Therapeutic repair of myelin disorders may be limited by the relatively slow rate of human oligodendrocyte differentiation. To identify appropriate pharmacological targets with which to accelerate differentiation of human oligodendrocyte progenitors (hOPCs) directly, we used CD140a/O4-based FACS of human forebrain and microarray to hOPC-specific receptors. Among these, we identified CHRM3, a M 3 R muscarinic acetylcholine receptor, as being restricted to oligodendrocyte-biased CD140a

The molecular mechanisms controlling human oligodendrocyte development are poorly characterized. Microarray analysis of human oligodendrocyte progenitor cells (OPCs) and immature oligodendrocytes revealed that specific-class I histone deacetylase (HDAC) target genes were actively repressed during oligodendrocyte commitment. Although epigenetic regulation of oligodendrocyte differentiation has been established in rodent development, the role of HDACs in human OPCs remains undefined. We used HDAC inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate to determine the importance of HDAC activity in human primary OPC differentiation. Treatment with either drug resulted in significant dose-dependent inhibition of O4(+) oligodendrocyte cell differentiation, reduction of oligodendrocyte morphological maturation, and downregulation of myelin basic protein mRNA. High dose TSA treatment was also associated with reduction in OPC proliferation. HDACi treatment prevented downregulation of SOX2, ID4, and TCF7L2 mRNAs but did not regulate HES5, suggesting that targets of HDAC repression may differ between species. These results predict that HDACi treatment would impair proliferation and differentiation by parenchymal oligodendrocyte progenitors, and thereby degrade their potential for endogenous repair in human demyelinating disease. © 2012 Wiley Periodicals, Inc.

Background Spinal cord injury (SCI) is a debilitating event with multiple mechanisms of degeneration leading to life-long paralysis. Biomaterial strategies, including bridges that span the injury and provide a pathway to reconnect severed regions of the spinal cord, can promote partial restoration of motor function following SCI. Axon growth through the bridge is essential to characterizing regeneration, as recovery can occur via other mechanisms such as plasticity. Quantitative analysis of axons by manual counting of histological sections can be slow, which can limit the number of bridge designs evaluated. In this study, we report a semi-automated process to resolve axon numbers in histological sections, which allows for efficient analysis of large data sets. New Method Axon numbers were estimated in SCI cross-sections from animals implanted with poly(lactide co-glycolide) (PLG) bridges with multiple channels for guiding axons. Immunofluorescence images of histological sections were filtered using a Hessian-based approach prior to threshold detection to improve the signal-to-noise ratio and filter out background staining associated with PLG polymer. Results Semi-automated counting successfully recapitulated average axon densities and myelination in a blinded PLG bridge implantation study. Comparison with Existing Methods Axon counts obtained with the semi-automated technique correlated well with manual axon counts from blinded independent observers across sections with a wide range of total axons. Conclusions This semi-automated detection of Hessian-filtered axons provides an accurate and significantly faster alternative to manual counting of axons for quantitative analysis of regeneration following SCI.

Oligodendrocytes are destroyed in demyelinating diseases such as Multiple Sclerosis. In a genomic study to identify novel drug targets, we identified the muscarinic cholinergic M3 receptor as highly expressed by human oligodendrocyte progenitor cells (OPCs). Human OPCs expressing CD140a antigen were isolated from 19–22 week fetal brain using immunomagnetic sorting. We treated OPCs with oxotremorine, a specific agonist of muscarinic receptors, for 4 days and assessed cell fate and proliferation using immunocytochemistry. Oxotremorine treatment resulted in a dose‐dependent decrease in oligodendrocyte differentiation (1‐way ANOVA, p<0.01). The proportion of O4+ oligodendrocytes was significantly reduced from 16.0±2.4% at 0μM to 8.3±1.5% at 40μM oxotremorine (n=3, Tukey's post‐hoc p<0.05). The proportions of dividing cells (BrdU) and oligodendrocyte lineage cells (Olig2) were not significantly altered suggesting that the effect of oxotremorine was to directly block oligodendrocyte differentiation. As such, we have identified an operative receptor expressed by human OPCs that may be targeted to regulate oligodendroctye differentiation. This work was supported by NYSTEM contracts (C026413 and C026428).