RNA-SEQ REVEALS NOVEL REGULATORS IN HUMAN STEM CELL DIFFERENTIATION
Open Access
- Author:
- Witmer, Evan Brooke
- Graduate Program:
- Bioengineering
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 03, 2017
- Committee Members:
- Xiaojun Lance Lian, Thesis Advisor/Co-Advisor
- Keywords:
- stem cell
stem cells
pluripotent
differentiation
mesoderm
mesodermal
RNA-seq
RNA-sequencing
FOXB1
ARL4A
PMAIP1 - Abstract:
- Many mesodermal cell fates such as cardiomyocytes cannot recreate themselves after significant damage or disease. The ability to generate mesoderm tissues from stem cells could offer a way to recover after this cell death. There exists protocols to induce this differentiation, but in order to improve their effectiveness a better understanding of the gene expression during differentiation is required. In this study, the first 21 hours of mesoderm differentiation are explored through RNA-sequencing. Wnt/β-catenin signaling is activated through CHIR99021 to start the differentiation process into prospective mesoderm. Every three hours, one well is collected and cells are lysed for their RNA. The RNA content at each time point is sequenced through Illumina RNA-sequencing and output in the form of a fastq file. Genes displaying significant rises and declines in transcription are isolated and displayed using parallel bioinformatics techniques and software (Kallisto, Python, GENE-E). Roles are proposed for these genes dependent on their time of expression. Eleven novel direct targets of Wnt/β-catenin signaling are suggested (ARL4A, NODAL, PMAIP1, GNAI1, F2RL1, CA2, MPPED2, SESN3, SALL3, LPAR6, & FOXB1) as well as further downstream targets. Upon experimental confirmation, these putative targets may prove useful reagents in future protocols for mesodermal differentiation.