2.2. Autophagy level declines in BMMSCs and bone with age

To compare autophagy levels of young and aged bone, we performed immunohistochemical (IHC) analysis and real‐time PCR on bone marrow. Immunohistochemical analysis of LC3 showed that both groups displayed positive expression of LC3 and aged bone marrow exhibited less positive staining than young bone marrow (Figure 2a,b). Accordingly, mRNA expressions of Beclin1, Atg7 and LC3 of aged bone marrow were significantly reduced compared with that of the young group (Figure 2c). Although from the results above we can tell autophagy‐associated genes and protein in aged bone marrow is reduced compared with that of young bone marrow, it is hard to tell which kind of cells contribute to this change mostly. To detect autophagy of BMMSCs specifically, different methods were applied to examine autophagy levels of young and aged BMMSCs. We analysed protein expression of key autophagy‐associated proteins Atg7, Beclin1, P62 and LC3II/I in young and aged BMMSC by Western blot. Significant reduction in protein expression of autophagy‐associated proteins Atg7, Beclin1 and LC3II/I and upregulation of P62 protein were observed in aged BMMSCs compared with that of young cells (Figure 2d,e). Real‐time PCR showed that autophagy‐associated genes were reduced in aged cells compared with that of young BMMSCs (Figure 2f). We then investigated autophagy flux in both young and aged BMMSCs by treating the cells with either vehicle or chloroquine (CQ), and analysing the levels of LC3 puncta by immunofluorescent assay. We found that young BMMSCs treated with either vehicle or CQ accumulate more LC3 dots compared with aged BMMSCs (Figure 2g,h). Autophagosomes could be identified by transmission electron microscope (TEM), and aged cells possess fewer autophagosomes than young cells (Figure 2i). Autophagosomes are spherical structures with double‐layer membranes that contain cytoplasmic material and/or organelles. Taken together, these data revealed that with aging, autophagy displayed a decreased tendency in both bone marrow and BMMSCs which might contribute to degenerative changes of bone and BMMSCs. Further investigation of the relationship between autophagy and degenerative changes of BMMSCs and bone is required to elucidate the possible mechanism.

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Figure 2

Comparison of autophagy of young and aged bone marrow and bone marrow‐derived mesenchymal stem cells (BMMSCs) showed that autophagy was decreased with aging. (a, b) LC3 of immunohistochemistry (IHC) of young and aged bone marrow. Scale bar = 200 μm. (c) Real‐time PCR analysis on whole bone marrow of young and aged femora. BM = bone marrow. (d, e) Western blot was performed to examine expressions of Atg7, Beclin1, P62 and LC3 in young and aged BMMSCs at protein level. (f) Real‐time PCR was performed to detect mRNA expression of Atg7, Beclin1 and LC3. (g, h) Immunofluorescence (IF) staining of LC3 in young and aged BMMSCs treated with CQ and PBS. Scale bar = 10 μm. (i) Transmission electron microscopy (TEM) was used to detect autophagosomes of young and aged BMMSCs. Scale bar = 500 nm. Results are presented as means ± SD. n = 3. *p < .05, **p < .01, ***p < .001

2.3. Downregulation of autophagy by 3‐MA caused senescence of young BMMSCs

As we observed that autophagy was significantly decreased in aged BMMSCs compared with young BMMSCs, we next investigated the relationship between autophagy and stem cell aging by manipulating autophagy levels of young and aged BMMSCs. The autophagy inhibitor 3‐MA was used in young cells to examine changes of osteogenesis, adipogenesis and proliferation. A concentration of 5 mm recommended in the literature could effectively reduce autophagy (Figure S2a,b). Alizarin Red staining showed that 3‐MA could significantly inhibit the osteogenic differentiation capacity of young BMMSCs (Figure 3a,b). As we had already observed that aged BMMSCs exhibited higher adipogenic differentiation capacity, Oil Red staining revealed that 3‐MA could significantly increase adipogenic differentiation capacity of young BMMSCs (Figure 3c,d). Cell cycle analysis showed that 3‐MA inhibited proliferation of young BMMSCs (Figure 3e,f). Overall, these results indicated that inhibition of autophagy by 3‐MA turned young BMMSCs into an aged state with decreased osteogenic differentiation capacity, increased adipogenic differentiation capacity and reduced proliferation, which suggested that autophagy might be a key regulator of stem cell senescence. However, further investigation of autophagy in aged BMMSCs is needed.

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Figure 3

Inhibition of autophagy reduced proliferation and caused imbalanced differentiation of young bone marrow‐derived mesenchymal stem cells (BMMSCs). (a, b) Alizarin Red staining and quantitative analysis of young BMMSCs and 3‐MA‐treated young BMMSCs. (c, d) Oil Red staining and quantitative analysis of young BMMSCs and 3‐MA‐treated BMMSCs. Scale bar = 100 μm. (e, f) Cell proliferation capacity was evaluated by cell cycle analysis. Results are presented as means ± SD. n = 3. *p < .05, **p < .01, ***p < .001

2.4. Upregulation of autophagy by rapamycin restored degenerative function of aged BMMSCs

Next, we used rapamycin as an inducer of autophagy to examine its effect on aged BMMSCs. A dose of 100 nm of rapamycin resulted in the greatest increase in autophagy levels (Figure S2c‐e). As shown previously, aged BMMSCs exhibited lower osteogenic differentiation capacity; we found that rapamycin could restore the osteogenic differentiation capacity of aged cells (Figure 4a,b). Meanwhile, rapamycin significantly reduced the adipogenic differentiation capacity of aged BMMSCs as shown by Oil Red staining (Figure 4c,d). Furthermore, rapamycin promoted proliferation of aged BMMSCs (Figure 4e,f). In conclusion, activation of autophagy by rapamycin could restore aged BMMSCs functions of differentiation and proliferation.

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Figure 4

Rapamycin as an autophagy inducer could restore biological properties of aged bone marrow‐derived mesenchymal stem cells (BMMSCs). (a, b) Alizarin Red staining and quantitative analysis of aged BMMSCs and rapamycin‐treated aged BMMSCs. (c, d) Oil Red analysis and quantitative analysis of aged BMMSCs and rapamycin‐treated BMMSCs. Scale bar = 100 μm. (e, f) Cell cycle analysis of aged BMMSCs and rapamycin‐treated aged BMMSCs. Results are presented as means ± SD. n = 3. *p < .05, **p < .01, ***p < .001

2.5. Autophagy regulated ROS and p53

We observed that activation of autophagy could alleviate aging of BMMSCs and restore aged BMMSCs' osteogenic differentiation and proliferation, while inhibition of autophagy could cause senescence of young BMMSCs. However, the underlying mechanism remained unclear. Therefore, we explored the possible mechanisms involved. Intracellular ROS levels of BMMSCs were detected based on primary fluorescence. First, we investigated ROS levels of young BMMSCs, H₂O₂‐treated young BMMSCs (100 μm), H₂O₂ (100 μm)‐ and rapamycin (100 nm)‐treated young BMMSCs, 3‐MA (5 mm)‐treated young BMMSCs and aged BMMSCs and rapamycin (100 nm)‐treated aged BMMSCs. The results showed that aged BMMSCs had higher ROS levels than young BMMSCs. Rapamycin could reduce ROS levels in both H₂O₂‐treated young BMMSCs and aged BMMSCs, while 3‐MA could induce more ROS in young BMMSCs (Figure 5a,b). Accordingly, protein levels of p53 had the same trend as ROS, which was that H₂O₂ increased expression of p53, while rapamycin could effectively reduce the expression of p53 in H₂O₂‐treated cells and aged cells. 3‐MA increased p53 expression of young BMMSCs. Therefore, autophagy might regulate BMMSC aging via the ROS and p53 pathways (Figure 5c,d). From these results, we concluded that activation of autophagy could reduce ROS levels and p53 expression, while inhibition of autophagy could increase ROS levels and p53 expression, causing senescence.

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Figure 5

Activation of autophagy could reduce reactive oxygen species (ROS) levels and p53 expression, while 3‐MA reversed these effects. (a) ROS assessment of different groups of BMMSCs (young bone marrow‐derived mesenchymal stem cells [BMMSCs], H₂O₂‐treated young BMMSCs, H₂O₂‐ and rapamycin‐treated young BMMSCs, 3‐MA‐treated young BMMSCs and aged BMMSCs and rapamycin‐treated aged BMMSCs) by confocal microscopy. Scale bar = 100 μm. (b) ROS assessment of each group of cells by flow cytometry. (c, d) The protein level of p53 of each group of cells. Results are presented as means ± SD. n = 3. *p < .05, **p < .01, ***p < .001

4.2. Micro‐computed tomography

Micro‐CT imaging was performed on the distal femora to analyse bone mass using Inveon micro‐CT (Siemens AG, Germany). X‐ray source was set at 80 kV, 500‐μA microfocus. We reconstructed image slices using micro‐CT image analysis software to produce three‐dimensional images. The region of interest was selected manually in the marrow cavity after threshold values were set. Ultra‐high‐resolution images of 11 μm of specimens were obtained. The relevant bone morphometric parameters, including BMD (mg/cc), bone volume relative to total volume (BV/TV, %), trabecular thickness (Tb. Th, mm), trabecular number (Tb. N, 1/mm) and trabecular spacing (Tb. Sp, mm) were assessed. The CT number for a Siemens phantom contained 4 bars of known densities was measured. The resulting Hounsfield unit (HU) values for each bar were then used to determine the HU‐to‐BMD conversion formula. BMD was calibrated as previously described according to the formula (Chityala, Pudipeddi, Arensten, & Hui, 2013).

4.3. BMMSC isolation and culture

Bone marrow‐derived mesenchymal stem cells were harvested from femora and tibiae of young C57BL/6J (3 months) and aged C57BL/6J (16 months) mice. Femora and tibiae were dissected out and cleaned of connective tissue. After clipping both ends of the bones, bone marrow was flushed out from femora and tibiae by a syringe to complete culture media that consisted of a‐MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 20% foetal bovine serum (FBS, Atlanta Biologicals, Atlanta, GA, USA), 100 U/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen) and 12 μm l‐glutamine (Invitrogen). The cell suspension was then plated in 10‐cm culture dishes. The medium was changed every 2–3 days. After digestion with 0.25% trypsin/1 mm ethylenediaminetetraacetic acid (EDTA; Sigma‐Aldrich, St. Louis, MO, USA), cells were passaged at confluence.

4.4. Senescence‐associated β‐galactosidase staining

A total of 1 × 10⁵ BMMSCs per well were seeded in 12‐well plates and cultured for 3 days. β‐gal activity was assessed with a β‐gal staining kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the protocol provided. The numbers of senescent cells that were stained blue were counted, and the percentage was calculated.

4.5. Western blot

Cells were harvested and lysed with lysis buffer (Beyotime Institute of Biotechnology), and total protein concentration was assessed with BCA protein assay reagent (Bio‐Rad, CA, USA). Thirty micrograms of each sample was loaded and subjected to SDS‐polyacrylamide gels and then transferred to immunoblot PVD membranes (Millipore, Billerica, MA, USA). The membrane was blocked in blocking buffer (5% milk and 2 mg/ml BSA in PBST) for 1 hr and then incubated in primary antibodies against p53 (1:500; Cell Signaling), p21 (1:1,000; Cell Signaling), p16 (1:10,000; Cell Signaling), Atg7 (1:1,000; Cell Signaling), Beclin1 (1:1,000; Abcam), P62 (1:1,000; Abcam), LC3 (1:1,000; Abcam) and β‐actin (1:2,000; Abcam) in blocking solution overnight. After being washed in PBST for 10 min, membranes were incubated with secondary antibody in blocking solution for 2 hr at room temperature. After incubation, membranes were washed in PBST. An enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ, USA) was applied for visualization. ImageJ was used to quantify the results.

4.6. Real‐time PCR

Total mRNA was extracted from cells using TRIzol (Sigma‐Aldrich) reagent according to the provided protocol. Then, 2,000 ng mRNA was reverse‐transcribed with a PrimeScript RT reagent kit (Takara Bio Inc., Shiga, Japan). Settings of the program were 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min. The primers for mRNA are listed below: Atg7 (F:ACCTCGCTGGGACTTGTGC;R:GGTGAATCCTTCTCGCTCGT), Beclin1 (F:CAGTACCAGCGGGAGTATAGTGA;R:TGTGGAAGGTGGCATTGAAGA), LC3 (F:CCTGTCCTGGATAAGACCAAGTT;R:CTCCTGTTCATAGATGTCAGCGAT), β‐actin (F:CTGGCACCACACCTTCTACA;R:GGTACGACCAGAGGCATACA).

4.7. Alizarin Red staining

Alizarin Red staining was applied for analysing osteogenesis. Cells were seeded at 50 cells/cm² in 12‐well plates. After cells reached 80% confluence, osteogenic medium (a‐MEM supplemented with 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mm l‐glutamine, 2 mm β‐glycerol phosphate [Sigma‐Aldrich], 10 nm dexamethasone [Sigma‐Aldrich] and 100 μm l‐ascorbic acid 2‐phosphate [Sigma‐Aldrich]) was added and changed every 2–3 days. Rapamycin (Sigma‐Aldrich) and 3‐MA (Sigma‐Aldrich) were added in different experiments. After 14 days, cells were fixed in 4% formaldehyde at room temperature for 30 min and then stained with 0.1% Alizarin Red S (Sigma‐Aldrich) for 20 min at room temperature. To quantify the stain, we used 2% cetylpyridinium chloride (Sigma‐Aldrich) to elute the stain for 30 min and measured the spectrophotometric absorbance at 540nm.

4.8. Oil Red staining

Oil Red staining was used for analysing adipogenesis. Cells were seeded at 50 cells/cm² in 12‐well plates. After cells reached 100% confluence, adipogenic medium (a‐MEM supplemented with 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mm l‐glutamine, 5 μg/ml insulin [Sigma‐Aldrich], 50 μm indomethacin [Sigma‐Aldrich], 1 × 10⁻⁶ m dexamethasone and 0.5 μm 3‐isobutyl‐1‐methylxanthine [IBMX, Sigma‐Aldrich]) was added and changed every 2–3 days. Rapamycin and 3‐MA were added in different experiments. After 7 days, cells were fixed in 4% formaldehyde at room temperature for 30 min and then stained with 0.5% Oil Red (Sigma‐Aldrich) in methanol for 20 min at room temperature. To quantify the stain, we used methanol to elute the stain for 30 min and measured the spectrophotometric absorbance at 510nm.

4.9. Immunohistochemical staining

Immunohistochemical staining was performed as described previously. Femora were dissected out from young and aged mice. Serial sections (9 μm) were obtained after samples had been fixed, decalcified and embedded. After deparaffinizing, rehydrating and antigen retrieval, sections were blocked in 10% normal serum with 1% BSA in TBS for 2 hr at room temperature followed by applying LC3 (1:200, Abcam) antibody to sections overnight at 4°C. After HRP polymer incubation for 30 min and development with chromogen for 10 min at room temperature, sections were dehydrated and mounted.

4.10. Transmission electron microscope

Both young and aged BMMSCs were harvested and fixed in 4% formaldehyde and 1% glutaraldehyde in 0.1 m PB (pH 7.4) overnight. After fixation, dehydration, embedding, sectioning and staining, samples were viewed with a TEM at an accelerating voltage of 80–120 kV.

4.11. Immunofluorescence analysis

Both young and aged cells were cultured on sterile coverslips and treated with 50 μM CQ (Sigma‐Aldrich) for 4 hr. The control group was treated with equal volumes of PBS. Cells grown on coverslips were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.03% Triton X‐100 for 20 min. After that, 3% bovine serum albumin was used for blocking and then cells were incubated with LC3 (1:100, Abcam) antibody at 4°C overnight. After incubation with anti‐rabbit second antibody for 1 hr, nuclei were counterstained with Hochest 33258. Images were taken by a Zeiss LSM 510 laser‐scanning confocal microscope (Gottingen, Germany).

4.12. Cell cycle assessment

Young and aged BMMSCs were treated with 3‐MA and rapamycin, respectively, for 24 hr. The control group was treated with equal volumes of PBS and DMSO, respectively. After 24 hr drug treatment, cells were harvested and then washed twice with cold PBS. After that, cells were fixed in cold 70% ethanol at 4°C overnight. Then, 400 μl of 50 μg/ml stock of PI (BD Biosciences, USA) and 50 μl of a 100 μg/ml stock of RNase (Sigma‐Aldrich) were added, and cells were incubated for 30 min at 4°C. Cells from each group were then analysed via flow cytometry. The percentages of cells in the G1, G2 and S phases were calculated by the BD FACS software.

4.13. Measurement of ROS

We performed both qualitative and quantitative assay of ROS levels on BMMSCs which were cultured on coverslips and in 25‐cm² flasks, respectively. After being cultured for 24 hr, cells were treated accordingly for 8 hr as young BMMSCs, H₂O₂‐treated young BMMSCs (100 μm), H₂O₂ (100 μm)‐ and rapamycin (100 nm)‐treated young BMMSCs, 3‐MA (5 mm)‐treated young BMMSCs and aged BMMSCs and rapamycin (100 nm)‐treated aged BMMSCs. An ROS test kit (Genmed, Shanghai, China) was used to detect intracellular ROS level. For cells cultured on coverslips, DCFH‐DA (50 μm) was added and the cells were incubated for 30 min at 37°C. Then, the reaction solution was removed and fluorescence of cells was detected by confocal microscopy. For cells cultured in 25‐cm² flasks, after being trypsinized and washed, the cells were incubated in DCFH‐DA (50 μm) for 30 min with gentle agitation. Then, the reaction was stopped, and flow cytometry with excitation at 488 nm was applied for quantitative analysis.

4.14. Rapamycin treatment in vivo

Sixteen‐month‐old male mice (n = 14) were randomly divided into two groups (7/each group) as a control group and a rapamycin group. In the rapamycin group, rapamycin was administered at 1.5 mg/kg every other day for 2 months through intraperitoneal injection. DMSO was injected in the control group. After 2 months of injections, mice from both groups were sacrificed and three femora from each group were collected for micro‐CT scanning. Bone marrow‐derived mesenchymal stem cells from femora and tibiae of the other mice were harvested and cultured for Western blot analysis, CFU analysis and Alizarin Red analysis. Bone marrow‐derived mesenchymal stem cells from these two groups were combined with a hydroxyapatite–tricalcium phosphate scaffold (HA/TCP) and then implanted into subcutaneous pockets on the backs of the 8‐week‐old NOD/SCID mice (Fourth Military Medical University). After 8 weeks, the implants were fixed with 4% paraformaldehyde and decalcified with 10% EDTA (pH 6.0). H&E staining was applied to detect histology.

4.15. CFU analysis

To assess the self‐renewal capacity of BMMSCs, 1 × 10⁵ primary cultured BMMSCs from control and experimental groups were seeded in 5‐cm dishes. After 10 days' culture, 4% paraformaldehyde was used to fix the cells, and then 0.1% toluidine blue was applied to stain the colonies. Colonies of more than 50 cells were counted and analysed.

This work was supported by National Natural Science Foundation of China (31571532, 31570991, 81670947). We thank Li Liao, Bei Li and Chenghu Hu for their intellectual help on the project and Xiaolin Xu, Hua Ni, Leilei Zhang and Wei Yuan for their technical help.