Flow Cytometry Analysis of the Differentiation of Stem Cells Using Staining Procedures with Various Markers

• Krisztina Sershen, Biological Sciences, University of Delaware

• Deni Galileo, Biological Sciences, University of Delaware

Glioblastoma (GBM) is the deadliest form of brain cancer that spreads aggressively throughout the human brain. The existence of Glioblastoma Stem Cells (GSCs) are believed to be the main reason why GBM recurs and is incurable. GSCs invade the surrounding brain tissue along with non-GSC tumor cells and drive the spread of the tumor. GSCs are thought to have the ability to transition between “differentiated” GBM cells, like GFAP+ astrocytic cells, and a stem-like phenotype, which could be a reason that contributes to recurrence. Our lab previously has made “mixed” tumors in the chick embryo brain using patient derived GSCs mixed with older, established GBM cell lines from other sources. To extend these studies, I am trying to differentiate GSCs to be able to make mixed tumors using cells from a single patient. To do this, a patient derived GSC line was cultured in 6 different conditions that might promote differentiation for 7 days, fixed, immunofluorescently stained for GFAP, Nestin, and Integrin a6, and analyzed by flow cytometry for expression levels of these markers. One condition is standard GSC media, and the other 5 include high serum media (DMEM+10%FBS+P/s+Glut) as a base with additives of N2 neural supplement, retinoic acid (RA), and B27 supplement with RA. Our method for assessing differentiation is to analyze astrocytic marker GFAP and GSC marker proteins Nestin and Integrin a6. An upregulation of GFAP expression and a downregulation of stem cell protein expression would be indicative of differentiation. Data gathered indicates that GSCs cultured with RA, B27, and N2+B27 showed an increase of GFAP expression. Decrease of stem cell marker expression was observed in Nestin and Integrin ⍺6, but not Sox-2. Tentatively, it appears that differentiation might be occurring in media with B27 as reflected by an increase in GFAP and decrease in Integrin ⍺6.