Talk abstracts

Talk on Saturday 04:45-05:00pm submitted by Matthew Murtha

Efficient Differentiation of ES Cells to Motorneurons by Developmental Transcription Factors

Matthew Murtha (MCDB, The Ohio State University), Mark Hester (Center for Gene Therapy, Research Institute at Nationwide Childrens Hospital), Meghan Rao (Center for Gene Therapy, Research Institute at Nationwide Childrens Hospital), Brian Kaspar (Center for Gene Therapy, Research Institute at Nationwide Childrens Hospital and The Ohio State University)

Abstract:
Motor neurons (MNs) derived from embryonic stem (ES) cell are being developed for stem cell-based models and therapies for degenerative diseases of motor neurons, such as Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy. Currently, ES cells are induced towards a MN fate by the addition of key extrinsic developmental cues including Retinoic Acid (RA) and Sonic Hedgehog (SHH) protein. However, the ability to control the type of MNs produced is difficult and the efficiency is low. Understanding the molecular basis of spinal MN development led us to identify genes intricately involved in MN differentiation in vivo that could be utilized to direct the fate of ES cells in vitro. These genes encode the neurogenic transcription factors (TF) Neurogenin 2 (Ngn2), Islet-1 (Isl1) and Lhx-3. We explored the potential of these genes by directly delivering them into ES cells to determine whether these developmental TF are sufficient to direct MN differentiation in vitro. Using an ES cell line with an early MN promoter (HB9) driving GFP, we delivered Ngn2, Isl1, and Lhx3 by single adenoviral infections and monitored for MN induction compared to the standard RA/SHH treatment. We found that treatment with TF increased the number of Hb9+ neurons over two-fold (21.2% of cells compared to 10.5% with RA/SHH). TF mediated approach also increased the rate of differentiation at twice the rate of RA/SHH treated cells. To demonstrate that these cells were developing into mature spinal MNs, we performed immunohistochemistry for MN markers and found that the cells were Smi31, HB9 and ChAT positive. To fully test the potential of the in vitro generated MNs, we transplanted the cells into the embryonic chick spinal cord. We show that TF directed MNs migrate to the appropriate dorso-lateral position, extend axonal projections, and enervate target muscle. Together these data demonstrate that by utilizing key transcription factors one can directly target the MN developmental pathway and focus the potential of ES cells to a highly specific fate. Thus, one can achieve efficient MN differentiation without the need for external cues, providing a population of defined cells for disease models and possible transplantation.

Keywords: Embryonic Stem Cells, Motorneurons