An investigation of the role of integrin alpha-6 in human induced pluripotent stem cell development and pluripotency
Thesis Final GW.pdf
The general functions of integrins in attachment, gene expression, motility, polarity, shape, proliferation, and survival are well known. These critical functions provide reason for their wide expression across cell types. In particular, integrins are expressed in varying stem cell populations as well as other cell populations throughout the human body. Integrin alpha-6 (ITGA6) is a particular isoform of the integrin family, which is expressed across stem cell populations and has been shown to play an integral role in embryonic pluripotent stem cell self-renewal (Villa-Diaz 2016). Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) share the same morphology and gene expression but they do not share the same origin, since hESCs are obtained from the inner cell mass of blastocyst embryos, while hiPSCs are derived from somatic cells via genetic reprogramming. This similarity is what makes hiPSCs so widely applicable to regenerative medicine as they are more ethically obtained and derived. In this research, hiPSCs were utilized as a model to determine the role of ITGA6 in hiPSC development and pluripotency. To address this objective, we knocked out ITGA6 in human gingival fibroblasts using 3 constructs of CRISPR-Cas9 single-guided (sg) RNAs that target specific sequences of nucleic acids in ITGA6, and we utilized a CRISPR-Cas9 sg control, which did not target any specific sequence of nucleic acids, and WT human gingival fibroblasts as control groups. These five groups of human gingival fibroblasts were then reprogrammed utilizing 4 crucial factors to generate iPSCs: Oct4, Sox2, Klf4 and c-Myc (Takahashi & Yamanaka 2006). Twenty-eight days post-infection the reprogrammed fibroblasts were passaged upon colony formation, or used for RNA and protein expression analysis by qRT-PCR and immunocytochemistry (ICC), respectively. Colony formation, RNA expression, and protein translation, were compared to the wildtype fibroblast-group. Pluripotency was determined by the expression of key markers of 2 An investigation of the role of integrin alpha-6 pluripotent stem cells such as Sox2, NANOG, KLF-4, and Oct4. Development of hiPSCs was tested through the proliferation of undifferentiated colonies over a two-week period. We confirmed that vectors targeting ITGA6 expressed significantly lower levels of ITGA6 mRNA, with Cas9 sgITGA6 15 consistently having the lowest expression of ITGA6 across the three independent replicates. As expected, formation of hiPSCs was obtained from the wild-type group. The groups from CRISPR-Cas9 sgITGA6 10 and 14 groups, as well as the control developed an average of 3-5 colonies per replicate, which expressed ITGA6 and were able to passage and survive. In the Cas9/15 group only one colony developed within the thee replicates, which was consistent with its lower ITGA6 mRNA expression. However, the one colony that did develop also expressed ITGA6. All together these data suggest that the knock-out of this gene did not have 100% efficiency because every colony that developed expressed ITGA6, which led us to the conclude that ITGA6 is crucial for the development and maintenance of hiPSCs.
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