Analysis of the characteristics and expression profiles of coding and noncoding RNAs of human dental pulp stem cells in hypoxic conditions

Abstract

BackgroundHuman dental pulp stem cell (DPSC)-mediated regenerative endodontics is a promising therapy for damaged teeth; however, the hypoxic environment in root canals can affect tissue regeneration. In this study, we investigate the characteristics and possible regulatory mechanisms of DPSC function under hypoxic conditions.MethodsHuman DPSCs were cultured under normoxia (20% O2) and hypoxia (3% O2). DPSC proliferation and osteo/odontogenic differentiation potential were assessed by Cell Counting Kit-8 (CCK8) assay, carboxyfluorescein succinimidyl ester (CFSE) assay, alkaline phosphatase (ALP) activity, Alizarin red staining, real-time RT-PCR assays, and western blot analysis. Microarray and bioinformatic analyses were performed to investigate the differences in the mRNA, lncRNA, and miRNA expression profiles of DPSCs.ResultsDPSCs exhibited a more powerful proliferation ability and lower osteo/odontogenic differentiation potential in hypoxic conditions. A total of 60 mRNAs (25 upregulated and 35 downregulated), 47 lncRNAs (20 upregulated and 27 downregulated), and 14 miRNAs (7 upregulated and 7 downregulated) in DPSCs were differentially expressed in the hypoxia group compared with the normoxia group. Bioinformatic analysis identified that 7 mRNAs (GRPR, ERO1L, ANPEP, EPHX1, PGD, ANGPT1, and NQO1) and 5 lncRNAs (AF085958, AX750575, uc002czn.2, RP3-413H6.2, and six-twelve leukemia (STL)) may be associated with DPSCs during hypoxia according to CNC network analysis, while 28 mRNAs (including GYS1, PRKACB, and NQO1) and 13 miRNAs (including hsa-miR-3916 and hsa-miR-192-5p) may be involved according to miRNA target gene network analysis. The depletion of one candidate lncRNA, STL, inhibited the osteo/odontogenic differentiation potentials of DPSCs.ConclusionsOur results revealed that hypoxia could enhance the proliferation ability and impair the osteo/odontogenic differentiation potential of DPSCs in vitro. Furthermore, our results identified candidate coding and noncoding RNAs that could be potential targets for improving DPSC function in regenerative endodontics and lead to a better understanding of the mechanisms of hypoxia’s effects on DPSCs.Electronic supplementary materialThe online version of this article (10.1186/s13287-019-1192-2) contains supplementary material, which is available to authorized users.

Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties

“…We have shown that the cell isolation and expansion at low [O 2 ] led to an increased proliferation capability of SCAPs and to a preserved expression of the classical MSC markers with a slight decrease of CD105, as already reported [43]. A recent report with DPSC isolated at 21% O 2 and expanded at either 21% or 3% O 2 (as in our EXP II) also demonstrated an advantage of cell growth under low [O 2 ] compared with ambient air [15]. Among the different markers analyzed, CD49f and SSEA4 remained highly expressed under low [O 2 ] in comparison with ambient air.…”

“…Further studies are required to better understand if there is a link between high CD49f and SSEA4 expression and proliferation in SCAPs. [15,62]. The media used for these experiments have been optimized under 21% O 2 and might lack important components essential for efficient differentiation at late passage.…”

“…The composition of SCAP secretome could change after many passages in vitro and further analyses will better characterize the differentiation restriction of aging SCAP cells [63]. In addition, it might be relevant to determine the expression profile of a new long non-coding RNA (lncRNA STL) in early and late UBx-SCAP according to the recent report showing its involvement in osteogenic differentiation in the DPSC model [15]. In addition, highly proliferative SCAPs grown at 3% O 2 should now be challenged for their potential to differentiate to neuronal or endodermal lineages.…”

“…Previous studies reported that DPSCs cultured for 24 h at 1% O 2 secrete FGF2 (Fibroblast Growth Factor 2) and display improved survival rates when injected into experimental animals [14]. Recently candidate coding and non-coding RNAs, induced upon hypoxia, were reported in the dental pulp stem cell (DPSC) model [15]. Furthermore, plating bone marrow (BM) extracts directly at 3% O 2 leads to the establishment of BM-MSCs, termed MIAMI cells, which have increased self-renewal and improved maintenance of osteoblastic differentiation capability [16][17][18].…”

Characterization of Dental Pulp Stem Cell Responses to Functional Biomaterials Including Mineralized Trioxide Aggregates

“…Expanding this study to include more DPSC isolates with variable expression of these pluripotency biomarkers will provide substantial information that could validate the findings of the current study. In addition, a more comprehensive evaluation of other potential biomarkers, such as non-coding microRNA, may provide more specific and targeted methods for bioengineering and biotechnology applications utilizing DPSC [ 40 , 41 , 42 ].…”