human embryonic stem cells.43,44 Surprisingly, Buttula et al. detected adipocyte differentiation in colonies derived from MSCA-1+/CD56− MSCs but not from MSCA-1+/CD56+ MSCs.44 This was consistent with the fact that CD56− OMSDCs show significantly higher adipocyte differentiation than CD56+ APDCs and PDLDCs.
HAPLN1 regulates cell growth in developing cartilage and heart valves and is expressed in mesoderm-committed cells derived from embryonic stem cells, cancer cells undergoing epithelial-mesenchymal transition (EMT), and metastatic melanomas.45 Mabarki et al. reported that HAPLN1 is expressed de novo in EPCAM1−/CD56 (NCAM1)+ mesoderm-committed progenitor cells and fibroblastic hepatocellular carcinoma cells and during the dedifferentiation of hepatocyte-like cells to liver progenitors and that silencing HAPLN1 downregulates the markers of EMT.45 As sphere-forming APDCs and PDLDCs expressed HAPLN1 and CD56, these markers may be related in MSCs, cells undergoing EMT, and NCSCs (Fig. 6c-e). Kim et al. reported that LRRC17, a member of the LRR superfamily, acts as a negative regulator of RANKL-induced osteoclast differentiation and is highly expressed in osteoblasts.46 This is consistent with its significantly high expression levels in APDCs and PDLDCs, which tend to differentiate more into mineralized cells in vitro than OMSDCs (Fig. 4a).
The roles of the other genes with significant differences in expression levels are unknown, and further investigations are warranted. Although it was difficult to identify stem cell-specific markers based only on the characteristic markers identified in this study, we successfully determined the specific markers for each tissue and tissue-derived cell.
The minimum defining criteria for MSCs are that they attach to a plastic cell culture dish, express MSC surface markers, and exhibit multipotency.47 Sacchetti et al. reported that MSCs obtained from different sites differ in their multilineage differentiation abilities and retain lineage-committed properties despite being isolated using certain MSC markers.48 Although previous reports suggest that human “NCSC-like cells” have been defined based on their sphere-forming capacity, expression of NCSC-related markers, and in vitro multipotential phenotype, this study also showed that the differentiation potential and expression of markers differed in distinct tissue-derived cells, depending on the tissue from which the cells were obtained, and that their characteristics should be considered during application in regenerative medicine.13,18,21,23,24,39 In the future, these distinct tissue-specific markers may play a crucial role in tooth regenerative medicine as they can clearly distinguish each tissue in the tooth organoids generated using induced pluripotent stem cells and tissue-derived stem/progenitor cells.