(402d) Live Cell Array for High-Throughput Study of Real-Time Gene Expression Dynamics: Towards Understanding of Mesenchymal Stem Cell Differentiation

Stem cells offer great potential for cell-based therapies and personalized medicine. However, the challenge still remains to understand regulatory mechanism of differentiation of adult or embryonic stem cells. Mesenchymal stem cell (MSC) differentiation involves precisely programmed gene expression that directs stem cells to tissue specific cell types such as fat, bone cartilage or muscle. Identification of temporal patterns of gene expression would potentially help uncover the regulatory mechanisms of mesenchymal stem cell (MSC) differentiation and would ultimately provide information to guide efficient differentiation of stem cells for cell-based therapies. To this end, we engineered a novel dual-promoter lentiviral vector that enables quantitative measurements of gene expression in real time and in a high-throughput manner. Using this vector, we developed scalable live-cell microarrays to quantitatively measure gene expression dynamics in real-time as MSC differentiate along the myogenic lineage. To do this, we generated a library of lentiviral vectors encoding promoters or transcription response elements of genes that are potentially involved in myogenic differentiation. Subsequently, we immobilized lentiviruses in a microarray format and used automated fluorescence microscopy to monitor real time gene expression as MSC differentiated toward smooth muscles. After identifying the optimal conditions that maintain MSCs in the undifferentiated versus differentiated state the kinetics of gene expression was interrogated in real time under differentiation promoting conditions. Our experiment identified temporal patterns of gene expression of myogenic activator e.g. myocardin or inhibitors e.g. Kruppel-like factor 4 (KLF4) as MSCs undergo differentiation. In contrast to standard methods, our studies provide rich dynamic information of gene expression over a period of several days, thereby providing real-time data that maybe useful in deciphering gene regulatory networks in stem cell differentiation.