| Ischemic stroke is the second leading cause of death and disability in the world. Due to the inability to replace damaged or lost neural cells, and current therapeutic strategies such as interventional procedures and surgery are limited, a bottleneck exists in ischemic stroke therapy.Recently, cell-based therapy using adult stem cells such as neural stem cells (NSCs) has provided hope for enhancing tissue repair and functional recovery after ischemic stroke. NSCs can differentiate into multiple functional neural cell types and hold the greatest potential for therapeutic applications. However, its clinical application has been greatly limited because of the risk of immune rejection and ethical controversy. Hence, it is necessary to find an alternative source of pluripotent cells to replace NSCs.In the last decade, induced pluripotent stem cells (iPSCs) were introduced as a new source of stem cells that can avoid the risk of immune rejection and ethical issues. iPSCs can be generated by transduction of defined transcription factors from adult somatic cells through reprogramming and have been differentiated in vitro into the early neural stem cell stage or the neural lineage, including neurons and glial cells. Moreover, Traditional Chinese medicine (TCM) is also an effective and unique approach to cure neurological illness (especially ischemic brain injury). As one of TCM, Qing-kai-ling (QKL) injection (modified from an-gong-niu-huang pill (AGNHP)) has great anti-pyretic, transforming phlegm, activating the brain, regaining consciousness, pain release detoxification, and anti-inflammatory effects. It is widely used to treat the acute stage of hemorrhagic cerebral apoplexy and cerebral ischemic injury. It can protect the cerebral ischemia injury through reducing the infarction area, improving the neurological function and increasing the expression of endothelial nitric oxide synthase (eNOS).However, to better understand the in vivo behavior and efficacy of iPSCs, a noninvasive, sensitive, and clinically applicable approach for tracking the transplanted iPSCs and monitoring the therapeutic response in living subjects is warranted. PET is one of the best-suited modalities to evaluate stem cell therapy, since it can be used in patients clinically for both cell trafficking and monitoring the response to therapy. 18F-FDG PET is a powerful tool to study the etiopathogenesis and progression of neurologic diseases.On the one hand, although NSCs, iPSCs and QKL have been able to improve functional recovery in the compromised central nervous system, no direct comparison had been made between these two different cell types, or the combination of stem cells and QKL in a single animal model of cerebral ischemia. Therefore, we directly compare the therapeutic effect of NSCs and iPSCs, and explore whether the combination approaches have better effect in present study. On the other hand, single molecular imaging technique can not provide comprehensive physiological information and is hard to get enough information of transplantated stem cells itself. Dual-mode or multimode molecular imaging has complementary advantages, and can provide more and comprehensive physiological information of transplantated stem cells etc.Part 1:Spatiotemporal PET imaging of dynamic metabolic changes after combined therapeutic approach of induced pluripotent stem cells (iPSCs), neural stem cells (NSCs), and Chinese patent medicine in a rat model of cerebral ischemic-reperfusion injuryWe use microPET imaging to investigate the dynamic metabolic changes after combined therapeutic approach of induced pluripotent stem cells (iPSCs), neural stem cells (NSCs) and tranditional Chinese medicine Qing-kai-ling (QKL) in a rat model of cerebral ischemic-reperfusion injury. Cerebral ischemia was established by middle cerebral artery occlusion (MCAO) approach. For QKL treatment, rats were injected with QKL injection from the day after MCAO to the last day. Transplantation of the iPSCs and NSCs labeled with GFP into the lateral ventricle were preformed at Day 3 after ischemic stroke, the control group was injected with the same volume of PBS. MicroPET scan was performed on the next day post-MCAO and 1,2,3,4 weeks after stem cell transplantation, and the neurological test was done at the same time. As gold standard of nuclear medicine, autoradiography detection was done at the end of the last neurobehavioral evaluation and F-FDG microPET scan to confirm microPET scan results. The remaining rats were perfused with 0.9% saline followed by 4% chilled paraformaldehyde in PBS (pH 7.4). The brains were immediately removed for immunohistochemistry and immunofluorescence staining to study the survival and differentiation of transplantated stem cells. Compared with PBS control group, significantly higher 18F-FDG accumulation in the ipsilateral cerebral infarction was observed in five treatment groups from Week 1 to Week 4.18F-FDG accumulation in the iPSCs+QKL group increased steadily in the 4-week period and was higher than that of iPSCs group at Week 3 (P< 0.01) and Week 4 (P< 0.01). At Week 1,18F-FDG accumulation increased in the NSCs+QKL group and was higher than that in other groups, then subsequently decreasing gradually.18F-FDG accumulation in the iPSCs+QKL group was higher than that in other groups at Week 4.The neurological scores in five treatment groups were significantly higher than those in the PBS group from Week 3 to 4. Furthermore, there was a significant correlation between the PET results and neurological score (P< 0.05) and the integral optical density (IOD) of GLUT-1 (P< 0.01). The iPSCs+QKL group expressed more GFAP than the iPSCs group according to immunohistochemical analysis. Immunofluorescent findings suggested that transplanted iPSCs survived and migrated to the ischemic region and their daughter cells expressed protein markers of nerve and endothelial cells. Immunofluorescent results also showed that transplanted NSCs expressed neurons, astrocytes markers in vivo.Spatiotemporal PET imaging with 18F-FDG demonstrated that the metabolic and functional recovery can be improved by iPSCs or NSCs, especially iPSCs combined with QKL which is better than the stem cells treatment alone, suggesting it is a promising strategy to promote recovery in a rat model of MCAO.Part 2:Dual-mode molecular imaging in vivo tracking transplanted iPSCs in a rat model of ischemic strokeiPSCs are a kind of totipotent stem cells which are similar to embryonic stem cells (ESCs) and have the potential to develop into any tissue or organ. iPSCs transplantation may become a safe, effective and important way to solve some medical challenges. An in vivo, noninvasive, repeated, long-term, real-time dynamic visualization technology to monitor the iPSCs will become a key technology to deepen the research of iPSCs. This study aims to transplant sr39tk-I-fluc (mutations in thymidine kinase and luciferase) reporter gene transfected iPSCs and moniter them in real time using noninvasive, dynamic, dual-mode molecular imaging of microPET and optical imaging in a rat model of cerebral ischemic-reperfusion injury. This study also aims to in vivo evaluate the distribution, survival, growth, and migration of transplanted iPSCs. We have developed and built a noninvasive, dynamic, real-time dual-mode molecular imaging which could be used to moniter transfected iPSCs in vivo. We also preliminarily clarify and reveal the dynamic condition of transplanted iPSCs which may provide a new strategy for iPSCs research. |
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