1Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul 03722, South Korea. 2Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, South Korea. 3Department of Internal Medicine, Brody School of Medicine at East Carolina University, Greenville, North Carolina 27834, USA. 4Severance Biomedical Science Institute, Yonsei University College of Medicine, 50-1 Yonsei -ro, Seodaemun-gu, Seoul 03722, South Korea. 5Lineberger Comprehensive Cancer Center, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599, USA. 6Department of Orthopaedic Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul 135-720, South Korea.
Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine. However, MSCs age rapidly during long-term ex vivo culture and lose their therapeutic potential before they reach effective cell doses (ECD) for cell therapy. Thus, a prerequisite for effective MSC therapy is the development of cell culture methods to preserve the therapeutic potential during long-term ex vivo cultivation. Resveratrol (RSV) has been highlighted as a therapeutic candidate for bone disease. Although RSV treatment has beneficial effects on bone-forming cells, in vivo studies are lacking. The current study showed that long-term (6 weeks from primary culture date)-cultured MSCs with RSV induction retained their proliferative and differentiation potential despite reaching ECD. The mechanism of RSV action depends entirely on the SIRT1-SOX2 axis in MSC culture. In a rat calvarial defect model, RSV induction significantly improved bone regeneration after MSC transplantation. This study demonstrated an example of efficient MSC therapy for treating bone defects by providing a new strategy using the plant polyphenol RSV.
Figure 1. 6w-MSCs with RSV induction preserve the proliferative capacity even after reaching effective cell dose (ECD) for MSC therapy
(A) Scheme for MSC isolation and long-term cultivation to obtain ECD-MSCs. First, 0.05% vehicle (EtOH) or 1 μM RSV was added to the medium for MSC culture and the vehicle or RSV-containing medium was exchanged every 2 days. The cells were cultured until vehicle-treated MSCs reached 1 × 107 cells, generally regarded as the ECD for bone regeneration. (B) The numbers of MSCs between 1-6 weeks of expansion in the absence or presence of RSV (n = 6 donors). The cells were counted every 7 days. (C) SA-β-gal assay was performed to compare cellular senescence between vehicle and RSV groups. 2w-MSCs refers to the MSCs cultured during 2 weeks from primary culture date, and 6w-MSCs refers to the MSCs cultured during 6 weeks from primary culture date in order to obtain 1 × 107 cells. SA-β-gal-positive cells were quantitated by ImageJ (n = 3, in triplicate per donor) (right). *p < 0.05 compared to vehicle-treated MSCs. 2w-MSCs, 3rd passage; 6w-MSCs, 13th passage. (D) Immunocytochemistry was performed to observe the bromodeoxyuridine-positive cell portion. Nuclei were stained with 4′,6-diamidino-2-phenylindole and images were captured by confocal microscopy. Scale bar = 100 µm. *, p < 0.05 compared to vehicle-treated 6w-MSCs (n = 3, in triplicate per donor). 6w-MSCs, 13th passage. (E) The proportion of 6w-MSCs treated with vehicle or RSV in each cell cycle phase was evaluated by flow cytometry with propidium iodide staining. *, p < 0.05 compared to vehicle-treated 6w-MSCs (n = 3, in triplicate per donor). 6w-MSCs, 13th passage. (F) Protein levels of P53, P21, P16, CASPASE-3, and cleaved CASPASE-3 were quantified by western blot analysis and normalized to that of HSP90. 2w-MSCs, 4th passage; 6w-MSCs, 13th passage. (G) Quantification of each protein level was determined by GraphPad Prism software (version 6.0). *, p < 0.05 compared to 2w- or vehicle-treated MSCs (n = 3, in triplicate per donor).
Figure 2. Long term-cultured MSCs with RSV induction preserve the self-renewal and multi-differentiation capacities even after reaching ECD for MSC therapy
(A) 2w- and 6w-MSCs (1 × 103 cells per well in 100-mm dishes) treated with vehicle or RSV were incubated in growth medium for 12 days. The colony-forming abilities of the cells were compared by crystal violet (CV) staining. The colony number was counted by three independent observers (n = 3, in triplicate per donor). *, p < 0.05. 2w-MSCs, 2nd passage; 6w-MSCs, 14th passage. (B) Alizarin red S staining was performed to detect mineral deposition and was quantified with ImageJ software (n = 3, in triplicate per donor). *, p < 0.05 compared to vehicle. 2w-MSCs, 2nd passage; 6w-MSCs, 14th passage. (C) The mRNA expression of RUNX2 and COL1A1 was determined in 2w- or 6w-MSCs treated with 0.05% vehicle (EtOH) or 1-μM RSV by real-time quantitative PCR (n = 3, in triplicate per donor). *, p < 0.05 compared to vehicle. 2w-MSCs, 3rd passage; 6w-MSCs, 14th passage. (D) Oil red O staining was performed to detect lipid droplets and were quantified with ImageJ software (n = 3, in triplicate per donor). *, p < 0.05 compared to vehicle. 2w-MSCs, 2nd passage; 6w-MSCs, 14th passage. (E) The mRNA expression of PPARγ and ADIPONECTIN was determined in 2w- or 6w-MSCs treated with 0.05% vehicle (EtOH) or 1-μM RSV by real-time quantitative PCR (n = 3, in triplicate per donor). *, p < 0.05 compared to vehicle. 2w-MSCs, 3rd passage; 6w-MSCs, 14th passage.
Figure 3. RSV treatment stabilizes SOX2 protein levels in MSCs depending on the presence of SIRT1
(A) Expected model of the mechanism of RSV action on the regulation and maintenance of MSC stemness via SIRT1-SOX2 axis. (B) Protein levels of SIRT1, SOX2, OCT4, and NANOG were quantified by western blot analysis and normalized to that of HSP90. (C) Quantitative analysis of SOX2 protein was performed by ImageJ software (n = 3, in triplicate per donor). *, p < 0.05, ns; not significant compared to control. MSCs of 4th passage were used for this western blot. (D) Immunoprecipitation was conducted in the presence of MG132. To confirm acetylated-lysine and SOX2, each protein was immunoprecipitated using antibodies targeting each protein followed by western blotting using an anti-SOX2 antibody (n = 3, in triplicate per donor). All study groups were treated with MG132 (10 µM), a proteasome inhibitor. MSCs of 4th passage were used for immunoprecipitation. (E) Immunocytochemistry was conducted to observe cellular localization of SOX2 from the nucleus to the cytoplasm, following SIRT1 knockdown in the presence of RSV (n = 3, in triplicate per donor). To inhibit proteasomal degradation of SOX2, all study groups were treated with MG132 (10 µM). Nucleus was stained with DAPI, and images were captured by confocal microscopy. Red arrowhead indicates nuclear exports of SOX2 protein. Scale bar = 50 μm. MSCs of 4th passage were used for immunocytochemistry. (F) Efficiency of SIRT1 knockdown or SOX2 overexpression was confirmed by western blot analysis in MSCs (n = 3, in triplicate per donor). MSCs of 4th passage were used for this western blot. (G) Colony-forming cells were detected by CV staining, and (H) the number was counted by three observers (lower panel) (n = 3, in triplicate per donor). *, p < 0.05. Alizarin red S (I) and oil red O (J) staining was conducted to compare mineralization and accumulation of lipid droplets. (K) Quantitative analysis for mineralization was measured at 595 nm absorbance (lower panel). *, p < 0.05. Lipid droplets were quantified with ImageJ software (n = 3, in triplicate per donor). Scale bar = 60 µm. *, p < 0.05. MSCs of 12th passage were used for CV, alizarin red S, and oil red O stains.
Figure 4. Time-course patterns of RSV-mediated SIRT1-SOX2 regulation in 6w-MSCs
(A) Immunofluorescence was performed to observe the time course patterns of RSV-mediated SIRT1-SOX2 regulation in the vehicle- and RSV-treated 6w-MSCs cultured under basal DMEM-low glucose medium containing 10% FBS and 1% antibiotic and antimycotic. SIRT1 and SOX2-stained cells were analyzed using Image J software. (B) Immunofluorescence was performed to observe the time course patterns of RSV-mediated SIRT1-SOX2 regulation in the vehicle- and RSV-treated 6w-MSCs cultured under osteogenic medium. SIRT1 and SOX2-stained cells were analyzed using Image J software. (C) Immunofluorescence was performed to observe the time course patterns of RSV-mediated SIRT1-SOX2 regulation in the vehicle- and RSV-treated 6w-MSCs cultured under adipogenic medium. SIRT1 and SOX2-stained cells were analyzed using Image J software. The nucleus was stained with DAPI, SIRT1was stained with phycoerythrin (PE, red)-conjugated secondary antibody, and SOX2 was stained with alexa fluor 568 (Yellow)-conjugated secondary antibody. Scale bar=10 μm. 6w-MSCs of 11th passage were used for this immunofluorescence.
Figure 5. RSV induction improves bone healing potential of 6w-MSCs
(A) Critical-sized calvarial defects (8-mm diameter) in rats were covered with fibrin glue, except for defect control treatment. Eight weeks after implantation, bone regeneration was measured by micro-computed tomography. A representative image is shown. (B) This graph shows the bone volume per mm3 (right panel) (n = 10). *, p < 0.05 compared to defect. #, p < 0.05 compared to 6w-MSCs treated with vehicle. (C) Hematoxylin and eosin staining was performed to observe new bone formation. The arrows show the edges of the host bone and line with asterisks indicates newly regenerated bone. Scale bar = 500 μm. (D) To confirm whether the newly regenerated bone was derived from a human origin, immunohistochemistry was performed using antibodies specific to human vimentin. The arrows indicate tissue derived from a human origin. Scale bar = 20 μm. (E) To confirm whether the transplanted 6w-MSCs contributed to bone regeneration of calvarial defects, immunohistochemistry was performed using antibodies against SIRT1, SOX2, RUNX2, and OCN as well as antibodies specific to human vimentin. The nucleus was stained with DAPI, and human VIMENTIN was stained with FITC-conjugated secondary antibody. SIRT1, SOX2, RUNX2, and OCN were stained with phycoerythrin (PE, red)-conjugated secondary antibody. Scale bar = 50 μm. (F) Effect of 6w-MSCs with RSV induction on tumorigenicity in 5-week-old female BALB/C nude mice. (G) Effect on the growth of MKN-74 cells, 6w-MSCs with vehicle induction, and 6w-MSCs with RSV induction xenografted in nude mice, showing no tumor growth in both 6w-MSC groups. (H) G-banding chromosome karyotype from 6w-MSCs with vehicle or RSV induction for 6 weeks. 6w-MSCs with 12th to 14th passages were used for animal experiments.
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