Transcriptional programming of spinal motor neurons from stem cells.

摘要

Motor neurons are the cholinergic cells of the spinal ventral horns responsible for mediating the central nervous system's control of voluntary muscle movement. Selective loss of these cells is a hallmark of the lethal diseases such amyotrophic lateral sclerosis and spinal muscular atrophy. As there are no effective treatments for these devastating disorders, studying the unique biology of these important cells has been a focus of intense research. Inhibiting these pursuits has been the inability to generate motor neurons efficiently and in large number in vitro. Furthermore, all motor neurons generated to date are typically of a single subtype and therefore fail to represent the diverse array of motor neuron types required for vertebrate locomotion. The aim of this dissertation is to create robust strategies for the derivation of motor neurons with subtype specificity at high efficiency in vitro. We have therefore undertaken a transcription factor mediated approach to direct stem cells to motor neuron fates at high efficiency with subtype specificity in vitro. This work is based on a central hypothesis stating that neuronal cell fate can be specified from stem cells through the rational expression of fate specifying transcription factors, i.e. 'transcriptional programming'.;Stem cells at various stages of commitment can be derived from different tissues and share the ability to self-renew and differentiate into multiple cell types. Embryonic stem-and induced pluripotent stem- (iPS) cells retain the potential to generate all cell types from the three germ layers endoderm, mesoderm and ectoderm. In contrast, adult-derived neural stem cells (NSCs) have a more limited differentiation capacity and differentiate into only neurons, astrocytes, and oligodendrocytes. It remains unclear whether they remain competent to generate specific neuronal subtypes. Here, we investigated the potential of programming embryonic stem cells to a motor neuron fate. We also investigated the ability of adult NSCs derived from either brain or spinal cord regions to be programmed to a cholinergic spinal motor neuron phenotype. We found that an embryonic transcription factor code---Isl1, Lhx3, and Ngn2---could instruct the differentiation of ES cells and adult NSCs from multiple origins into functional motor neurons. This work highlights the potential of directly programming stem cells to a defined phenotype as it is more efficient and generates motor neurons from cell populations typically refractory to motor neuron development by canonical approaches.;We also demonstrate the ectopic expression of the atypical zinc finger protein, Nolz1, directs ES derived motor neurons to a limb innervating lateral motor column (LMC) fate. Motor neurons transduced with Nolz1 express LMC character markers such as Lhx1 and Raldh2. Furthermore, LMC-induced motor neurons extend projections to limb muscles, and possess electrophysiological properties characteristic of LMC motor neurons. Together these data present a novel direct programming approach for the generation of an LMC motor neuron subtype from mES cells, which will greatly aid in the analysis of those motor neurons more susceptible to neurodegenerative disease.