파킨슨병의 질병연구모델 및 세포치료제 공급원으로써의 사람 전분화 줄기세포에 관한 연구

Abstract

Human pluripotent stem cells as typified by human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) with their self-renewal and differentiation properties hold great promise for understanding and treating human disease. Especially Human pluripotent stem cells derived neural stem cells (hNSCs) are useful to offer direct information to understand human brain development and to develop therapies for intractable human brain disorders. However, due to the inherent sensitivity of hNSCs, culturing hNSCs in a controlled, biorelevant and repeatable manner is known to be difficult. A major challenge in hNSCs-mediated regenerative medicine is the development of defined culture systems for the long-term expansion of clinical-grade hNSCs. Here we demonstrate that long term exposure of insulin was unexpectedly toxic to developing neural precursor cells and differentiated neurons of human origin. The insulin-mediated cell death is specific to hNSCs, as the similar treatment of insulin did not cause cell death, but rather enhanced cell survival in neural stem cells derived from rodent brain tissues. The species dependent toxicity of insulin is mediated by decreased availability of insulin receptor substrate 1 (IRS-1), the molecule downstream to insulin-insulin receptor binding in the intracellular signaling of insulin, and in turn reduced Akt-mediated signal activation. These findings provide a possible scenario supporting previous epidemiologic studies for increased prevalence of neurodegenerative disorders in hyperinsulinemia. We next examined whether transplantable neural stem cells can be generated from hiPSCs with this optimized insulin conditions. Unlike hESCs, hiPSCs can be created from the tissue of the same patient that will receive the transplantation, thus avoiding immune rejection, and the lack of ethical implications. Hence hiPSCs can offer a human disease model and promise for autologous cell transplantation in neurodegenerative disorders such as Parkinson’s disease (PD). To explore this possibility, here we have examined dopamine (DA) neuron differentiation of the hiPSCs established by different methods of reprogramming factor delivery such as those using lentiviral (Lenti-hiPSCs), retroviral vectors (Retro-iPSCs), and direct protein delivery (Pro-hiPSCs). All eight hiPSC lines tested could be induced to yield uniform populations of neural precursor cells (NPCs), which subsequently differentiated into high proportions of DA neurons. However, NPCs derived from Pro-hiPSCs (Pro-hiPSC-NPCs) are the safer and stable DA neuronal source over those of viral vector-mediated hiPSCs. Pro-hiPSC-NPCs were highly expandable for at least 8 passages without losing DA neurogenic potential. This is in a clear contrast to Lenti- and Retro-hiPSC-derived NPCs which underwent rapid cellular senescence with increased p53 protein levels. Furthermore, expression of exogenous reprogramming factors was continued in differentiated Lenti-hiPSCs. DA neurons derived from Pro-hiPSC-NPCs exerted functions as presynaptic DA neurons and were capable of reversing motor deficits in a rat PD model upon transplantation. In conclusion, we propose an optimal insulin treatment ensuing cell survival, proliferation and differentiation in the cultures for human neural stem cells. In addition, we demonstrate the potential clinical use of protein-based hiPSCs for personalized cell therapy of PD and other degenerative disorders.