HDPSC isolation and culture

The dental pulp was obtained from surgically extracted intact human third molars from patients aged 16 to 70 years. Based on findings from previous literatures12,55,56 and proteomic clustering from our earlier study,57 participants were categorized into three groups: young (<23 years), middle-aged (23–40 years), and old (>40 years). The study was approved by the Biomedical Ethics Committee of Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (No. SH9H‒2022‒TK510‒1), and informed consent forms were obtained from the participants or their guardians.

The teeth were rinsed with sterile phosphate-buffered saline (PBS) before isolating pulp tissue. The collected pulp tissue was minced into pieces and digested with 4 mg/mL dispase (Gibco, Grand Island, NY, USA) and 3 mg/mL collagenase type I (Invitrogen Life Technology, Carlsbad, CA, USA) at 37 °C for 1 h. Then, the digested single cells were collected and plated into a 10 cm culture dish with α-modified minimum essential medium (α-MEM) supplemented with 1% penicillin-streptomycin (Beyotime, China) and 20% fetal bovine serum (FBS, Biolnd, Israel). The dishes were incubated at 37 °C in 5% CO2.

Flow cytometry

Isolated HDPSCs were analyzed for surface marker expression using flow cytometry. HDPSCs at passage 3 were collected through trypsin-EDTA digestion and resuspended in PBS. A total of 5 × 105 cells dispersed in 600 μL of PBS were transferred to test tubes, incubated with immunophenotype antibodies (Table S1) for 30 min, and then analyzed using a flow cytometer (BD, LSR Fortessa X‒20). The data were further analyzed with FlowJo (version 10.4) software.

Lentiviral transfection of HDPSCs

NUP62 overexpression was induced in HDPSCs using adenoviral NUP62 constructs at a multiplicity of infection (MOI) of 10, with empty vector (Vector) used as a control, in the presence of polybrene (Hanheng, 8 ng/mL). Stable cells were generated selecting with puromycin (Geman, 2 μg/mL). The efficiency of NUP62 overexpression was assessed using Western blotting.

Knockdown of NUP62

HDPSCs were infected with a lentiviral vector containing control or NUP62-targeting short hairpin RNA (shRNA) at MOI of 20. Following infection, the cells were selected with puromycin (Geman, 2 μg/mL). The efficiency of NUP62 depletion was assessed using Western blotting. The specific shRNA sequences are listed in Table S2.

siRNA transfection

Small interfering RNAs (siRNAs) targeting NSD2 or E2F1 were purchased from RiboBio Co., Ltd (Guangzhou, China) and transfected into HDPSCs at a concentration of 25 nmol/L with JetPrimer (No. 101000046, France). The siRNA sequences are listed in Table S2.

Cell proliferation and migration assay

HDPSCs were seeded in 96-well plates (2 × 103 cells per well). 24 h after seeding HDPSCs into 96-well plates was recorded as Day 0. A Cell Counting Kit-8 (CCK8) (Dojindo, Japan) was used to measure the cell proliferation rate over 7 days. Absorbance was measured at 450 nm using a multiskan (GO, Thermo Scientific). HDPSCs were centrifuged at 1 000 r/min for 5 min to prepare a 1 × 105 cells per mL cell suspension. In the Transwell (Costar, Transwell BD Matrigel, 3422) migration assay, 200 μL of the cell suspension was added into the upper chamber, while 500 μL of culture medium containing FBS was added into the lower chamber. After 24 h of incubation, the culture medium in the upper chamber was replaced with an FBS-free culture medium. Following an additional 24 h of incubation, crystal violet ammonium oxalate solution (Solarbio, China) was added for staining. The cells were then counted and photographed under an inverted microscope (Leica). All the experiments were repeated three times.

Senescence‐associated beta-galactosidase assay (SA β-gal)

Senescent cells were detected using a β-gal staining kit (Solarbio, China). The culture medium was removed from the six-well plates, which were then washed once with PBS. The plates were fixed for 15 min with 1 mL of β-gal staining fixative buffer. After fixing, the cells were washed with PBS 3 times, and 1 mL of staining solution was added to each well. Six well plates were incubated at 37 °C, and the cells were counted under an ordinary light microscope (DP73, Olympus).

Differentiation Induction

HDPSCs were cultured in osteogenic differentiation medium containing 1% penicillin‒streptomycin, 10% FBS, 0.2% ascorbate, 1% glutamine, 1% β-glycerophosphate, and 0.01% dexamethasone.

HDPSCs were grown in an adipogenic medium consisting of 10% FBS, 100 μg/mL isobutyl-methylxanthine, 1 μmol/L dexamethasone, and 10 μg/mL insulin. After 28 days of cultivation, lipid droplet formation was detected using Oil Red O staining (Cyagen, USA).

For chondrogenic differentiation, HDPSCs were grown in a medium consisting of 10% FBS, 2 ng/mL transforming growth factor-β (TGF-1β), 50 μmol/L ascorbic acid-2-phosphate, and 10 nmol/L dexamethasone. After 21 days of cultivation, the cell pellets were fixed and cut into 5 μm thick paraffin sections. Alcian blue staining (Cyagen, USA) was performed to assess the deposition of glycosaminoglycans.

Additionally, HDPSCs were grown in neurobasal Medium (Gibco, Life Technologies, Carlsbad, CA) consisting of 1% B27 (Gibco, Life Technologies), 20 ng/mL epidermal growth factor (Thermo), 1% penicillin-streptomycin, and 40 ng/mL fibroblast growth factor 2 (Thermo).

Alizarin red staining and mineralization assay

After 21 days of osteogenic differentiation induction, the cells were fixed with 4% paraformaldehyde and stained with 1% Alizarin Red S solution for 10 min (Cyagen, USA). The mineralized matrix was destained with 10% cetylpyridinium chloride for 30 min. The calcium concentration was evaluated by determining the optical density at 562 nm with a multiscan (GO, Thermo Scientific).

Immunofluorescence staining

A cell suspension at a concentration of 2 × 104 cells per mL was prepared and then seeded into 24-well plates loaded with 15 mm glass slides. The plates were incubated at 37 °C for 24 h. Following this, the cells were washed with PBS, fixed in 4% paraformaldehyde for 20 min, and then treated with 0.5% Triton X-100 (Sigma‒Aldrich, #SLCF3053) to enhance cell membrane penetrability. After blocking in 5% BSA for 30 min, the cells were incubated overnight at 4 °C with primary antibodies against NUP62 (Abcam, ab96134, 1:200), γH2AX (Cell Signaling Technology, #9718T, 1:200), H3K36me2 (Active Motif, 39056, 1:200), H3K36me3 (Cell Signaling Technology, #4909S, 1:200) or E2F1 (Cohesion, CQA8351, 1:100). The next day, the cells were incubated for 1 h at 37 °C with fluorescently labeled secondary antibodies (Invitrogen, 1:200). Nuclei were stained with DAPI (Sigma-Aldrich, 1:1 000) for 10 min. Images were collected with a microscope (DP73, Olympus), and further analyses was performed using ImageJ software. To quantify immunofluorescence microscopy images, five random fields images were used to determine intensity of NUP62, γH2AX, H3K36me2 and H3K36me3 per cell. To quantify the Nuclear/Cytoplasmic fluorescence ratio of E2F1, images were used to determine the distribution of E2F1 in the nucleus and cytoplasm per cell (n = 10). To ensure reliable quantification, all samples in the same experiment were imaged under the same scanning conditions.

Nuclear cytoplasmic protein fractionation

Nuclear cytoplasmic protein fractionation was performed using NE-PER® Nuclear and Cytoplasm Extraction Reagents (Thermo Scientific, #78833).

Western blotting

HDPSCs were lysed in cell lysis buffer (Cell Signaling Technology, #9803) supplemented with 1 mmol/L phenylmethylsufonyl fluoride (Beyotime, China) and protease inhibitor cocktail (Millipore, #539134). Denatured proteins (20 μg) were subjected to SDS‒polyacrylamide gel electrophoresis and then transferred on to a polyvinylidene difluoride (Millipore, USA) membrane at 80 V for 1.5 h. After blocking for 2 h in 5% nonfat milk dissolved in Tris-buffered saline Tween 20 (TBST), the membranes were incubated with primary antibodies against P21 (Cell Signaling Technology, #2947S, 1:1 000), GAPDH (Abcam, ab8245, 1:5 000), NUP62 (Abcam, ab140651, 1:1 000), P53 (Santa Cruz, sc126, 1:500), RUNX2 (Cell Signaling Technology, #12556, 1:1 000), Osterix (Invitrogen, PA5-40509, 1:1 000), DSPP (Santa Cruz, sc73632, 1:500), DMP1 (Invitrogen, PA5-88069, 1:1 000), SOX2 (Cell Signaling Technology, #3579S, 1:1 000), GFAP (Abcam, ab207165, 1:1 000), MAP2 (Affinity, AF4081, 1:1 000), H3 (Cell Signaling Technology, #4499T, 1:1 000), H3K36me2 (Active Motif, 39056, 1:1 000), H3K36me3 (Cell Signaling Technology, #4909S, 1:1 000), H3K9me3 (Abcam, ab75359, 1:1000), H3K27ac (Active Motif, 39085, 1:1 000), H3K4me3 (Cell Signaling Technology, 9751 T, 1:1 000), NSD2 (Abcam, ab75359, 5 μg/mL), E2F1 (Cell Signaling Technology, #3742S, 1:1 000) or β-tubulin (Huaxingbio, HX1984, 1:5 000) overnight at 4 °C. The membranes were washed with TBST 3 times and then incubated with goat-anti-rabbit or goat-anti-mouse secondary antibodies for 2 h. After the membranes were washed 3 times with TBST, they were visualized using a chemiluminescence imaging system (ChemiDoc Systern, BioRad).

Real-time polymerase chain reaction

Total RNA was extracted from HDPSCs using TRIzol (Takara). A SuperScript Reverse Transcriptase Kit (Takara) was utilized to synthesize complementary DNA (cDNA) for analysis of mRNA expression. Quantitative PCR (qPCR) was performed with a Light Cycler (Roche) using specific primers (Table S3). The relative gene expression levels were analyzed using the 2-∆∆CT method and normalized to that of GAPDH.

Luciferase reporter assay

293 T cells were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). These cells were seeded into 24-well plates (5 × 104 cells per well). Once the cells reached 70% to 80% confluence, they were transfected with JetPrimer (No. 101000046, France). The transfection mixture contained 160 ng of pGL3 basic-promoter, 10 ng of pRL-Renilla, and 160 ng of either an E2F1 expression vector or red fluorescent protein control. Luciferase activity was measured with a dual-luciferase assay kit (Yeasen, 11405ES60) 48 h after transfection. Specifically, 80 μL of lysate was mixed with 80 μL of luciferase buffer, and the luciferase activity was analyzed with an automatic luminometer (Tecan Infinite Lumi). Then, 80 μL of Stop & Glo reagent was added, and Renilla luminescence was measured after 10 min of incubation. The ratio of Firefly to Renilla luciferase activity was calculated to determine promoter activity. The promoter region sequences of NSD2 were provided in Table S4.

Transcription profiling of NUP62-overexpressing HDPSCs

We sequenced the RNA from HDPSCs 48 h after transfection with adenoviral constructs containing an empty vector or those overexpressing NUP62 (n = 9). RNA was extracted using the RNeasy Plus Kit from QIAGEN. Total RNA samples were subjected to poly (A)-selected sequencing library preparation with the TruSeq RNA Sample Prep Kit version 2 (Illumina). The abundance of library scripts (fragments per kilobase of exons per million mapped reads) was calculated using TopHat and Cufflinks software in conjunction with the human reference genome (hg19). GSEA software (version 4.3.2) was used for GSEA analysis.

Cleavage Under Targets and Tagmentation (CUT&Tag) Sequencing

HDPSCs were transfected with adenoviral constructs containing either an empty vector or overexpressing NUP62. The cells were then counted, harvested, and centrifuged for 5 min at 300 × g. Following this, they were washed twice with wash buffer. Concanavalin A magnetic-coated beads (Vazyme) were activated by washing twice in binding buffer. The beads were separated using a magnet, and the supernatant was removed. Five microliters of H3K36me2 (Active Motif, 39056) antibody or 1 μL of H3K36me3 (Cell Signaling Technology, #4909S) antibody was added to 200 μL of antibody buffer and incubated overnight at 4 °C. The Hyperactive In-Situ ChIP Library Pre Kit for Illumina (pG-Tn5) (Vazyme) was used for CUT&Tag. The data were visualized using IGV (version 2.16.2) along with the human reference genome (hg19).

Chromatin immunoprecipitation

Chromatin from crosslinked HDPSCs transfected with adenoviral constructs containing either an empty vector or overexpressing NUP62 was sonicated, precleared, and incubated overnight with antibodies in RIPA buffer. This mixture was then precipitated with protein G magnetic beads (Cell Signaling Technology, #9006) for 2 h. The antibodies used for the ChIP assay were H3K36me2 (Active Motif, 39056, 3 μg per test), H3K36me3 (Cell Signaling Technology, #4909S, 3 μg per test), NSD2 (Millipore Sigma, MABE191, 4 μg per test), E2F1 (Cell Signaling Technology, #3742S, 4 μg per test) and IgG (Cell Signaling Technology, #3900S, 4 μg per test). The DNA-protein-antibody complexes were washed once with Low Salt Wash Buffer (LS), once with High Salt Wash Buffer (HS), once with LiCl Wash Buffer, and twice with TE Buffer. The cross-linking of the co-precipitated DNA-protein complexes was reversed with Dr. GenTLETM Precipitation Carrier (TaKaRa, #9094). The immunoprecipitated DNA was then analyzed by RT‒PCR with primers listed in Table S5. The data are presented as the percentage of input DNA or the fold enrichment of the promoter (target/IgG).

Rat calvarial bone defects were reconstructed by HDPSC scaffolds

Six- to eight-week-old male Sprague-Dawley rats were used to evaluate bone regeneration in vivo. Ethical approval was received from Shanghai Ninth People’s Hospital (No. SH9H‒2022‒TK510‒1). After exposing the calvarial bone, a critical-size defect was created with an 8 mm dental trephine. A total of 5 × 105 HDPSCs were seeded into a commercialized collagen sponge (6 mm × 6 mm × 3 mm, Helistat 1690ZZ) and inserted into each defect. After 8 weeks, the animals were euthanized with CO2, and the repaired skulls were removed. Micro‒CT (Skyscan1176, USA Bruker) was used for the examination of calvarial bone defect repair. Images were 3D reconstructed with ctvox (Bruker micro‒CT, Kontich, Belgium). Bone mineral density (g/cc) was measured and analyzed. Sections (5 μm) were prepared using a microtome for hematoxylin-eosin (H&E) staining.

Tumorigenic capacity

Female BALB/c nude mice (4 weeks old) were used to evaluate the tumorigenic capacity in vivo. Ethical approval for the animal experiments was received from Shanghai Ninth People’s Hospital (No. SH9H‒2022‒TK510‒1). A total 5 × 105 Y-HDPSCs or O-HDPSCs transfected with an empty vector or overexpression of NUP62 were seeded into a collagen sponge (3 mm×3 mm×3 mm, Helistat 1690ZZ) and subsequently subcutaneously transplanted into both flanks of the nude mice (n = 6). After 6 weeks, the animals were euthanized with CO2. The transplants were surgically removed, fixed in 4% paraformaldehyde, and prepared for histological analysis.

Immunohistochemical analysis

Sections (5 μm) of calvarial bone were prepared with a microtome for immunohistochemical staining. After deparaffinization and rehydration, the slices were processed according to the manufacturer’s protocols. The anti-Coll1A1 antibody and anti-OCN antibody were purchased from Santa Cruz and Servicebio. Images were collected with a microscope (Leica, Germany).

Proteomic analysis and bioinformatic analysis

Proteomic analysis of HDPSC samples was conducted at the Institute of Human Phenome, Fudan University, Shanghai, China. The raw mass spectrometry files were processed using “Firmiana” (a one-stop proteomic cloud platform) against the human National Center for Biotechnology Information (NCBI) RefSeq protein database (updated on April 7, 2013; 32 015 entries).

Statistical Methods

All quantitative data are shown as the mean ± standard deviation (SD). Analyses and graphical presentations were performed with the GraphPad Prism 8 software. One-way ANOVA analysis was used to indicate differences among multiple groups, while two-tailed Student’s t tests were used for comparisons between two independent groups. All results represent two or more independent repeats. The following p-value indication scheme was used: ns: P > 0.05, *P < 0.05; **P < 0.01; ***P < 0.001.