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Proliferation and differentiation of human stem cells
Department of Molecular Cell Biology, Institute of Biochemistry, Vilnius University Life Sciences Center
Prof. Rūta Navakauskienė, PhD
Aistė Zentelytė, PhD Student
Type of Research Project
- Basic science
What is the background of the project?
Amniotic fluid is a novel and promising source of stem cells. These stem cells are considered to be multipotent but it is also shown to have some pluripotency markers like the expression of embryonic transcription factors and ability to differentiate to cell lineages from all three germ layers. There are practically no ethical restrictions for isolation of these stem cells since they can be isolated from samples obtained during amniocentesis. But a lot needs to be done before amniotic fluid stem cells could be used for a therapeutic approach.
What is the aim of the project?
The aim of the project is to evaluate the proliferation and differentiation of human stem cells.
What techniques and methods are used?
Amniotic fluid is obtained through amniocentesis at Santaros Hospital Centre for Medical Genetics. Stem cells are extracted, multiplied and frozen for storage. Some of the cells are thawed and analyzed using flow cytometry. First, cells are labelled with fluorescent markers and then characterized by their surface antigen expression. Cells are sorted by their immunophenotypes. Gene expression analysis is carried out with the selected cells in order to evaluate differentiation potential. RNR is extracted from the cell and PCR is performed to multiply genetic material. This helps to evaluate the quantity of expressed genes (qPCR) and by analyzing relative gene expression data in SPSS we can evaluate proliferating cell characteristics. We also compare the differentiation potential of healthy and non-healthy cell lines with genetic disorders, for example, with Trisomy 21. Both groups of pluripotent stem cells are induced to differentiate into neural cells by adding different concentrations and combinations of induction agents. By performing the same gene expression analysis protocol we try to understand the best environment for stem cells proliferation and differentiation into selected tissues. We also compare the difference in differentiation potential between control (healthy stem cells) and affected (Trisomy 21) stem cells. This is done by comparing different data recorded during qPCR.
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
- The tasks will be done under supervision
- The tasks of the student will be performed on his/her own
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
What are the tasks expected to be accomplished by the student?
The student will assist the tutor during the lab work of this project. The student will participate in tasks, such as stem cell cultivation, stem cell preparation for further experiments. Also, the student will perform techniques for phenotypic characterization of sample stem cells, for example, light microscopy and flow cytometry. Student will assist the tutor while doing gene expression analysis (RNA isolation, complementary DNA synthesis, real-time quantitative polymerase chain reaction) and collecting and interpreting the data.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
There will be no theoretical teaching provided.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic knowledge and understanding of aseptic technique. We also expect that the student will attend at least 80% of the research exchange, be responsible for his/her actions at the practice and be accountable to the supervisor and LORE/NORE.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields - Dental medicine students (IADS members)
- Jūratė Savickienė; Sandra Baronaitė; Aistė Zentelytė; Gražina Treigytė; and Rūta Navakauskienė; “Senescence-Associated Molecular and Epigenetic Alterations in Mesenchymal Stem Cell Cultures from Amniotic Fluid of Normal and Fetus-Affected Pregnancy;” Stem Cells International; vol. 2016; Article ID 2019498; 13 pages; 2016. https://doi.org/10.1155/2016/2019498
- Monika Glemžaitė and Rūta Navakauskienė; “Osteogenic Differentiation of Human Amniotic Fluid Mesenchymal Stem Cells Is Determined by Epigenetic Changes;” Stem Cells International; vol. 2016; Article ID 6465307; 10 pages; 2016. https://doi.org/10.1155/2016/6465307.
- Savickienė; J. ; Matuzevičius; D. ; Baronaitė; S. ; Treigytė; G. ; Krasovskaja; N. ; Zaikova; I. ; Navakauskas; D. ; Utkus; A. and Navakauskienė; R. (2017); Histone Modifications Pattern Associated With a State of Mesenchymal Stem Cell Cultures Derived From Amniotic Fluid of Normal and Fetus‐Affected Gestations. J. Cell. Biochem.; 118: 3744-3755. doi:10.1002/jcb.26022
- Gasiūnienė; M; Zubova; A; Utkus; A; Navakauskienė; R. Epigenetic and metabolic alterations in human amniotic fluid stem cells induced to cardiomyogenic differentiation by DNA methyltransferases and p53 inhibitors. J Cell Biochem. 2019; 120: 8129‐ 8143. https://doi.org/10.1002/jcb.28092
- Gasiūnienė; M; Zentelytė; A; Wojtas; B; et al. DNA methyltransferases inhibitors effectively induce gene expression changes suggestive of cardiomyogenic differentiation of human amniotic fluid‐derived mesenchymal stem cells via chromatin remodeling. J Tissue Eng Regen Med. 2019; 13: 469– 481. https://doi.org/10.1002/term.2800
- Gasiūnienė; M. ; Zentelytė; A. ; Treigytė; G. ; Baronaitė; S. ; Savickienė; J. ; Utkus; A. and Navakauskienė; R. (2019); Epigenetic alterations in amniotic fluid mesenchymal stem cells derived from normal and fetus‐affected gestations: A focus on myogenic and neural differentiations. Cell Biol Int; 43: 299-312. doi:10.1002/cbin.11099
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