Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Exhibit Hall
Kiersten Pena - Indiana University-Purdue University Indianapolis
Co-Author(s): Melody Hernandez, Stark Neurosciences Research Institute, Indianapolis, IN; Jade Harkin, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN; Jason Meyer, Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN
Human pluripotent stem cells (hPSCs) are a useful tool for modeling disease in vitro, as well as for cell replacement in vivo. hPSCs have the unique ability to be differentiated into many different cell types, one being retinal organoids, which are hPSC-derived tissue structures that contain many key cell types present in the human retina. hPSCs, and by association the generated organoids, retain the genetic background of the patient they were derived from. While many cell lines contain green fluorescent protein (GFP), some may cease to fluoresce green due to the cell types they have differentiated into, hindering their ability to be used as models. To remedy this, we synthesized a plasmid that contains an EGFP reporter at the CLYBL genomic locus, which is a safe harbor site that should allow for the constitutive expression of the reporter, along with the expression of Thy1 at the BRN3b genomic locus. This process is permanent and thus the green fluorescence is permanently turned on, while the expression of the cell surface antigen Thy1 at the BRN3b locus allows for positive selection of retinal ganglion cells. DNA plasmids were created using DNA purification and ligation, and validated via restriction digest and gel electrophoresis with CLYBL-eGFP expression in hPSCs verified via fluorescent microscopy. Subsequent differentiation of these cells to a retinal lineage demonstrated the maintained expression of the eGFP signal. Taken together, this study introduces a method that can be used to allow eGFP-containing cells to fluoresce green regardless of their condition. Future research involves taking the organoids further into retinal development, isolating the retinal ganglion cells, and testing if they continue to fluoresce green at that point.
Funder Acknowledgement(s): This material is based upon work supported by the National Science Foundation under Grant No. HRD 1618408, 2016-2023. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
Faculty Advisor: Jason Meyer, firstname.lastname@example.org
Role: I was involved in cell culture tasks, such as stem cell maintenance and differentiation of stem cells into specialized cell types. Outside of that, I performed many immunostainings on stem cells to confirm the presence of desired markers, such as pluripotency markers and transcription factors. Additionally, I did gel electrophoreses to separate and analyze several samples of DNA. I also screened and imaged cells at different stages of differentiation to confirm that they were expressing the green fluorescent protein that was inserted into them.