scholarly journals Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Christian Bucher ◽  
Amiq Gazdhar ◽  
Lorin M. Benneker ◽  
Thomas Geiser ◽  
Benjamin Gantenbein-Ritter
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Intervertebral Disc ◽  
Organ Culture ◽  
Nonviral Gene Delivery ◽  
Human Mscs ◽  
Differentiation Factor ◽  
Gdf5 Gene

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved byin vitroelectroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

scholarly journals Effect of lentivirus-mediated growth and differentiation factor-5 transfection on differentiation of rabbit nucleus pulposus mesenchymal stem cells

2022 ◽  
Vol 27 (1) ◽  
Author(s):  
Kun Zhu ◽  
Rui Zhao ◽  
Yuchen Ye ◽  
Gang Xu ◽  
Changchun Zhang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nucleus Pulposus ◽  
Lentiviral Vector ◽  
The Other ◽  
Cell Phenotype ◽  
Mrna Levels ◽  
Qrt Pcr ◽  
Differentiation Factor ◽  
Gdf5 Gene

Abstract Background Intervertebral disc degeneration (IDD) is a natural progression of age-related processes. Associated with IDD, degenerative disc disease (DDD) is a pathologic condition implicated as a major cause of chronic lower back pain, which can have a severe impact on the quality of life of patients. As degeneration progression is associated with elevated levels of inflammatory cytokines, enhanced aggrecan and collagen degradation, and changes in the disc cell phenotype. The purpose of this study was to investigate the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs)—a key factor in IDD—and to determine the effect of the growth and differentiation factor-5 (GDF5) on the differentiation of rabbit NPMSCs transduced with a lentivirus vector. Methods An in vitro culture model of rabbit NPMSCs was established and NPMSCs were identified by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Subsequently, NPMSCs were randomly divided into three groups: a transfection group (the lentiviral vector carrying GDF5 gene used to transfect NPMSCs); a control virus group (the NPMSCs transfected with an ordinary lentiviral vector); and a normal group (the NPMSCs alone). FCM, qRT-PCR, and western blot (WB) were used to detect the changes in NPMSCs. Results The GDF5-transfected NPMSCs displayed an elongated shape, with decreased cell density, and significantly increased GDF5 positivity rate in the transfected group compared to the other two groups (P < 0.01). The mRNA levels of Krt8, Krt18, and Krt19 in the transfected group were significantly higher in comparison with the other two groups (P < 0.01), and the WB results were consistent with that of qRT-PCR. Conclusions GDF5 could induce the differentiation of NPMSCs. The lentiviral vector carrying the GDF5 gene could be integrated into the chromosome genome of NPMSCs and promoted differentiation of NPMSCs into nucleus pulposus cells. Our findings advance the development of feasible and effective therapies for IDD.

scholarly journals Enhanced PRL-1 expression in placenta-derived mesenchymal stem cells accelerates hepatic function via mitochondrial dynamics in a cirrhotic rat model

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jae Yeon Kim ◽  
Jong Ho Choi ◽  
Ji Hye Jun ◽  
Sohae Park ◽  
Jieun Jung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Cell Therapy ◽  
Delivery Systems ◽  
Nonviral Gene Delivery ◽  
Early Gene ◽  
Next Generation ◽  
Atp Production ◽  
Mitochondrial Functions

Abstract Background Placenta-derived mesenchymal stem cells (PD-MSCs) have been highlighted as an alternative cell therapy agent that has become a next-generation stem cell treatment. Phosphatase of regenerating liver-1 (PRL-1), an immediate early gene, plays a critical role during liver regeneration. Here, we generated enhanced PRL-1 in PD-MSCs (PD-MSCsPRL-1, PRL-1+) using lentiviral and nonviral gene delivery systems and investigated mitochondrial functions by PD-MSCPRL-1 transplantation for hepatic functions in a rat bile duct ligation (BDL) model. Methods PD-MSCsPRL-1 were generated by lentiviral and nonviral AMAXA gene delivery systems and analyzed for their characteristics and mitochondrial metabolic functions. Liver cirrhosis was induced in Sprague-Dawley (SD) rats using common BDL for 10 days. PKH67+ naïve and PD-MSCsPRL-1 using a nonviral sysyem (2 × 106 cells/animal) were intravenously administered into cirrhotic rats. The animals were sacrificed at 1, 2, 3, and 5 weeks after transplantation and engraftment of stem cells, and histopathological analysis and hepatic mitochondrial functions were performed. Results PD-MSCsPRL-1 were successfully generated using lentiviral and nonviral AMAXA systems and maintained characteristics similar to those of naïve cells. Compared with naïve cells, PD-MSCsPRL-1 improved respirational metabolic states of mitochondria. In particular, mitochondria in PD-MSCsPRL-1 generated by the nonviral AMAXA system showed a significant increase in the respirational metabolic state, including ATP production and mitochondrial biogenesis (*p < 0.05). Furthermore, transplantation of PD-MSCsPRL-1 using a nonviral AMAXA system promoted engraftment into injured target liver tissues of a rat BDL cirrhotic model and enhanced the metabolism of mitochondria via increased mtDNA and ATP production, thereby improving therapeutic efficacy. Conclusions Our findings will further our understanding of the therapeutic mechanism of enhanced MSCs and provide useful data for the development of next-generation MSC-based cell therapy and therapeutic strategies for regenerative medicine in liver disease.

scholarly journals Nonviral Gene Delivery to Mesenchymal Stem Cells Using Cationic Liposomes for Gene and Cell Therapy

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
C. Madeira ◽  
R. D. Mendes ◽  
S. C. Ribeiro ◽  
J. S. Boura ◽  
M. R. Aires-Barros ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Cationic Liposome ◽  
Nonviral Gene Delivery ◽  
Great Promise ◽  
Full Potential ◽  
Genetically Engineer ◽  
Or Gene ◽  
First Time

Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from ~2% to ~35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and ~20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.

scholarly journals Enhanced PRL-1 expression in placenta-derived mesenchymal stem cells accelerates hepatic function via mitochondrial dynamics in a cirrhotic rat model

2020 ◽  
Author(s):  
Jae Yeon Kim ◽  
Jong Ho Choi ◽  
Ji Hye Jun ◽  
Sohae Park ◽  
Jieun Jung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Cell Therapy ◽  
Delivery Systems ◽  
Nonviral Gene Delivery ◽  
Early Gene ◽  
Next Generation ◽  
Atp Production ◽  
Mitochondrial Functions

Abstract Background: Placenta-derived mesenchymal stem cells (PD-MSCs) have been highlighted as an alternative cell therapy agent that has become a next-generation stem cell treatment. Phosphatase of regenerating liver-1 (PRL-1), an immediate early gene, plays a critical role during liver regeneration. Here, we generated enhanced PRL-1 in PD-MSCs (PD-MSCsPRL-1, PRL-1+) using lentiviral and nonviral gene delivery systems and investigated mitochondrial functions by PD-MSCPRL-1 transplantation for hepatic functions in a rat bile duct ligation (BDL) model. Methods: PD-MSCsPRL-1 were generated by lentiviral and nonviral AMAXA gene delivery systems and analyzed for their characteristics and mitochondrial metabolic functions. Liver cirrhosis was induced in Sprague-Dawley (SD) rats using common BDL for 10 days. PKH67+ naïve and PD-MSCsPRL-1 were intravenously administered into cirrhotic rats using a nonviral system (2x106 cells/animal). The animals were sacrificed at 1, 2, 3, and 5 weeks after transplantation and engraftment of stem cells, and histopathological analysis and hepatic mitochondrial functions were performed. Results: PD-MSCsPRL-1 were successfully generated using lentiviral and nonviral AMAXA systems and maintained characteristics similar to those of naïve cells. Compared with naïve cells, PD-MSCsPRL-1 improved respirational metabolic states of mitochondria. In particular, mitochondria in PD-MSCsPRL-1 generated by the nonviral AMAXA system showed a significant increase in the respirational metabolic state, including ATP production and mitochondrial biogenesis (*p<0.05). Furthermore, transplantation of PD-MSCsPRL-1 using a nonviral AMAXA system promoted engraftment into injured target liver tissues of a rat BDL cirrhotic model and enhanced the metabolism of mitochondria via increased mtDNA and ATP production, thereby improving therapeutic efficacy. Conclusions: Our findings will further our understanding of the therapeutic mechanism of enhanced MSCs and provide useful data for the development of next-generation MSC-based cell therapy and therapeutic strategies for regenerative medicine in liver disease.

scholarly journals Therapeutic Effects of rAAV-Mediated Concomittant Gene Transfer and Overexpression of TGF-β and IGF-I on the Chondrogenesis of Human Bone-Marrow-Derived Mesenchymal Stem Cells

2019 ◽  
Vol 20 (10) ◽  
pp. 2591 ◽  
Author(s):  
Stephanie Morscheid ◽  
Ana Rey-Rico ◽  
Gertrud Schmitt ◽  
Henning Madry ◽  
Magali Cucchiarini ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Growth Factor ◽  
Gene Transfer ◽  
Cartilage Defects ◽  
Therapeutic Effects ◽  
Human Mscs ◽  
Lacz Gene ◽  
Igf I

Application of chondroreparative gene vectors in cartilage defects is a powerful approach to directly stimulate the regenerative activities of bone-marrow-derived mesenchymal stem cells (MSCs) that repopulate such lesions. Here, we investigated the ability of combined recombinant adeno-associated virus (rAAV) vector-mediated delivery of the potent transforming growth factor beta (TGF-β) and insulin-like growth factor I (IGF-I) to enhance the processes of chondrogenic differentiation in human MSCs (hMSCs) relative to individual candidate treatments and to reporter (lacZ) gene condition. The rAAV-hTGF-β and rAAV-hIGF-I vectors were simultaneously provided to hMSC aggregate cultures (TGF-β/IGF-I condition) in chondrogenic medium over time (21 days) versus TGF-β/lacZ, IGF-I/lacZ, and lacZ treatments at equivalent vector doses. The cultures were then processed to monitor transgene (co)-overexpression, the levels of biological activities in the cells (cell proliferation, matrix synthesis), and the development of a chondrogenic versus osteogenic/hypertrophic phenotype. Effective, durable co-overexpression of TGF-β with IGF-I via rAAV enhanced the proliferative, anabolic, and chondrogenic activities in hMSCs versus lacZ treatment and reached levels that were higher than those achieved upon single candidate gene transfer, while osteogenic/hypertrophic differentiation was delayed over the period of time evaluated. These findings demonstrate the potential of manipulating multiple therapeutic rAAV vectors as a tool to directly target bone-marrow-derived MSCs in sites of focal cartilage defects and to locally enhance the endogenous processes of cartilage repair.

scholarly journals Enhanced PRL-1 expression in placenta-derived mesenchymal stem cells accelerates hepatic function via mitochondrial dynamics in a cirrhotic rat model

2020 ◽  
Author(s):  
Jae Yeon Kim ◽  
Jong Ho Choi ◽  
Ji Hye Jun ◽  
Sohae Park ◽  
Jieun Jung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Cell Therapy ◽  
Delivery Systems ◽  
Nonviral Gene Delivery ◽  
Early Gene ◽  
Next Generation ◽  
Atp Production ◽  
Mitochondrial Functions

Abstract Background: Placenta-derived mesenchymal stem cells (PD-MSCs) have been highlighted as an alternative cell therapy agent that has become a next-generation stem cell treatment. Phosphatase of regenerating liver-1 (PRL-1), an immediate early gene, plays a critical role during liver regeneration. Here, we generated enhanced PRL-1 in PD-MSCs (PD-MSCsPRL-1, PRL-1+) using lentiviral and nonviral gene delivery systems and investigated mitochondrial functions by PD-MSCPRL-1 transplantation for hepatic functions in a rat bile duct ligation (BDL) model.Methods: PD-MSCsPRL-1 were generated by lentiviral and nonviral AMAXA gene delivery systems and analyzed for their characteristics and mitochondrial metabolic functions. Liver cirrhosis was induced in Sprague-Dawley (SD) rats using common BDL for 10 days. PKH67+ naïve and PD-MSCsPRL-1 were intravenously administered into cirrhotic rats using a nonviral system (2x106 cells/animal). The animals were sacrificed at 1, 2, 3, and 5 weeks after transplantation and engraftment of stem cells, and histopathological analysis and hepatic mitochondrial functions were performed.Results: PD-MSCsPRL-1 were successfully generated using lentiviral and nonviral AMAXA systems and maintained characteristics similar to those of naïve cells. Compared with naïve cells, PD-MSCsPRL-1 improved respirational metabolic states of mitochondria. In particular, mitochondria in PD-MSCsPRL-1 generated by the nonviral AMAXA system showed a significant increase in the respirational metabolic state, including ATP production and mitochondrial biogenesis (*p<0.05). Furthermore, transplantation of PD-MSCsPRL-1 using a nonviral AMAXA system promoted engraftment into injured target liver tissues of a rat BDL cirrhotic model and enhanced the metabolism of mitochondria via increased mtDNA and ATP production, thereby improving therapeutic efficacy.Conclusions: Our findings will further our understanding of the therapeutic mechanism of enhanced MSCs and provide useful data for the development of next-generation MSC-based cell therapy and therapeutic strategies for regenerative medicine in liver disease.

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