Major and Classification
Biomedical Engineering (Biochemical Engineering)
Faculty Mentor
Wange Lu, Ph.D.
Department
Viterbi School of Engineering
McNair Project
“Pitx2 Transcription Factor Inhibits Reprogramming of Human Dermal Fibroblast Cells”
Project Abstract
Embryonic stem (ES) cells are derived from the inner cell mass of embryos and they have the capacity to grow indefinitely while maintaining pluripotency. Previous studies have demonstrated successful reprogramming of somatic cells into a pluripotent embryonic stem cell-like state via transduction or direct transfection. These induced pluripotent stem cells (iPSCs) can be generated by forced expression of the four sets of transcription factors, Oct4, Sox2, Klf4, and c-Myc (OSKM). Expression profiling during ES cell differentiation suggests that the gene, Pitx2, is highly expressed in differentiating cells, which are no longer in an ES cell-like state. Therefore, we hypothesized that Pitx2 is a candidate gene for inhibiting pluripotency gene expression and that Pitx2 knockdown might enhance reprogramming in fibroblast cells. We utilized retrovirus transduction to reprogram human dermal fibroblast cells. Retroviral vectors packed with the four OSKM factors were introduced into fibroblast cells. Preliminary reprogramming was assessed by the detection of red fluorescent in cells infected with red fluorescent protein. Cell morphology was monitored at day 8 post-infection. We found that after infection, long and thin fibroblast cells transformed into round ES cell-like colonies and were more transparent 11 days after reprogramming. Using alkaline phosphatase staining to mark ES cells, we identified ES-like colonies that were reprogrammed from fibroblast cells in the plate where OSKM factors were present and the Pitx2 gene was silenced. This study demonstrated that it is possible to reprogram human dermal fibroblast cells to a pluripotency state although further studies are needed using different reprogramming methods and other pluripotency genes. A thorough understanding of the Pitx2 gene will lead to more efficient reprogramming strategies, enabling large-scale production of iPS cells and producing an alternative to embryonic stem cells for use in research and therapy for diabetes, heart failure, Parkinson’s disease and other neurodegenerative diseases.