The Effect Of TRPM7Suppression On The Proliferation,Migration And Osteo-/Odontogenic Differentiation Of Human Dental Pulp Stem Cells

Background and objection:Stem cells are a kind of undifferentiated cells with self-renewable capacity.They can differentiate into various cell types and have organogenesis potential under certain circumstance.Human dental pulp stem cells(hDPSCs), a unique precursor population of mesenchymal stem cells (MSC),were firstly isolated from human wisdom teeth and named by Gronthos S.The clonogenic cells were demonstrated to possess both self-renewal and multilineage differentiation capability.hDPSCs has specific competence to form dentin-like structures in both ex vivo and and in vivo conditions.Therefore,they are an important source for tissue repair and therapy in regenerative dentistry and it is of great significance to understand the physiology regulation mechanism of hDPSCs for regenerative dentistry.TRPM7, a widely expressed bifunctional protein belonging to TRPM family, is composed of a Ca2+-Mg2+permeable ion channel and an inherent serine/threonine kinase. and it has been regarded as a most important cellular Mg2+homeostasis regulator.TRPM7has been associated with important cell functions including cell survival proliferation,migration,differentiation and organogenesis.Calcium ion and magnesium ion has been shown to play an important role in cell proliferation,migration,and differentiation.TRPM7,as a potent regulator of calcium and magnesium homeostasis at cellular level,might be an indispensable controller of these vital processes.To date,little information was obtained about the role of TRPM7in regulation of hDPSCs.TRPM7, as a intracellular Ca2+-Mg2+homeostasis regulator,might play a important role in manipulation of hDPSCs biological function.Thus,the research mainly focused on the role of TRPM7in regulation of hDPSCs proliferation,migration and differentiation.The thesis is composed of the following four chaptersChapter1The isolation and identification of hDPSCsWe used modified tissue explant collagenase method to isolate human dental pulp stem cells from young permanent teeth.Limiting dilution analysis was used to purified hDPSCs.Flow cytometry analysis was used to evaluate cell surface molecule expression pattern.The colony forming capacity and multilineage differentiation capability of hDPSCs were examined.Chapter2The expression and location of TRPM7in hDPSCs and dental pulpRT-PCR and Western blot was used to detect TRPM6mRNA, TRPM7mRNA and TRPM7protein in hDPSCs respectively.TRPM7protein was located by immunocytochemistry in hDPSCs and immunohistochemistry was performed to reveal its distribution in dental pulp tissue.Chapter3the role of TRPM7in regulation of hDPSCs proliferation and migrationSpecific TRPM7shRNA lentivirus was constructed to inhibit the expression of TRPM7. Realtime PCR and Western blot were used to verified the inhibition efficiency of TRPM7shRNA lentivirus.hDPSCs were exposed to50μM,100μM 2-aminoethoxydiphenyl borate(2-APB) and the effect of2-APB on the cell proliferation was evaluated with MTT assay after72h culture.The growth rate of TRPM7shRNA hDPSCs and Scrambled shRNA hDPSCs was examined by MTT assay at4different time points(day2, day4, day6, day8after culture).The cell cycle distribution of both groups was revealed by flow cytometry analysis.The migration capacity of TRPM7shRNA hDPSCs and Scrambled shRNA hDPSCs was evaluated by Transwell assay.Chapter4The effect of TRPM7suppression on osteo-/odontogenic differentiation of hDPSCs.Realtime PCR was used to detected the dynamic change of TRPM7during osteo-/odontogenic differentiation of hDPSCs. We used realtime PCR to reveal the expression level of osteo-/odontogenic differentiation related genes in both groups including ALP,DSPP,BSP,RUNX2,OSX following3weeks of mineralization induction.Materials and methodshDPSCs isolation,culture and purificationHuman dental pulp was obtained from complete wisdom teeth of healthy subjects at the Department of Stomatology, Nanfang Hospital, China. All protocols were reviewed by the Medical Ethics Committee of Nanfang Hospital, and written informed consent was provided by all subjects.The pulp tissue were isolated from the crown and root and then washed with sterile PBS at least3times.The pulp tissue was minced to1mm×1mm×1mm size,then digested in4g/L collagenase type I at37℃for10min,followed by centrifugation at800rpm for10min.Transferred the cells and tissues to a6-well plate and placed a slide to make sure that the tissues sticked to the plate firmly.The explants were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with15%fetal bovine serum (FBS),100units/mL penicillin and100mg/mL streptomycin, and incubated at37℃with5%CO2in humidified incubator.Single cell was obtained by limitation dilution analysis.The morphology of cell colony was observed under microscope.Flow cytometry analysisAt least1×106cells were prepared in cold PBS for each test. Cell phenotype analysis was performed by flow cytometric detection of CD29/PE, CD34/PE CD90/PE,CD45/PE,CD44/FITC and CD105/FITC according to the manufacturers’ instructions.In vitro analysis of hDPSCs multilineage differentiation potentialFor osteogenic differentiation, hDPSCs were seeded at5×104/35mm plate and cultured to70%confluence. Differentiation was induced by culturing cells in complete medium supplemented with10mM β-glycerol phosphate,50μg/mL ascorbic acid, and10-7M dexamethasone for3weeks. The induced cells were fixed in4%paraformaldehyde for10min at room temperature (RT) and then stained with2%Alizarin Red for10min.For adipogenic differentiation, hDPSCs were seeded into5×104/35mm plate and cultured to70%confluence. Differentiation was induced by culturing cells in complete medium supplemented with0.5mM methylisobutylxanthine,0.5mM hydrocortisone, and60mM indomethacin for3weeks. The cells were fixed in4%paraformaldehyde for10min at RT and then stained with Oil Red O for10min.For chondrogenic differentiation, hDPSCs were prepared as described for adipogenic differentiation. However, the cells were incubated with chondrogenic differentiation medium supplemented with10ng/mL transforming growth factor-β3(TGF-P3) for3weeks. The cells were then fixed in4%paraformaldehyde (PFA) for 10min at RT and then stained with Alcian Blue for2h.Realtime PCR analysisTotal RNA was isolated from hDPSCs using TRIzol according to the manufacturer’s instructions. First-strand cDNA was generated using the PrimeScript(?) RT reagent Kit. The cDNA was amplified with SYBR(?) Premix DimerEraserTM using an ABI7500realtime PCR system glyceraldehydes-3-phosphatedehydrogenase (GAPDH) was used as internal control to normalize for the amount of RNA in each sample.Western blot analysisCell extracts were separated by SDS-PAGE and transferred onto PVDF at200mA for2h. Polyclonal rabbit anti-human TRPM7was used as the primary antibody and HRP-conjugated anti-rabbit IgG as used as the secondary antibody. Immunoreactive proteins were visualized using ECL Plus.ImmunocytochemistryCells grown in48-well plates were fixed in4%paraformaldehyde for15min, permeabilized with PBS containing0.25%(v/v) Triton X-100for10min, and incubated sequentially with PBS containing5%BSA for30min. Cells were then incubated overnight with goat anti-human TRPM7polyclonal antibody(1:100) at4℃. After cells were washed in PBS, they were covered with Cy3-labeled rabbit anti-goat IgG antibody(1:1000)and DAPI.Immunofluorescence images were recorded with an Olympus AX70epifluorescence microscope.ImmunohistochemistryFormalin-fixed paraffin-embedded sections were deparaffinized by sequential washing with xylene,100%ethanol,90%ethanol,80%ethanol,70%ethanol and distilled water. The sections were treated with0.3%H2O2in methanol for5min. The slides were washed with PBS and blocked with PBS containing5%BSA for30min. They were then incubated overnight with a1:100dilution of anti-TRPM7primary antibody at4℃. After sections were rinsed with PBS, they were incubated with FITC-conjugated mouse anti-goat IgG (1:500) for1h at room temperature. For the negative controls, the primary antibody was replaced with PBS.Construction of TRPM7shRNA lentivirus and cell infectionThe TRPM7-targeting sequence of oligonucleotides was designed as previously described.The negative control consisted of a scrambled sequence with no homology to any human gene. The oligonucleotides were cloned into GV118-GFP (GeneChem, Shanghai, China) to generate the lentiviral vectors. Recombinant lentiviral vectors and packaging vectors were then transfected into293T cells. Supernatants containing lentiviruses expressing TRPM7shRNA or Scrambled shRNA were harvested72h after transfection. The lentiviruses were then purified by ultracentrifugation, and the titer of lentiviruses was determined. hDPSCs were infected with the TRPM7shRNA lentivirus at different multiplicity of infection (MOI), and mock-infected hDPSCs cells were used as negative controls.Cell proliferation assay by MTTFor MTT assays, the cells were seeded in96-well plates at2×103in100μL medium per well. Cell proliferation was assessed at the indicated time points.20μL of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide at a concentration of5mg/mLwas then added to each culture well. After incubation for4h at37℃, the supernatant was discarded, and200μL dimethyl sulfoxide was added to each well. The absorbance of each well was measured using a microculture plate reader with a test wavelength of490nm.Cell cycle analysishDPSCs infected with TRPM7shRNA lentivirus or scrambled shRNA lentivirus were collected and mixed with1mL cold70%alcohol at-20℃for48h to fix cells. The cells were then washed twice with PBS, stained with1mg/mL propidium iodide at4℃for30minutes, and analyzed on fluorescence activated cell sorter by reading on cytometer at488nm. The fraction of cells in the G1, S, and G2/M phases of cell cycle was determined.Transwell migration assayThe migration activities of TRPM7shRNA hDPSCs and Scrambled shRNA hDPSCs were assayed using transwell cell culture chambers. Following overnight serum starvation, cells were harvested and resuspended in DMEM containing0.1%FBS. Cells were added to the upper transwell chamber.The migration of TRPM7knockdown cells and Scrambled shRNA hDPSCs were stimulated by the addition of DMEM+10%FBS to the lower chamber. After24h the cells that migrated through the membrane were fixed in methanol and stained with0.1%crystal violet. Cells were counted in four random fields visualized under a light microscope and expressed as the average number of cells per field.Statistical analysisThe data were analyzed and expressed as the mean±tandard deviation. Statistical significance was evaluated by independent samples t-test using SPSS v13.0software. Statistical significance was set at P<0.05.Results1. The cell phenotype characteristics and biological property of hDPSCs.We successfully expanded hDPSCs from human dental pulp.hDPSCs are able to form colonies in culture.A typical colony of hDPSCs was identified after seeding as a single cell suspension.The cells were spindle-shaped and fibroblastic-like.We performed hDPSCs surface antigen analysis using flow cytometry.The rate of CD34,CD45(hematopoietic surface molecule)was0.07%,0.19%respectively,However, the positive rate of CD29,CD44,CD90,CD105(MSC-specific cell surface markers) was97.77%,99.09%,99.12%,79.15%respectively.To evaluate the multipotent differentiation capacity of hDPSCs,we treated hDPSCs with different differentiation inducing medium.For osteogenic differentiation,mineralization nodules could be detected in the presence of mineralization medium after21d of culture.For adipogenic differentiation,hDPSCs were cultured in the adipogenic induction media for3weeks,the accumulation of lipid-rich vacuoles was visualized within cells by Oil Red O staining.Chondrogenic differentiation assayed by the accretion of sulfated matrix stained with Alcian Blue was apparent.Thus,the hDPSCs we isolated from human dental pulp had multilineage differentiation ability.2. TRPM7expression on human dental pulp tissue and hDPSCsOur findings showed that TRPM6and TRPM7expressed in human dental pulp stem cells at mRNA level,and TRPM7was mainly expressed in the cytomembrane and cytoplasm of hDPSCs.Moreover,The human dental pulp widely expressed TRPM7.3.Silencing of TRPM7inhibited proliferation and migration of hDPSCsRealtime PCR and Western blot showed that the TRPM7shRNA lentivirus was constructed successfully.In order to determine the importance of TRPM7in the proliferation of hDPSCs.The growth rate of hDPSCs reduced when the cells were exposed at50μM,100μM2-APB, MTT assays were performed with cells infected with specific shRNA lentivirus against TRPM7at days2,4,6,8.The results showed that the growth rate of TRPM7knockdown cells was reduced significantly than control cells infected with scramble shRNA lentivirus at days2,4,6,8.The cells were retarded in G1phase after TRPM7suppression. Compared with scrambled control shRNA cells,TRPM7knockdown cells showed a38%reduction in the number of cells that crossed the membrane, indicating that TRPM7channels were required for migration of hDPSCs. 4.TRPM7knock-down inhibited osteo-/odontogenic differentiation of hDPSCsThe TRPM7mRNA was elevated during osteo-/odontogenic of hDPSCs.Both TRPM7shRNA group and Scrambled shRNA group were cultured in mineralized medium for21days. The expression level of ALP,DSPP,BSP,OSX,RUNX2mRNA was detected by quantitive realtime PCR analysis.The data showed that the osteo-/odontogenic differentiation markers ALP,DSPP,BSP,OSX,RUNX2expressed lower in TRPM7shRNA group than Scrambled shRNA group. These finding suggested that inhibiting TRPM7signaling could inhibited osteo-/odontogenic differentiation of hDPSCs.ConclusionIn summary, the isolated clonogenic cells were demonstrated to possess both MSC-specific markers and multilineage differentiation capability.We showed that both human dental pulp tissue and hDPSCs expressed TRPM7.TRPM7down-regulation inhibits the proliferation and migration capacity of hDPSCs and tends to inhibited hDPSCs osteo-/odontogenic differentiation under osteogenic differentiation-inducing conditions. These findings suggested that TRPM7plays an important role in regulating biological function of hDPSCs.