Pengpai News reporter Lu Xinwen
According to the WeChat public account of the Center for Excellence in Brain Science and Intelligent Technology, the Journal of Clinical Investigation published an online research paper entitled "Targets of Differentiation of Dopaminergic Neurocytes in Human Midbrain" (Diagnostic Cell Treatment Results of Parkinson's Disease Cells")
In this study, scientists analyzed the single-cell transcriptome map of the dopaminergic nerve differentiation of the midbrain, discovered specific surface protein molecules of the dopaminergic nerve progenitor cells of the midbrain, and used them to obtain high-purity target donor cells to achieve stable and predictable results of Parkinson's disease cell therapy.
This research was completed by Chen Yuejun's research group of the Center for Excellence in Brain Science and Intelligent Technology, Chinese Academy of Sciences (Institute of Neuroscience), Key Laboratory of Primate Neurobiology, and Shanghai Brain Science and Brain-like Research Center.
Global Burden of Disease Statistics show that there are approximately 9.3 million Parkinson’s Disease (PD) patients worldwide in 2030, of which about 4.94 million are present in China. Parkinson's disease brings great pain to patients themselves and also puts heavy burdens on families and society. Parkinson's disease is mainly caused by the degenerative lesions and losses of dopamine neurons in the midbrain of midbrain substantia nigra, causing a significant reduction in the dopamine content of striatal body, resulting in increased tone, slow motion, static tremor, and abnormal gait. There is no effective solution to cure Parkinson's disease in clinical practice. The current treatment methods are mainly symptomatic treatment, including drug treatment represented by levodopa and deep brain stimulation. However, drug treatment is only effective in the early stages, while deep brain stimulation is only suitable for some patients. The follow-up cost is high and it will cause side effects such as depression and . Therefore, new treatment strategies and methods are urgently needed.
cell replacement therapy (cell therapy) is considered a promising PD treatment strategy. Cell therapy uses transplantation of exogenous nerve cells to supplement lost dopaminergic neurons, thereby restoring the brain dopamine release level and relieving the patient's motor dysfunction. Human pluripotent stem cells, including human embryonic stem cells and human -induced pluripotent stem cells , can produce transplantable midbrain dopaminergic neural progenitor cells through in vitro differentiation, providing a large number of renewable cellular resources for cell therapy. At present, many countries including China, the United States, Japan and Australia have successively carried out clinical trials of PD cell therapy based on human pluripotent stem cell technology, but there are still many problems that need to be solved. (1) The donor cells for transplantation obtained by in vitro differentiation are highly heterogeneous; (2) After the donor cell transplantation, the proportion of target neurons (dopaminergic neurons) in the brain graft is usually low (no more than 10%); (3) The cell composition of the graft is unknown. Therefore, how to obtain standardized neural cell products to ensure stable and predictable therapeutic effects after transplantation remains the main challenge facing Parkinson's disease cell therapy in clinical wider and safer applications.
To solve the above problem, the researchers first drew a single-cell transcriptome map of the differentiation of human pluripotent stem cells into mesencephalic dopaminergic nerve cells, and found that the in vitro differentiation process of dopaminergic nerve cells simulates the development process of multiple but adjacent human fetal brain regions, including the ventral midbrain, the midbrain-hindbrain boundary (MHB, also known as the isthmus) and the ventral hindbrain. Only some ventral midbrain cells can further differentiate into midbrain dopaminergic neurons, while cells in other brain regions are mainly differentiated into GABAergic neurons, glutamategic neurons, motor neurons, and serotonergic neurons. This result explains the reason why human pluripotent stem cells differentiate into midbrain dopaminergic nerve cells to produce heterogeneous cell populations.
researchers further reconstructed the differentiation trajectory of human midbrain dopaminergic nerve cells and found specific surface protein molecules, CLSTN2 and PTPRO, representing early or late dopaminergic nerve progenitor cells. These surface protein molecules can be used to predict the efficiency of the terminal differentiation of heterogeneous neural progenitor cell populations obtained by in vitro differentiation into midbrain dopaminergic neurons (Figure 1A).The researchers sorted and enriched neural progenitor cells expressing these surface molecular proteins from heterogeneous neural progenitor cells, and transplanted them into the brain of PD model mice. They found that the transplantation of the sorted neural progenitor cells can produce highly enriched midbrain dopaminergic neurons, with a proportion of up to 80%. Most (about 90%) of the dopaminergic neurons are substantia nigra midbrain dopaminergic neurons subtype - the dopaminergic neuron subtype that is mainly missing in the brain of patients with PD. Dopaminergic neurons in grafts can be integrated into the host circuit and specifically innervate the dorsal striatum of the host. Further studies found that dopaminergic nerve cells in the sorted group had higher therapeutic efficacy than those in the unsorted group, manifested as stronger dopaminergic innervation of the host striatum and only a small number of cells transplanted (10% of conventional therapeutic cell dose) could achieve therapeutic effect (Figure 1B).
Figure 1 | (A) Through the time-course single-cell transcriptome of the human midbrain dopaminergic differentiation process in vitro, the cell type heterogeneity of cultures was analyzed and the surface molecular markers of novel dopaminergic progenitor cells were found. (B) Unsorted neural progenitor cells and marker sorted neural progenitor cells (CLSTN2+ or PTPRO+) were transplanted into Parkinson's disease mouse model, and the transplant results were evaluated by behavioral , electrophysiology, histological staining and other means. (C) By dissecting the grafts in the brains of Parkinson's mice and performing single-cell transcriptome analysis, it was found that the grafts after the novel marker had stable and predictable graft results, and the target neurons (i.e., midbrain dopaminergic neurons) were highly enriched, and most of the non-target neurons were eliminated.
single-cell sequencing of the grafts simultaneously found that the proportion of dopaminergic neurons in the unsorted group of grafts was low and contained multiple types of non-purpose neurons, including serotonergic neurons that clearly could cause side effects. In the sorting group, the dopaminergic neurons in the midbrain were highly enriched, and most non-target neuron types were eliminated (Figure 2A). Meanwhile, grafts sorted with different molecular markers had highly consistent neuronal composition (Fig. 2B), suggesting that transplantation of highly enriched dopaminergic neural progenitors can obtain stable and predictable transplant results.
Figure 2 | (A) Unsorted progenitor cells, CLSTN2+ progenitor cells and PTPRO+ progenitor cell-derived grafts, typical plots of immunostaining with human nucleus (hN) and dopaminergic neuron marker TH and quantification of the proportion of TH+ neurons in the graft. scale bar , 100μm. (B) By dissecting the grafts in the brains of Parkinson's mice and performing single-cell transcriptome analysis, it was found that the grafts after the novel marker had stable and predictable graft results, and the target neurons (i.e., midbrain dopaminergic neurons) were highly enriched, and most of the non-target neurons were eliminated.
The single-cell transcriptome maps of in vitro differentiation and in vivo grafts provided a more comprehensive understanding of the generation of cellular heterogeneity during the differentiation of midbrain dopaminergic nerve cells and the source of non-target cells in grafts. At the same time, the researchers established a method to obtain highly purified donor cells that can obtain stable and predictable cell treatment results after transplantation. This study has taken an important step toward achieving more effective and safer cell replacement therapy for Parkinson's disease.
This study was completed by Xu Peibo, doctoral student at the Center for Excellence of Brain and Intelligence, He Hui, and postdoctoral student Gao Qinqin under the guidance of Researcher Chen Yuejun. Zhou Yingying, Wu Ziyan, Zhang Xiao, Sun Linyu, Hu Gang, Guan Qian, You Zhiwen, Zhang Xinyue, Zheng Wenping and Researcher Xiong Man from the Institute of Brain Science of Fudan University, provided important help to this research. Professor Aaron Gitler of Stanford University and Researcher Liang Zhifeng of Brain Intelligence Center gave valuable suggestions for revision of the paper. The research was supported by the Ministry of Science and Technology, , Chinese Academy of Sciences, , Shanghai, and the Foundation Committee. This result has applied for intellectual property protection , and signed a technology transfer agreement with a third-party company for milestone payment model, with a total amount of more than RMB 40 million.
Editor in charge: Tang Yubing Photo editor: Li Jingyun
Proofreading: Luan Meng
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