Direct Reprogramming of Neural Stem Cells into Oligodendrocyte Progenitors by Defined Factors
Keller, Alexandra Corrine
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Multiple Sclerosis is an autoimmune disease of the CNS in which the protective myelin sheaths composed of myelinating cells called oligodendrocytes which surround the axons of the brain and spinal cord are damaged, leading to demyelination and scarring. As a result of demyelination, conduction of action potentials carrying messages from the brain and spinal cord are blocked, leading to subsequent axonal degradation and reduced or lost bodily function. Unlike other neurodegenerative diseases, myelin may be regenerated via a process known as remyelination, yet it is unknown what regulates human oligodendrocyte differentiation and this prevents the development of effective treatment. We have therefore investigated which transcription factors (TFs) contribute to the transition from a neural stem cell to an oligodendrocyte progenitor cell (OPC) fate in humans. Previously, through a combination of cell sorting techniques and whole genome microarray, we identified TFs specifically expressed by human OPCs and oligodendrocytes. We cloned several of these into a lentiviral vector to drive overexpression in human neural stem cells (NSCs) and determine their ability to instruct OPC fate. Among six TFs tested, we found that overexpression of SOX10, NKX2.2, PRRX1, and ASCL1 significantly increased the expression of OPC-specific markers A2B5 and NG2 by between 10 and 25 fold (1-way ANOVA, p<0.0001, n=3) suggesting that individual factor expression was sufficient to induce OPC fate. As OPC induction was not 100% efficient using individual factors, we combined all four factors and asked whether together they were capable of driving OPC specific gene expression using a SOX10 enhancer-driven GFP reporter lentivirus. We found that the combinatorial action of SOX10, NKX2.2, PRRX1, and ASCL1 was sufficient to induce 71% GFP via flow cytometry, suggesting strong induction of OPC fate (1-way ANOVA, p<0.0001, n=3). In addition, we found that only when ASCL1 was removed from this pool, the percentage of GFP + cells was significantly reduced to 38.67 ± 9.40% (Dunnett's post-hoc test, n=3, p<0.01), demonstrating that ASCL1 was necessary for optimal OPC fate induction. Furthermore, this data suggests that the action of each of the other three factors could be effectively compensated by the remaining TFs. To determine which TFs might regulate oligodendrocyte differentiation and maturation from human OPCs, we used a similar genomic approach to identify TFs that were highly expressed by human oligodendrocytes. Of these, we identified thyroid hormone receptor alpha (THRA) as a promising candidate. We tested the capacity of THRA to prompt differentiation and maturation of OPCs to oligodendrocytes using a lentiviral-based approach. THRA overexpression in the absence of growth factors significantly increased the proportion of complex and sheet forming O4 + oligodendrocytes (2-way ANOVA, p<0.0003, n=3). In the presence of T3, a potent THRA ligand, the proportion of complex and sheet forming O4 + oligodendrocytes among THRA infected cells was further increased (Bonferroni's post-hoc test, n=3, p<0.001). This data shows that THRA and T3 act cooperatively to encourage OPC commitment to mature oligodendrocyte cell fate, and that endogenous levels of THRA were rate limiting in human OPCs. In summary, we have identified several factors that contribute to OPC fate from NSCs and THRA as a possible rate-limiting factor in oligodendrocyte maturation. Ultimately these targets either directly or via small-molecule induction could be used to induce oligodendrocyte commitment and myelination from human stem cells, as well as inspire future efforts to understand myelination repair and remyelination in individuals suffering from Multiple Sclerosis or other demyelinating diseases.