Parkinson's disease, which has been known as a refractory disease, is caused by the degeneration of dopaminergic neurons in the substantia nigra of the midbrain. The disease is a fatal geriatric disease, since it frequently occurs and gradually causes chronic movement disorders. Therefore, it is necessary to develop a method for treating the disease. Until now, there have been known treatments such as drug therapy using several drugs, and surgery, which implants a deep brain stimulator. However, drug therapy has short term-effects, including side effects after continuous administration, thereby not being easily applied. Furthermore, surgical therapy for Parkinson's disease imposes physical and economic burdens on the patient. Accordingly, an alternative treatment for Parkinson's disease is absolutely needed.
Recently, cell replacement therapy, in which depleted or damaged cells can be replaced with new healthy ones, has been considered as an effective treatment for the disease. In particular, as studies on human stem cells have rapidly developed, many studies for using the stem cells to restore damaged tissues or cells which are hard to be repaired have been actively conducted widely in several applications. More specifically, since adult stem cells, which are used to restore damaged brain and neural tissues, are hard to obtain and supply, a study has been actively conducted on the differentiation of brain and neural cells from embryonic stem cells. Embryonic stem cells can be isolated from the inner cell mass of the blastocyst during the embryonic development stage, and can proliferate indefinitely in an undifferentiated state under specific culture conditions. Furthermore, since embryonic stem cells are pluripotent, they can differentiate into every cell type according to conditions. Therefore, embryonic stem cells can be a source of cells used in cell therapy of all kinds of the tissues.
Cell replacement therapy for Parkinson's disease has been studied for a long time. However, cell replacement therapy using human embryonic stem cells has lately been studied, and many research institutes worldwide are currently engaged in this research.
The current studies on human embryonic stem cells are described in the following publications:                Human embryonic stem cells were induced to differentiate into neural precursors and various types of neurons (Su-Chun Zhang et al., 2001).        The efficiency of generating dopaminergic neurons was increased by coculturing human embryonic stem cells with PA6 cells and then adding a glial cell derived neurotrophic factor, GDNF (Kimberley et al., University of Colorado, USA, 2004).        It was confirmed that dopaminergic neurons were generated from embryonic stem cell aggregates by coculturing human embryonic stem cells with a stromal cell, PA6 cell, and the dopaminergic neurons generated a dopaminergic neuron specific marker. Unfortunately, it was reported that after transplantation, only a small portion of the cells generated were the dopaminergic neurons, and other type of cells still remained (Zeng et al., National Institute on Drug Abuse, USA, 2004).        Embryonic stem cell aggregates were confirmed to differentiate into dopaminergic neurons in serum-free suspension culture without any other factors, in which the neurons released dopamine to respond electrophysiologically, and even after transplantation, the cells released dopamine (Schulz et al., BresaGen Inc. 2004).        Human embryonic stem cells were induced to form a cluster of neural progenitors using a different stromal cell, MS5 cell, and then various growth factors and differentiation-inducing factors were added, so that 70% or more of the neurons were differentiated into dopaminergic neurons (Perrier et al., Sloan-Kettering Institute, 2004).        A behavior disorder caused by Parkinson's disease was found to be improved, in which neural progenitors derived from human embryonic stem cells were transplanted into a Parkinsonian rat, and the transplanted cells spontaneously were differentiated into dopaminergic neurons (Tamir et al., Hadassah University Hospital, Israel, 2004).        In Korea, there are a few research institutes that are engaged in the study on the differentiation of dopaminergic neurons from human embryonic stem cells. It was published that almost 20% of neurons derived from Human embryonic stem cells were differentiated into dopaminergic neurons with various differentiation-inducing factors, in which function of the differentiated cells and other makers, however, were not be confirmed, the efficiency was lower than that in other publications, and the survival rate after transplantation was not reported (Sepill Park et al., Maria Infertility Hospital, 2004).        Human embryonic stem cells were cocultured with PA6 stromal cells, so as to differentiate into neural progenitors. The neural progenitors were cultured in the form of a spherical neural mass or a single cell with a combination of differentiation factors, thereby differentiating into dopaminergic neurons. Unfortunately, the differentiation efficiency and the result after transplantation were not impressive (Sanghoon Lee, et al., Hanyang University, Korea).        
Even though many research institutes worldwide are currently engaged in the research, there are still problems in that the efficiency of generating pure dopaminergic neurons from total cells is very low, and the survival rate and functionality after transplantation have not been improved. Therefore, the present inventors have established a method for obtaining neural progenitors, neurons, and pure dopaminergic neurons from embryonic stem cells with high efficiency of 80% or more, in which the cells at the stage of neural progenitor are subcultured to produce a large number of progenitors, neurons, and dopaminergic neurons, thereby completing the present invention.
USPTO Backgrounds
USPTO Backgrounds