Gene-modified Bone Marrow Mesenchymal Stem Cells Research Articles

COL6A3 enhances the osteogenic differentiation potential of BMSCs by promoting mitophagy in the osteoporotic microenvironment.

Bone marrow mesenchymal stem cells (BMSCs) have been widely recognized as a highly promising option for cell-based tissue engineering therapy targeting osteoporosis. However, the osteogenic differentiation of BMSCs is impeded by the limited viability and diminished capacity for bone formation within the osteoporotic microenvironment. In this study, the COL6A3 gene was confirmed through an extensive analysis of the preceding single-cell sequencing database. The generation of an inflammatory microenvironment resembling osteoporotic cell transplantation was achieved by employing lipopolysaccharide (LPS). A lentivirus targeting the COL6A3 gene was constructed, and a Western blotting assay was used to measure the marker proteins of osteogenesis, adipogenesis, and mitophagy. Immunofluorescence was utilized to observe the colocalization of mitochondria and lysosomes. The apoptosis rate of each group was evaluated using the TUNEL assay, and the mitochondrial membrane potential was assessed using JC-1 staining. This investigation discovered that the impaired differentiation capacity and decreased viability of BMSCs within the inflammatory microenvironment were markedly ameliorated upon overexpression of the specific COL6A3 gene. Moreover, the administration of COL6A3 gene overexpression successfully mitigated the inhibitory impacts of LPS on mitophagy and the expression of inflammatory mediators, specifically inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in BMSCs. To clarify the underlying mechanism, the role of mitophagy during the differentiation of COL6A3 gene-modified BMSCs in the inflammatory microenvironment was evaluated using the mitophagy inhibitor Mdivi-1. In the context of lipopolysaccharide (LPS) stimulation, COL6A3 enhances the differentiation of BMSCs into osteogenic and adipogenic lineages through the promotion of mitophagy and the maintenance of mitochondrial health. Our findings may provide a novel therapeutic approach utilizing stem cells in the treatment of osteoporosis.

Transplantation of ERK gene-modified bone marrow mesenchymal stem cells ameliorates cognitive deficits in a 6-hydroxydopamine rat model of Parkinson's disease

The study aimed to investigate bone marrow mesenchymal stem cells (BMSCs) and extracellular signal-regulated kinase (ERK) gene-modified BMSCs (ERK-BMSCs) transplantation in ameliorating cognitive deficits in Parkinson’s disease (PD). The PD rat model was built by 6-hydroxydopamine (6-OHDA) injection into the right striatum for 8 weeks, then successful PD rats were randomly divided into three groups and respectively transplanted in the same position of striatum as modeling with PBS, BMSCs and ERK-BMSCs for another 8 weeks. The 6-OHDA-induced PD rat model was successfully established, as demonstrated by reduced active avoidance response (AAR) times, percentage of time exploring in the light area (Ltime%) and platform quadrant time (PQT), as well as p-ERK expression. Compared with PBS rats, both BMSCs and ERK-BMSCs transplantation significantly reduced the left turn number, while increased AAR, Ltime%, PQT and p-ERK expression, suggesting improved cognitive abilities through restoring p-ERK expression. In addition, ERK-BMSCs injection exhibited higher therapeutic efficacy against cognitive deficits compared with BMSCs injection. These results demonstrated that BMSCs transplantation ameliorated cognitive deficits, and ERK-BMSCs exerted synergistic effects, which may prove beneficial against cognitive impairments in PD.

Experimental Study on the Bone Morphogenetic Protein 1-Modified Bone Marrow Mesenchymal Stem Cell Sheets to Promote Mandibular Distraction Osteogenesis.

ObjectiveThe present study aims to increase the concentration of genetically modified bone marrow mesenchymal stem cells (BMSCs) in the distraction osteogenesis (DO) interstitial space and induce the conversion of BMSCs to osteoblasts to improve the osteogenic efficiency in DO and shorten the treatment period.MethodsBone morphogenetic protein 1 (BMP-1) and green fluorescent protein (GFP) gene-modified cell sheets of BMSCs were constructed by tissue engineering. Thirty-six New Zealand white rabbits were randomly divided into three groups: group A (the blank control group), group B (the GFP group) with the injection of GFP gene-modified BMSC sheets into the DO gap, and group C (the BMP-1 group) with the injection of BMP-1 gene-modified BMSC sheets into the DO gap. Rabbits in all three groups were distracted for 5 days at a distraction rate of 2.0 mm/d, once/day. After distraction, the above-mentioned cell sheet suspension was injected into the distraction gap to observe osteogenesis, which was observed by gross specimen observation, micro-computed tomography (Micro-CT) scanning, and histomorphology.ResultsThe gross specimen observation showed that all animals had smooth and continuous bone cortex in the distraction region with relatively high hardness. The osteogenesis quality or hardness was ranked from the highest to the lowest, as Group C > Group B > Group A. Micro-CT and histomorphological observation revealed that group C had better maturation and bone volume of the new bone in the DO region at weeks 3 and 6 than groups B and A.ConclusionBMP-1 gene-modified BMSC sheets could effectively promote the formation of new bone during rapid DO in the mandible, compensating for the poor osteogenesis caused by rapid distraction and providing a new approach to shorten the DO treatment period in clinical practice.

Transplantation of BMP-7 gene-transfected bone marrow mesenchymal stem cells for the treatment of spinal cord injury in rats

Background: Spinal cord injury (SCI) is a serious traumatic disease of the central nervous system, and there is currently no effective treatment for SCI because of its complicated pathophysiology. Bone marrow mesenchymal stem cells (BMSCs) have multidirectional differentiation abilities. Our study aims to explore the effects of bone morphogenetic protein 7 (BMP-7)-modified BMSCs transplantation on the repair of SCI in rats. Methods: In this study, a rat spinal cord injury model was established with the modified Allen method. Then, BMSCs transfected with the BMP7 gene were transplanted to treat the spinal cord injury in rats. Forty Sprague-Dawley rats were randomly divided into the sham operation group (sham group), spinal cord injury group (model group), BMSC treatment group (BMSC group) and LV-BMP7-BMSC treatment group (LV-BMP7-BMSC group). The Basso, Beattie, and Bresnahan (BBB) score was used to evaluate the recovery of hindlimb function in the rats. The levels of neurofilament protein NF-200 (NF-200) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence, RT-PCR and Western blotting. Results: At 14 d, 21 d, and 28 d after treatment, the BBB score of the rats in the LV-BMP7-BMSC group was higher than that of the rats in the model group and BMSC group. The results showed that NF-200 was expressed at the local spinal cord injury site. Compared with that of the sham group, the NF-200 expression level of the BMSC group and LV-BMP7-BMSC group was increased (P < 0.05). The results showed that the mRNA expression levels of NF-200 in the spinal cord tissue of the BMSC group and LV-BMP7-BMSC group were increased compared with those of the sham group (P < 0.05). The western blotting results further confirmed the PCR results. Conclusion: BMP-7 gene-modified BMSC transplantation can promote the repair of spinal cord functions after SCI in rats.

Antitumor effect of IL-12 gene-modified bone marrow mesenchymal stem cells combined with Fuzheng Yiliu decoction in an in vivo glioma nude mouse model

BackgroundGlioma is a complex cancer with a high morbidity and high mortality. Bone marrow mesenchymal stem cells (BMSCs) have shown promise as an excellent cell/drug delivery vehicle for gene-targeted therapy; however, maintaining genetic stability and biological activity remains difficult. Furthermore, whether BMSCs support or inhibit tumor growth remains debated. This study investigated whether a traditional Chinese medicine fomular, Fuzheng Yiliu decoction (FYD) had a synergistic antitumor effect with IL-12 gene-modified BMSCs in glioma-bearing nude miceMethodsThe lentivirus-mediated IL-12 gene was transfected into primarily cultured BMSCs. A total of 72 BALB/c nude mice were used to establish xenograft models with glioma U251 cells and were divided into groups (n = 12) including blank control group, nude mouse model group (model group), lentiviral transfection of BMSC group with no gene loading (BMSC group), IL-12 lentivirus-transfected BMSC group (IL-12 + BMSC group), FYD treatment group (FYD group), and FYD treatment in IL-12 lentivirus-transfected BMSC group (FYD + IL-12 + BMSC group).. After treatment for 14 days, all mice were sacrificed to collect tumor tissue and serum for more detection, such as distribution of BMSCs, cell apoptosis in xenograft tumors, serum IL-12 and INF-γ levels, mouse weight and tumor volume were measuredResultsThere were significantly more apoptotic cells in tumor tissue in IL-12 gene transfected group, FYD treatment group and FYD combining with IL-12 gene transfected group than that in the model group (P < 0.05). The FYD + IL-12 + BMSC group showed significantly higher Bax and lower Bcl-2 expression (P < 0.05), and serum IL-12 and INF-γ levels (P < 0.05) were higher than that in all other groups. After the intervention, this group also showed a strong inhibitory effect against tumor growth (P < 0.05)ConclusionsThis study suggested FYD treatment combined with IL-12 gene-modified BMSCs shows synergistic antitumor effect in glioma-bearing nude mice.

Promoting Osseointegration of Dental Implants in Dog Maxillary Sinus Floor Augmentation Using Dentin Matrix Protein 1-Transduced Bone Marrow Stem Cells.

Beta-tricalcium phosphate (β-TCP) has been employed successfully as a synthetic graft material in maxillary sinus floor augmentation (MSFA) for placing dental implants. However, the lack of osteogenic and osteoinductive properties of this substitute invariably results in bone regeneration of low quality and quantity. The purpose of this study was to determine whether loading dentin matrix protein-1 (DMP1) gene-modified bone marrow mesenchymal stem cells (BMSCs) onto β-TCP promoted bone regeneration and osteointegration of dental implants in MSFA of dogs. BMSCs were transduced with a lentiviral vector overexpressing the DMP1 gene (Lenti-DMP1) and with a lentiviral vector overexpressing enhanced green fluorescent protein (Lenti-EGFP) in vitro and were loaded into β-TCP scaffolds for autologous sinus grafting. Beagles received bilateral MSFA with four biomaterials (① Lenti-DMP1-transduced BMSCs/β-TCP, ② Lenti-EGFP-transduced BMSCs/β-TCP, ③ BMSCs/β-TCP, ④ β-TCP) and simultaneous implant placement at each sinus. Twelve weeks post operation, the maxillae were explanted, and every sinus was evaluated by radiographic observation, micro-CT and histological analysis. The osteogenic outcomes of bone regeneration and osseointegration were compared between the four groups. The sinuses grafted with Lenti-DMP1-transduced BMSCs/β-TCP constructs presented a significantly higher increase in compact radiopaque area, higher local bone mineral densities, greater bone-implant contact and greater bone density when compared to other three groups. These results demonstrated that combinations of β-TCP and DMP1 gene-modified BMSCs could be used to construct tissue-engineered bone to enhance mineralization of the regenerated bone and osseointegration of dental implants in MSFA.

Human Brain-Derived Neurotrophic Factor Gene-Modified Bone Marrow Mesenchymal Stem Cells Combined With Erythropoietin Can Improve Acute Spinal Cord Injury.

Objective:To assess the effect as well as mechanism of bone marrow mesenchymal stem cells (BMSCs) modified by the human brain–derived neurotrophic factor gene combined with erythropoietin (EPO) in the treatment of acute spinal cord injury (SCI) in rats.Methods:The Brain-derived neurotrophic factor (BDNF) gene was transected by a virus vector. Rats with SCI were randomly split into following groups: The normal saline (NS) group, the EPO group, The Basso, Beattie, and Bresnahan scores, messenger RNA BDNF expression, and apoptosis rates were compared between the 4 groups at 1, 3, 7, 14, and 21 days after SCI.Results:At 7, 14, and 21 days after operation, the expression of the BDNF gene in the other 3 groups was higher than that of the NS group, and the difference was statistically significant (P < .05). The apoptosis rate in the combined group was less than that of NS, EPO, and BDNF/BMSC groups, and the differences were statistically significant (P < .05).Conclusion:Brain-derived neurotrophic factor gene-modified BMSC transplantation combined with EPO can promote the repair of nerve function after SCI in rats.

Klotho gene-modified BMSCs showed elevated antifibrotic effects by inhibiting the Wnt/β-catenin pathway in kidneys after acute injury.

Klotho is a protein primarily expressed in renal tubular epithelial cells. Studies have suggested that Klotho is an antiaging protein that reduces renal fibrosis after acute kidney injury (AKI) and inhibits stem cell senescence. Bone marrow mesenchymal stem cells (BMSCs) have consistent proliferation ability and multidirectional differentiation ability and have been used to treat tissue injury. Thus, we hypothesized that Klotho expressed in BMSCs could increase the renal protective effects of BMSCs. To verify the hypothesis, we isolated BMSCs from C57BL/6 mice, transfected them with Klotho-GFP-adenovirus and investigated the change in BMSC proliferation. We then transplanted Klotho-GFP-BMSCs into mice with AKI and investigated the therapeutic effect compared with that of sham-treated mice and GFP-BMSC-transplanted mice. Kidney fibrosis after ischemia/reperfusion injury (IRI) was relieved by BMSC transplantation, and the antifibrotic effect of BMSCs was significantly enhanced by overexpressing the Klotho gene. Mechanistic studies showed that Klotho increased pluripotency gene expression in BMSCs. Klotho produced by Klotho-GFP-BMSCs inhibited the Wnt/β-catenin pathway in renal tubular epithelial cells (TECs). Klotho-GFP-BMSCs showed increased proliferative ability and more potent immuno-regulation ability than did GFP-BMSCs. Our findings suggested that Klotho gene-modified BMSCs may be a better choice for cell therapy after AKI.

Effect of neuroglobin gene-modified bone marrow mesenchymal stem cells on spinal cord injury in rats

Objective To observe the protective effect of neuroglobin gene-modified bone marrow mesenchymal stem cells(BMSCs)on spinal cord injury(SCI)in rats and explore the mechanism. Methods Sixty SD rats were randomly divided into saline group, BMSCs group, Neuroglobin(Ngb)/BMSCs group and blank group, given saline, BMSCs-culture medium, BMSCs-culture medium infected with Ngb after SCI,and nothing respectively.Measurements were evaluated by the Basso,Beattie and Bresnahan (BBB)scores at 1st,3rd,7th,14th and 21st day separately Results The BBB scores at 1st and 3rd day in each group were as follows:blank group(2. 76±0. 50,and 3. 70±0. 76),saline group(2. 72±0. 53,and 4. 06±0. 77), BMSCs group(2. 76±0. 71,and 4. 13±0. 76),and Ngb/BMSCs group(2. 79±4. 20,and 4. 20±0. 82). There was no significant difference among groups. At 7th, 14th and 21st day, the BBB scores in each group were gradually increased:blank group(5. 22±0. 64,6. 16±0. 78,and 7. 54±0. 90), saline group(5. 72±0. 94, 7. 61±0. 76, and 10. 87±0. 91), BMSCs group(5. 87±0. 84, 8. 33±0. 94, and 11. 45±1. 02), and Ngb/BMSCs group(6. 52±0. 95, 9. 78±0. 84, and 13. 33±1. 05).There were statistically significant difference between Ngb/BMSCs group and others(P< 0. 05). Conclusion Neuroglobin gene-modified BMSCs can make injured spinal cord supply stable and a high level Ngb in time, which can obviously help recovery of neural function after SCI in rats. Key words: Neuroglobin; Gene modified; Bone marrow mesenchymal stem cells; Spinal cord injury

Fibrin patch-based insulin-like growth factor-1 gene-modified stem cell transplantation repairs ischemic myocardium.

Bone marrow mesenchymal stem cells (BMSCs), tissue-engineered cardiac patch, and therapeutic gene have all been proposed as promising therapy strategies for cardiac repair after myocardial infarction. In our study, BMSCs were modified with insulin-like growth factor-1 (IGF-1) gene, loaded into a fibrin patch, and then transplanted into a porcine model of ischemia/reperfusion (I/R) myocardium injury. The results demonstrated that IGF-1 gene overexpression could promote proliferation of endothelial cells and cardiomyocyte-like differentiation of BMSCs in vitro. Four weeks after transplantation of fibrin patch loaded with gene-modified BMSCs, IGF-1 overexpression could successfully promote angiogenesis, inhibit remodeling, increase grafted cell survival and reduce apoptosis. In conclusion, the integrated strategy, which combined fibrin patch with IGF-1 gene modified BMSCs, could promote the histological cardiac repair for a clinically relevant porcine model of I/R myocardium injury.

Interferon-β gene-modified human bone marrow mesenchymal stem cells attenuate hepatocellular carcinoma through inhibiting AKT/FOXO3a pathway

Objective:This study aims to investigate the using of bone marrow mesenchymal stem cells (BMSCs) genetically engineered to produce interferon-β (IFN-β) as a gene delivery system to treat hepatocellular carcinoma (HCC) in vitro and in vivo.Methods:To measure the effects on tumour cell growth in vitro, IFN-β-producing BMSCs (BMSC/IFN-β) were co-cultured with the HCC cell line HepG2 and Huh7. Enzyme-linked immunosorbent assay (ELISA) was used to detect the IFN-β secretion in the BMSC culture condition medium (CM). The effect of BMSC/IFN-β on HCC cells proliferation was examined both in vitro and in vivo by using MTT, colony formation assay, BrdU staining, cell cycle analysis, and xenografted NOD/SCID mouse tumour model. To examine the impact of BMSC/IFN-β on the AKT/FOXO3a signalling, RT–PCR and western blotting were performed.Results:The BMSC/IFN-β cells can stably secrete high levels of IFN-β. Both MTT and colony forming assay showed that HCC cells had a lower growth rate when cultured in BMSC/IFN-β-CM as compared with that in BMSC/vector-CM or DMEM culture group. Co-culture with BMSC/IFN-β-CM dramatically decreased the percentages of cells with incorporated BrdUrd. In BMSC/IFN-β-CM-treated HCC cells, the proportion of G1-phase cells increased but it decreased in the S phase of the cell. The BMSC/IFN-β inhibited HCC growth in NOD/SCID mice and proved the survival period of these mice. Compared with the control group, p21 and p27 expression of hepatoma cells increased, whereas cyclin D1 and phosphorylation of Rb expression decreased when co-cultured with BMSC/IFN-β-CM. It was associated with suppression of Akt activity and enhanced transcriptional activity of FOXO3a.Conclusion:The IFN-β gene-modified BMSCs can effectively inhibit the proliferation of HCC cells in vitro and in vivo through inhibiting AKT/FOXO3a pathway. These results indicate that BMSC/IFN-β are a powerful anticancer cytotherapeutic tool for HCC.

Effect of Neuroglobin Genetically Modified Bone Marrow Mesenchymal Stem Cells Transplantation on Spinal Cord Injury in Rabbits

ObjectiveThis study aims to investigate the potentially protective effect of neuroglobin (Ngb) gene-modified bone marrow mesenchymal stem cells (BMSCs) on traumatic spinal cord injury (SCI) in rabbits.MethodsA lentiviral vector containing an Ngb gene was constructed and used to deliver Ngb to BMSCs. Ngb gene-modified BMSCs were then injected at the SCI sites 24 hours after SCI. The motor functions of the rabbits were evaluated by the Basso–Beattie–Bresnahan rating scale. Fluorescence microscopy, quantitative real-time PCRs, Western blots, malondialdehyde (MDA) tests, and terminal deoxynucleotidyltransferase-mediated UTP end labeling assays were also performed.ResultsNgb expression in the Ngb-BMSC group increased significantly. A more significant functional improvement was observed in the Ngb-BMSC group compared with those in the other groups. Traumatic SCI seemingly led to an increase in MDA level and number of apoptotic cells, which can be prevented by Ngb-BMSC treatment.ConclusionThis study demonstrates that Ngb gene-modified BMSCs can strengthen the therapeutic benefits of BMSCs in reducing secondary damage and improving the neurological outcome after traumatic SCI. Therefore, the combined strategy of BMSC transplantation and Ngb gene therapy can be used to treat traumatic SCI.

Inhibition of liver fibrosis by IL-10 gene-modified BMSCs in a rat model

To investigate the potential curative effects of bone marrow mesenchymal stem cells (BMSCs) overexpressing IL-10 for treating liver fibrosis by using a CCl4-induced rat model. BMSCs were cultured and marked by DAPI staining of the cells' nuclei. Meanwhile, 60 Wistar rats were treated with CCl4 to induce liver fibrosis and randomly divided into three groups: A: injected with DAPI-marked BMSCs transfected with pcDNA3.0-IL-10; B: injected with DAPI-marked BMSCs; C: injected with phosphate-buffered saline. Histological analysis of excised livers was conducted to observe BMSCs (by DAPI marker) and fibrosis (by Masson's stain). Western blotting was used to detect IL-10 expression in BMSCs. Effects on expression of tumor necrosis factor-alpha (TNFa) were analyzed by enzyme-linked immunosorbent assay, and on hydroxyproline (HYP) levels were analyzed by the acid hydrolysis method. Following CCl4-inducement, rat livers showed the characteristic pathological changes of fibrosis and DAPI-marked cells (BMSCs) were observed. Compared with group C (pulmonary fibrosis score: 3.15+/-0.96; HYP level: 1.89+/-1.03 mug/mg), the extent of liver fibrosis was significantly lower in group A (pulmonary fibrosis score: 1.01+/-0.35; HYP level: 0.73+/-0.29 mug/mg) and group B (pulmonary fibrosis score: 1.34+/-0.65; HYP level: 1.21+/-0.78 mug/mg). The therapeutic effects were significantly greater in group A than group B (q=-4.73, P less than 0.05). TNFa protein expression was significantly lower in group A (275.21+/-86.35) and group B (321.76+/-98.49) than in group C (476.23+/-126.43). The TNFa expression was significantly lower in group A than group B (q=-7.43, P less than 0.05). IL-10 gene-modified BMSCs are effective for treating liver fibrosis in a CCl4-induced rat model.

Treatment of osteonecrosis of the femoral head with hBMP-2-gene-modified tissue-engineered bone in goats

The efficacy of beta-tricalcium phosphate (beta-TCP) loaded with bone morphogenetic protein-2 (BMP-2)-gene-modified bone-marrow mesenchymal stem cells (BMSCs) was evaluated for the repair of experimentally-induced osteonecrosis of the femoral head in goats. Bilateral early-stage osteonecrosis was induced in adult goats three weeks after ligation of the lateral and medial circumflex arteries and delivery of liquid nitrogen into the femoral head. After core decompression, porous beta-TCP loaded with BMP-2 gene- or beta-galactosidase (gal)-gene-transduced BMSCs was implanted into the left and right femoral heads, respectively. At 16 weeks after implantation, there was collapse of the femoral head in the untreated group but not in the BMP-2 or beta-gal groups. The femoral heads in the BMP-2 group had a normal density and surface, while those in the beta-gal group presented with a low density and an irregular surface. Histologically, new bone and fibrous tissue were formed in the macropores of the beta-TCP. Sixteen weeks after implantation, lamellar bone had formed in the BMP-2 group, but there were some empty cavities and residual fibrous tissue in the beta-gal group. The new bone volume in the BMP-2 group was significantly higher than that in the beta-gal group. The maximum compressive strength and Young's modulus of the repaired tissue in the BMP-2 group were similar to those of normal bone and significantly higher than those in the beta-gal group. Our findings indicate that porous beta-TCP loaded with BMP-2-gene-transduced BMSCs are capable of repairing early-stage, experimentally-induced osteonecrosis of the femoral head and of restoring its mechanical function.