This array technique is based on a library of lentiviral reporters of transcription factor activation that drive expression of luciferase. Extracellular cues initiate complex intracellular signaling cascades that terminate in translocation of multiple transcription factors to the nucleus to directly modulate gene expression. Bioluminescence imaging is then performed in live, 3D cultures to quantitatively monitor transcription factor activation in real-time, which represents the current "functional state" of a cell and ultimately drives differentiation.
Dynamic Measurements of Intracellular Signaling Pathways during Oligodendrocyte Differentiation
A major factor contributing to the failure of stem cell therapies for SCI is an inhibition of oligodendrocyte differentiation by the local, in vivo microenvironment. As the process of oligodendrocyte differentiation is not well understood, there is a need to identify the microenvironmental parameters required to direct oligodendrocyte differentiation. To date, a handful of protocols describing differentiation of human oligodedendrocytes in vitro have been reported. However, there is little consistency among the different methods and terminally differentiated yields of oligodendrocytes. Moreover, none of these methods has been effective, simple or consistent enough to be adopted by the field as a standard protocol. In order to effectively employ cell-based therapies for axon remyelination, it is imperative to gain a better fundamental understanding of the mechanisms that drive human oligodendrogenesis. To this end, our lab is employing arrays of transcription factor activity to identify common set(s) of master transcription factors that are uniquely activated in human neural stem/progenitor as they undergo differentiation in real-time. Once characterized, strategies to induce simultaneously activation of this core set of transcription factors can be developed to substantially increase researchers’ abilities to efficiently and directly generate oligodendrocytes. Moreover, this core set of transcription factors can be experimentally monitored to identify extrinsic parameters that further drive differentiation.
Identification of Novel Drug Targets for Treatment of Glioblastoma Multiforme
Gliobastoma multiforme is an extremely aggressive cancer that is typically unresponsive to currently available pharmacological agents. The ultimate goal of these studies is to identify key differences in the extracellular environment surrounding glioblastomas that impart drug resistance with the goal of exploiting these differences to develop novel, more effective pharmaceutical agents. Using our transcription factor reporter library, we are screening for intracellular pathways that are differentially activated in response to varying microenvironmental cues, as presented by 3D hydrogels. Furthermore, we are quantifying the real-time, dynamic changes in transcription factor activity as glioblastoma cells acquire resistance to alkylating agents and tyrosine kinase inhibitors. Acquisition of systems biology level data will provide key insights into the early, conserved events that initiate this resistance. Moreover, characterization of key transcriptional pathways that confer apoptotic resistance will identify promising new targets for adjunct therapies aimed at rendering glioblastomas sensitive to chemotherapeutic agents. This project is in collaboration with Dr. David Nathanson, Assistant Professor in Medical and Molecular Pharmacology at UCLA.