Projects
Name
Culture and in vitro differentiation of embryonic stem cells.
University
China (IFMSA-China) - Soochow University, Suzhou
Domain
Biology
Departement
Department of Cell Biology
Head
Tutor
Jing QU
Languages
English, Chinese
Duration
4 weeks
Availability
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
No No No No No No Yes Yes Yes No No No
Type of Research Project
- Basic science
What is the background of the project?
Human pluripotent stem cells (hPSCs), including mainly human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the capacity to differentiate to all cell types in the human body. Human pluripotent stem cells (hPSCs) have been used in many applications in basic research and translational medicine, including disease modeling, drug screening and cell therapy. In order to maintain their pluripotency, hPSCs require proper combinations of extrinsic signal stimuli to establish a stem cell niche in cell culture systems. Maintenance of high quality hPSCs is dependent on consistent in vitro cell culture conditions and handling techniques. Traditionally, hPSCs are cultured as a 2-dimensional (2D) monolayer on mouse embryonic fibroblast feeder cells (MEFs) in medium supplemented with either fetal bovine serum (FBS) or components extracted from serum.
What is the aim of the project?
In this study, we aim to understand the effect of microenvironment on in vitro differentiation of hPSCs, which might contribute to new techniques to culturing and inducing differentiation of hPSCs.
What techniques and methods are used?
1. Maintenance and cell culture of hPSCs: Transgene free human iPSCs (6-9-9 and 19-9-11), lentiviral integrated human iPSC (IMR90C4) and hESCs (H9, H13, H14) were maintained on Matrigel (BD Biosciences) or Synthemax plates (Corning) in mTeSR1 medium (STEMCELL Technologies). 2. Differentiation of hPSCs: hPSCs were dissociated into single cells with Accutase (Invitrogen) at 37°C for 5 min and then seeded onto a Matrigel-coated cell culture dish at 100,000 – 200,000 cell/cm2 in mTeSR1 supplemented with 5 μM ROCK inhibitor (Y-27632, Stemgent). Cells were then cultured in mTeSR1 containing cytokines. 3. Assesment of gene expression λ Real-time Polymerase Chain Reaction (RT-PCR) and Quantitative RT-PCR: Total RNA was prepared with the RNeasy mini kit (QIAGEN) and treated with DNase (QIAGEN). 1 μg RNA was reverse transcribed into cDNA via Oligo (dT) with Superscript III Reverse Transcriptase (Invitrogen). RT-PCR was done in triplicate with iQ SYBR Green SuperMix (Bio-Rad). λ Flow cytometry: Cells were dissociated into single cells and then fixed with 1% paraformaldehyde for 20 min at room temperature and stained with primary and secondary antibodies in PBS plus 0.1% Triton X-100 and 0.5% BSA. λ Western Blot Analysis: Cells were lysed in M-PER Mammalian Protein Extraction Reagent in the presence of Halt Protease and Phosphatase Inhibitor Cocktail. Proteins were separated by 10% Tris-Glycine SDS/PAGE (Invitrogen) under denaturing conditions and transferred to a nitrocellulose membrane. After blocking with 5% milk in TBST, the membrane was incubated with primary antibody overnight at 4°C. The membrane was then washed, incubated with an anti-mouse/rabbit peroxidase-conjugated secondary antibody at room temperature for 1 hr, and developed by SuperSignal chemiluminescence. Statistics analysis: Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined by Student’s t-test (two-tail) for two groups or one-way ANOVA for multiple groups.1. Maintenance and cell culture of hPSCs: Transgene free human iPSCs (6-9-9 and 19-9-11), lentiviral integrated human iPSC (IMR90C4) and hESCs (H9, H13, H14) were maintained on Matrigel (BD Biosciences) or Synthemax plates (Corning) in mTeSR1 medium (STEMCELL Technologies). 2. Differentiation of hPSCs: hPSCs were dissociated into single cells with Accutase (Invitrogen) at 37°C for 5 min and then seeded onto a Matrigel-coated cell culture dish at 100,000 – 200,000 cell/cm2 in mTeSR1 supplemented with 5 μM ROCK inhibitor (Y-27632, Stemgent). Cells were then cultured in mTeSR1 containing cytokines. 3. Assesment of gene expression λ Real-time Polymerase Chain Reaction (RT-PCR) and Quantitative RT-PCR: Total RNA was prepared with the RNeasy mini kit (QIAGEN) and treated with DNase (QIAGEN). 1 μg RNA was reverse transcribed into cDNA via Oligo (dT) with Superscript III Reverse Transcriptase (Invitrogen). RT-PCR was done in triplicate with iQ SYBR Green SuperMix (Bio-Rad). λ Flow cytometry: Cells were dissociated into single cells and then fixed with 1% paraformaldehyde for 20 min at room temperature and stained with primary and secondary antibodies in PBS plus 0.1% Triton X-100 and 0.5% BSA. λ Western Blot Analysis: Cells were lysed in M-PER Mammalian Protein Extraction Reagent in the presence of Halt Protease and Phosphatase Inhibitor Cocktail. Proteins were separated by 10% Tris-Glycine SDS/PAGE (Invitrogen) under denaturing conditions and transferred to a nitrocellulose membrane. After blocking with 5% milk in TBST, the membrane was incubated with primary antibody overnight at 4°C. The membrane was then washed, incubated with an anti-mouse/rabbit peroxidase-conjugated secondary antibody at room temperature for 1 hr, and developed by SuperSignal chemiluminescence. Statistics analysis: Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined by Student’s t-test (two-tail) for two groups or one-way ANOVA for multiple groups.
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?
The student will: 1. Learn and prepare cell culture, especially related knowledge about hPSCs, which benefits for future study in all areas; 2. Use molecular biology techniques, including Polymerase Chain Reaction (PCR), Western blotting, to study gene expression influenced under different media. 3. Observe the microenvironment of cells by flow cytometry and immunohistochemistry; Analyzing experiment results with IBM SPSS ® Statistics 20 software.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
There are 2-3 seminars together with other students and teachers, provided by Department of Cell Biology.
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
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic knowledge of molecular cell biology; Subjects passed: Molecular Biology / Cell Biology
Are there any legal limitations in the student’s involvement
No
Hours
6
Type of students accepted
This project accepts:
- Medical students
- Pre-Medical students from the American-British system
- Students in biomedical fields
Articles
- Qu J and Zhang H. Roles of mesenchymal stem cells in spinal cord injury. Stem Cells International 2017