Therapeutic potential of hepatocyte growth factor overexpressing human adipose tissue derived mesenchymal stem cells and its mechanism study in an amyotrophic lateral sclerosis mouse model : 간세포성장인자 유전자 도입 사람지방유래 중간엽줄기세포를 이용한 근위축성측삭경화증 마우스 모델에서 치료효능 및 기전연구

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수의과대학 수의학과
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서울대학교 대학원
amyotrophic lateral sclerosisapoptosishepatocyte growth factorhuman adipose tissue derived mesenchymal stem cellsmotor neuron proliferation
학위논문 (박사)-- 서울대학교 대학원 수의과대학 수의학과, 2017. 8. 윤화영.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder and seriously affects lower and upper motor neurons, resulting in progressive weakness and atrophy of skeletal muscles. Although several mechanisms have been proposed to likely contribute to sporadic disease pathogenesis, the etiology of motor neuron death remains elusory and there exists no method for effective treatment of ALS. Recently, stem cell therapy has been a promising therapeutic approach for this devastating disorder and has raised great expectations.
In this study, hepatocyte growth factor overexpressing human adipose tissue derived mesenchymal stem cells (HGF-hATMSCs) were generated by liposomal transfection with pMEX expression vector and their therapeutic potential for ALS was assessed in vitro. To assess the bioactivity of HGF-hATMSCs engineered to overexpress HGF following pMEX expression vector-mediated transduction, expression of the human HGF was evaluated by RT-PCR and ELISA. The secretion of HGF protein in HGF-hATMSCs increased by 7.7-fold compared to the control. These results demonstrate that HGF-hATMSCs were successfully generated by liposomal transfection. Immunophenotypic characteristics of HGF-hATMSCs were confirmed by positivity to CD29, CD44, CD73, CD90, and CD105 markers and negativity to CD31, CD34, CD45, and HLA-DR using flow cytometry. There was no difference in expression of surface markers between HGF-hATMSCs and control (unmodified hATMSCs). Thus, the results of flow cytometry analysis indicate that expression of stem cell surface markers did not change due to HGF gene transfection. To investigate the effect of HGF-hATMSCs on motor neuron proliferation, NSC34 cells and HGF-hATMSCs were co-cultured for 1, 2, and 3 days under transwell co-culture system. The WST-1 cell proliferation assay and trypan blue exclusion assay showed that proliferation of NSC34 cells was significantly promoted in a time-dependent manner in the HGF-hATMSCs groups. To assess the stimulatory effect of HGF-hATMSCs on motor neuron proliferation, cell cycle analysis was performed after culturing NSC34 cells and HGF-hATMSCs under indirect co-culture system. NSC34 cells were harvested for cell cycle analysis by flow cytometry. Cell cycle analysis showed that 73.27±4%, 10.68±2%, and 15.23±1.5% of control (NSC34 cells alone) were in G0/G1, S, and G2/M phases, respectively. For NSC34 cells after co-culturing with hATMSCs, G0/G1, S, and G2/M phases represented 63.1±2.1%, 15.1±1.83%, and 20.4±2% of the cell population, respectively. In the NSC34 cells co-cultured with HGF-hATMSCs, G0/G1, S, and G2/M phases represented 57.12±1.5%, 17.23±1.1%, and 24.1±1.3% of NSC34 cell population, respectively. The results of cell cycle analysis indicate that HGF-hATMSCs contribute to proliferation of NSC34 cells via the increase of S and G2/M phases during NSC34 cell cycle. Results from western blot analysis showed marked increases in the expression of cyclin D1 involved in S phase and mitosis, when NSC34 cells was co-cultured with HGF-hATMSCs or hATMSCs, and revealed that HGF produced from HGF-hATMSCs activates c-Met known as its receptor in NSC34 cells. These data show that HGF-hATMSCs can contribute to enhancing motor neuron proliferation. To investigate whether HGF-hATMSCs have a stimulatory effect on survival of motor neurons after induction of endoplasmic reticulum (ER) stress, NSC34 cells pre-treated with thapsigargin were cultured with HGF-hATMSCs in an indirect co-culture system. Survival rate of NSC34 cells was assessed by the WST-1 cell proliferation assay. After co-culture with HGF-hATMSCs, NSC34 cells viability (76±2% at Day 1 and 68.4±3% at Day 2) was highly increased compare to that of control (NSC34 cells alone, 52.1±2% at Day 1 and 25.2±1% at Day 2) or that of co-culture with hATMSCs (65.1±3% at Day 1 and 48.3±2% at Day 2). The results indicate that HGF-hATMSCs can indirectly stimulate survival of NSC34 cells when they were received ER stress.
In addition, to determine inhibitory effect of HGF-hATMSCs on apoptosis of motor neurons, NSC34 cells, after co-culture with HGF-hATMSCs, were analyzed by Annexin V and Propidium Iodide (PI) staining and flow cytometry. Annexin V positive cells were prominently decreased in NSC34 cells co-cultured with HGF-hATMSCs (8.6±0.2%) compared with control (28.6±0.1%). NSC34 cells co-cultured with hATMSCs showed decrease of Annexin V positive cells (22.6±0.1%) than control. Consistent with inhibitory effect of HGF-hATMSCs on apoptosis of motor neurons, the results of western blot analysis demonstrated that activation of PARP and caspase 3 was markedly lower in NSC34 cells co-cultured with HGF-hATMSCs than control. Taken together, these results show that HGF-hATMSCs can strongly inhibit apoptosis of NSC34 cells.
Therapeutic potential of HGF-hATMSCs for ALS was assessed in vivo. To investigate therapeutic effects of HGF-hATMSCs in a mouse model of ALS, the SOD1 G93A transgenic mice were divided into three groups including treatment with HGF-hATMSCs, hATMSCs, and saline as control, and were administrated by intra-spinal cord injection. After transplantation of HGF-hATMSCs, diseases onset point and rotarod failure were assessed to test progression of symptoms and the motor function. Treatment of HGF-hATMSCs significantly delayed symptom onset (111±2.7 days of age) in the SOD1 G93A transgenic mice compared to that of control (101±2.4 days of age). Symptom onset of the hATMSCs-treated group (108±1.6 days of age) began slower than that of control group. Average time of rotarod failure in HGF-hATMSCs-treated group, hATMSCs-treated group, and control group was 131.7±3.2, 128.1±3.1, and 120.8±2.9 days after birth, respectively. These results indicate that HGF-hATMSCs contribute to improvement of motor function and retardation of symptom onset in the SOD1 G93A transgenic mice. Furthermore, the lifespan of the mice was remarkably prolonged in HGF-hATMSCs-treated group (141.6±4.1 days) compare to the control group (127±4.1 days). The lifespan of hATMSCs-treated group was longer (135.8±2.7 days) than that of control group.
In conclusion, these data show that HGF-hATMSCs have a clear therapeutic potential in slowing down the disease progression in mice model with ALS and their application present the possibility as a novel approach for ALS treatment.
A new and useful alternative gene based stem cell therapy that can enable motor neuron regeneration or cell transplantation with which the limitation of ALS therapy for future use in human clinical applications as well as veterinary medicine may be overcome.
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College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Theses (Ph.D. / Sc.D._수의학과)
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