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Differentiation of Midbrain Dopaminergic Neurons from Primate Pluripotent Stem Cells
China (IFMSA-China) - Nanjing Medical University, Nanjing
Institute for Stem Cell and Neural Regeneration, School of Pharmacy
Type of Research Project
- Basic science
What is the background of the project?
Human pluripotent stem cells, including embryonic stem cells and human induced pluripotent stem cells (hiPSCs), can be expanded in vitro and retain their capacity to differentiate into any cell type of the three germ layers. Hypothetically, they represent an unlimited source of cells for several applications including drug screening and cell replacement therapy for treatment of neurological disorders. Parkinson´s disease (PD), the second most common neurodegenerative disorder, is characterized by the selective loss of dopaminergic neurons of the substantia nigra of the midbrain. However, better understanding of the pathogenesis of PD is still needed. Thereby, a homogeneous, robust and rapid method to derive dopaminergic neurons from hiPSC for addressing gene function, for drug screening or eventual cell replacement therapy would be of great significance.
What is the aim of the project?
To fabricate a homogeneous, robust and rapid method to derive dopaminergic neurons from hiPSC of normal people and people with Parkinson's disease
What techniques and methods are used?
1. Human induced pluripotent stem cell culture: They were maintained on Matrigel in six-well plates and passaged enzymatically with ethylene diamine tetraacetic acid (EDTA) every 4–5 days. 2. Dopaminergic neuron differentiation: Dopaminergic neuron differentiation was induced using modifications to the floor plate progenitors method. 3. Immunostaining for cells: the cells are first incubated with Primary antibodies, and then incubated with the appropriate secondary antibodies. Images were acquired using an Eclipse 80i fluorescence microscope. 4. Quantitative real-time polymerase chain reaction (RT-PCR): Total RNA was extracted in Trizol reagent (Invitrogen) and cDNA was reverse-transcribed by using the SuperScript III First-Strand kit (Invitrogen). RT-PCR was performed using the Bio-Rad MyiQ real-time PCR detection system.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
1. Maintain human induced pluripotent stem cell on Matrigel in six-well plates and passaged enzymatically with Accutase every 4–5 days. 2. Know the process of the floor plate progenitors method to derive dopaminergic neuron differentiation. 3. Observe the morphology of the cells and cell-cell connection with fluorescence microscope by immunostaining. 4. Perform RT-PCR by using the Bio-Rad MyiQ RT-PCR detection system. Analyze the images obtained by a fluorescence microscope and the data by PCR and conduct the statistical comparisons.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Preliminary readings will be provided. In addition, the tutor will discuss and explain in detail about the project that students will be conducting. Students are encouraged to take part in the group meeting and seminars.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Cell culture, Statistical data analysis
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Students in biomedical fields
- Chen Y; Xiong M; Dong Y; et al. Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson’s Disease. Cell Stem Cell; 2016; 18: 817-826.
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