Directed differentiation of mouse and human embryonic stem cells into motor neurons.

摘要

Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder in which motor neurons specifically die. One possible treatment is motor neuron replacement. Lines of embryonic stem cells (ESCs) with both their immortality and pluripotency offer a renewable source for transplantable motor neurons. In mouse, ESCs can be directed to differentiate into the motor neuron lineage in response to retmoic acid (RA) and sonic hedgehog (Shh). However, the high levels of Shh used suggest the presence of an inhibitory factor. Here, we show that cells in embryoid bodies treated with RA and Shh produce bone morphogenetic proteins (BMPs) that inhibit Shh directed ventral differentiation.;Through mimicking the signals of the anterior visceral endoderm (AVE), we were able to induce neural differentiation in human embryonic stem cells (hESCs). The AVE induces neural plate formation through inhibition of wnt, BMP and nodal signaling. Similarly, treatment of hESCs with these inhibitors induced neural differentiation, with the BMP inhibitor noggin giving the greatest effect. When treated with noggin, fgf-2 and fgf-4 in suspension, hESCs form columnar epithelial structures reminiscent of epiblast and neural plate. Within two weeks, these structures down regulated the pluripotent marker oct-4 and up-regulated expression of the neural progenitor marker PAX6. Human ESC derived neural progenitors were able to differentiate into neurons, astrocytes and oligodendrocytes. Finally, the neurons were able to fire tetrodotoxin sensitive action potentials.;In the final section of the thesis work, motor neurons were successfully differentiated from hESCs. Neural progenitors, formed through exposure to noggin, fgf-2 and fgf-4 were exposed to RA and neurotrophins on matrigel. After two weeks, many islet-1 and hb9 co-staining cells were observed. Further, these cells were found to be in the proximity of olig2 staining cells, and were not found to express various interneurons markers including lim-1/2. Motor neurons are formed when these progenitors are plated onto matrigel in the presence of retinoic acid. Finally, co-culture with c2c12-derived myotubes suggested that these motor neurons are indeed functional. This work is a first step toward the differentiation and isolation of motor neurons for transplantation based therapies.