Histone deacetylases are required for human glial progenitor cell differentiation
Conway, Gregory D.
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Demyelinating diseases, such as Multiple Sclerosis are characterized by a failure of the regenerative process known as remyelination. Remyelination involves the differentiation of glial progenitor cells (GPC) into myelinating oligodendrocytes leading to the restoration of fast axon conduction and functional recovery. The role of epigenetic regulation of oligodendrocyte differentiation in human cells has not been established. In genomic studies of human glial progenitors, we identified regulation of histone deacetylation as a possible rate limiting step in human oligodendrocyte differentiation. We examined the role of histone deacetylase (HDAC) in oligodendrocyte maturation and myelination in GPCs isolated by CD140a-directed magnetic activated cell sorting from dissociated 18-22 week fetal human brains. Upon removal of growth factor from the media, GPCs were pulsed daily with the HDAC inhibitors trichostatin A (TSA) (0-33.3 nM, IC 50 =5 nM) or sodium butyrate (SB) (0–5.0 mM, IC 50 =140 μM) for 1 or 3 days. O4 + oligodendrocytes and OLIG2 + glial lineage cells were quantified using immunocytochemistry. Both drug treatments produced a dose dependent reduction in the proportion of O4 + oligodendrocytes (1-way ANOVA, p<0.05; n=3) although OLIG2 + glial lineage cells remained unaltered. With a dose of 3.3 nM TSA for 3 days, the proportion of O4 + cells was, 16.0±2.9% compared to 33.7±5.7% O4 + cells in the control (Tukey’s post-hoc test, p<0.05). Interestingly, all doses of TSA tested, and SB equal to and above 0.5 mM limited the formation of highly branched or complex O4 + cell morphology; for example, 10 nM TSA reduced the proportion of complex O4 + cells from 10.3 ± 2.0% to 0.7 ± 0.7% in treated cells. In conjunction with the reduction of oligodendrocytic differentiation, TSA treated GPCs also demonstrated an 8.04 fold down regulation of myelin basic protein mRNA and a significant up regulation of mRNA of the transcriptional regulator ID4 (1.88 fold) and neural stem cell marker SOX2 (2.21 fold). Thus, the dose-dependent reduction of committed O4+ oligodendrocyte cells suggests that HDAC activity is required in GPC differentiation. Further, the loss of complex morphology associated with the maturation of oligodendrocytes supports the requirement for HDACs in the maturation of GPCs to functional myelinating cells. To better understand the molecular mechanisms of human GPC differentiation, we sought to develop a model of human myelination utilizing CD140a-sorted GPCs and re-aggregates of cortical human neurons. We found that neuronal re-aggregates produced a dense network of neurofilament that could be maintained up to 42 days. Despite differentiating as O4 + oligodendrocytes by 7 days and more mature O1 + oligodendrocytes after 14 to 21 days in culture, no myelination was observed in our co-culture system even when maintained for 42 days. Previous work in mouse models had shown that inhibition of notch signaling resulted in the increased formation of myelin. Attempts to dis-inhibit myelination by notch signaling blockade in our human model had no effect on myelination in our co-culture. The addition of serum to the co-culture, in order to improve long term survival of GPCs, showed no effect on GPC survival however resulted in degeneration of the neuronal re-aggregate cultures. As such, we have created a coculture of stable human neuronal re-aggregates which allow for the differentiation of human GPCs to oligodendrocytes, and therefore form the basis for the development of an entirely humanized myelination culture system.