Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

Low sulfated heparan sulfate mimetic differentially affects repair in immune-mediated and toxin-induced experimental models of demyelination.

There is an urgent need for therapies that target the multicellular pathology of central nervous system (CNS) disease. Modified, nonanticoagulant heparins mimic the heparan sulfate glycan family and are known regulators of multiple cellular processes. In vitro studies have demonstrated that low sulfated modified heparin mimetics (LS-mHeps) drive repair after CNS demyelination. Herein, we test LS-mHep7 (an in vitro lead compound) in experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. In EAE, LS-mHep7 treatment resulted in faster recovery and rapidly reduced inflammation which was accompanied by restoration of animal weight. LS-mHep7 treatment had no effect on remyelination or on OLIG2 positive oligodendrocyte numbers within the corpus callosum in the cuprizone model. Further in vitro investigation confirmed that LS-mHep7 likely mediates its pro-repair effect in the EAE model by sequestering inflammatory cytokines, such as CCL5 which are upregulated during immune-mediated inflammatory attacks. These data support the future clinical translation of this next generation modified heparin as a treatment for CNS diseases with active immune system involvement.

Generation of Rat Neural Stem Cells to Produce Different Astrocyte Phenotypes.

Striatum-derived neural stem cells have been used to generate a variety of neural cell populations. They are composed of free-floating clusters of clonal neural stem cells, termed neurospheres, and can be expanded under growth factor stimulation in vitro. The multipotent nature of neurospheres means that under certain growth conditions they can differentiate into neuronal and glial progenitors of the central nervous system (CNS).Here, we describe a method for creating a population of astrocytes derived from rat striatum neurospheres, which in turn can be used to generate astrocytes with different reactivity phenotypes. Several methods and techniques are already available for the generation of neurospheres, but the method detailed herein provides an accessible, reproducible protocol for large numbers of astrocyte cultures, that can then be manipulated in an experimental format for further investigation.

Myelinated axons are the primary target of hemin-mediated oxidative damage in a model of the central nervous system.

Iron released from oligodendrocytes during demyelination or derived from haemoglobin breakdown products is believed to amplify oxidative tissue injury in multiple sclerosis (MS). However, the pathophysiological significance of iron-containing haemoglobin breakdown products themselves is rarely considered in the context of MS and their cellular specificity and mode of action remain unclear. Using myelinating cell cultures, we now report the cytotoxic potential of hemin (ferriprotoporphyrin IX chloride), a major degradation product of haemoglobin, is 25-fold greater than equimolar concentrations of free iron in myelinating cultures; a model that reproduces the complex multicellular environment of the CNS. At low micro molar concentrations (3.3 - 10 µM) we observed hemin preferentially binds to myelin and axons to initiate a complex detrimental response that results in targeted demyelination and axonal loss but spares neuronal cell bodies, astrocytes and the majority of oligodendroglia. Demyelination and axonal loss in this context are executed by a combination of mechanisms that include iron-dependent peroxidation by reactive oxygen species (ROS) and ferroptosis. These effects are microglial-independent, do not require any initiating inflammatory insult and represent a direct effect that compromises the structural integrity of myelinated axons in the CNS. Our data identify hemin-mediated demyelination and axonal loss as a novel mechanism by which intracerebral degradation of haemoglobin may contribute to lesion development in MS.

Transmission Electron Microscopy and Morphometry of the CNS White Matter.

Transmission electron microscopy of central nervous system white matter has provided unparalleled access to the ultrastructural features of axons, their myelin sheaths, and the major cells of white matter; namely, oligodendrocytes, oligodendrocyte precursors, astrocytes, and microglia. In particular, it has been invaluable in elucidating pathological changes in axons and myelin following experimentally induced injury or genetic alteration, in animal models. While also of value in the examination of human white matter, the tissue is rarely fixed adequately for the types of detailed analyses that can be performed on well-preserved samples from animal models, perfusion fixed at the time of death. In this chapter we describe methods for obtaining, processing, and visualizing white matter samples using transmission electron microscopy of perfusion fixed tissue and for unbiased morphometry of white matter, with particular emphasis on axon and myelin pathology. Several advanced electron microscopy techniques are now available, but this method remains the most expedient and accessible for routine ultrastructural examination and morphometry.