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Identifying the correlation of embryonal neuroblastoma initiation with maternal stress-derived cellular condition
Korea (KMSA) - KyungHee University, Seoul
Department of Pathology, School of Medicine
Yong Jun Kim
Type of Research Project
- Basic science
What is the background of the project?
Neuroblastoma wihch is the most common malignancy under 1 year of age is originating from neural crest stem cells (NCSC). This type of cancer is known for correlation with temporal hypoxic condition which occurs in condition of maternal stress, however, details about pathologic mechanism of initiation and progression of neuroblastoma during embryonic development such stages of pre-/peri-/neo-natal is largely unkwon due to scarcity of proper research models. Recent advances in stem cell research give rise to successful modeling of developmental diseases and further precise analysis for pathologic mechanism of certain diseases. Although human NCSC has been differentiated from hESCs(human embryonic stem cell) for disease modeling studies including genetic disorders, it has not been yet employed in a study which associated with embryonic irritation from maternal conditions such as emotional trauma and physical stress.
What is the aim of the project?
The purpose of our project is to identify the correlation of initiation of embryonal neuroblastoma with maternal stress-derived ischemic/hypoxic embryonic cellular condition.
What techniques and methods are used?
In this study, we employ the human embryonic stem cells to achieve the neural crest stem cell (NCSC) which is origin of fetal neuroblastoma. To model pathologic cues for tumorigenesis, we stimulate NCSC with hypoxia in a various cellular conditions. To assess that cellular transition bound to cancer occur in NCSC under certain irritant condition, we perform the following methods; FACS(Fluorescence-activated cell sorting) analysis with CRISPR/Cas9* technique based PHOX2B::GFP(Green Fluorescent Protein) reporter hESC(human embryonic stem cell) system, FACS-sorting(fluorescence-activated cell sorting) and further assays for cellular features, and other methods in molecular biology. * CRISPR are direct repeats found in the DNA of many bacteria and archaea. The name is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats. 1) We generated GFP reporter hESC line by using the CRISPR/Cas9 technique. This line report with green fluorescence when the cells express endogenous PHOX2B protein which is known to be a marker protein in the neuroblatoma. FACS analysis after various hypoxic stimulation simply recognize the combinatorial conditions for cancer initiation with GFP signals. 2) We purify the population of PHOX2B expressing NCSC after critical hypoxic stimulation by FACS sorting. Isolated PHOX2B expressing cells is in vitro cultured and further tested for cancer cell features including proliferation, chromosomal instability, and lineage differentiation for neurons. 3) All the results from each step and method are confirmed with performing the molecular biological experiments such as quantitative PCR(Polymerase Chain Reaction), western blotting, and immunoflourescent staining. In terms of statistical analysis, SPSS** or Graphpad software will be used for one-way/two-way repeated measures ANOVA(analysis of variance). ** SPSS is the abbreviation of Statistical Package for Social Sciences and it is used by researchers to perform statistical analysis.
What is the role of the student?
- The student will mainly observe
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
1) Students are expected to maintain human embryonic stem cells and differentiate them into NCSCs(neural crest stem cell). The student will be educated basic skills for hESC(human embryonic stem cell) and NCSC culture, and have opportunity for single differentiation which takes about 14 days. 2) Students are expected to perform couple of assays for analyze cellular features such as migration, differentiation, and measuring DNA (deoxyribonucleic acid) damages. 3) Students are expected to validate molecular biological character of cultured cells by using quantitative real time PCR(Polymerase Chain Reaction), western blotting, and immunostaining for microscope-based imaging works. 4) Students are expected to conduct a statistical analysis using SPSS or Graphpad statistics software. 5) Students are expected to summarize data and illustrate figures. 6) Students are expected to present data in front of lab members.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
As we have lab meeting, students will have the opportunity to participate in presentations, ask questions and discuss topics related to the project with the professor and other researchers. Students can also attend other seminars and observe or attend various experiments. Students are expected to participate in lab meetings and actively ask questions and discuss the research project.
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 scientific report - The student will have the opportunity to present the results together with the supervisor at a conference
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
No particular skill is required. Basic labatory skills (e.g. pipetting, PCR) are appreciated. Motivation and willingness to learn is required. Subjects passed: Developmental biology, Pathology
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Pre-Medical students from the American-British system
- Kurapati; S.; Sadaoka; T.; Rajbhandari; L.; Jagdish; B.; Shukla; P.; Kim; Y.J.; Lee; G.; Cohen; J.I.; and Venkatesan; A. Role of JNK pathway in varicella-zoster virus lytic infection and reactivation. J. Virol. (2017)
- Nyffleler; J.; Karreman; C.; Leisner; H.; Kim; Y.J.; Lee; G.; Waldmann; T.; and Leist; M. Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants. ALTEX ( 2017)
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