Maya N. Hatch

Derivation of High-Purity Oligodendroglial Progenitors

Oligodendrocytes are a type of glial cells that play a critical role in supporting the central nervous system (CNS), in particular insulating axons within the CNS by wrapping them with a myelin sheath, thereby enabling saltatory conduction. They are lost, and myelin damaged - demyelination - in a wide variety of neurological disorders. Replacing depleted cell types within demyelinated areas, however, has been shown experimentally to achieve remyelination and so help restore function. One method to produce oligodendrocytes for cellular replacement therapies is through the use of progenitor or stem cells. The ability to differentiate progenitor or stem cells into high-purity fates not only permits the generation of specific cells for transplantation therapies, but also provides powerful tools for studying cellular mechanisms of development. This chapter outlines methods of generating high-purity OPCs from multipotent neonatal progenitor or human embryonic stem cells.

Endogenous remyelination is induced by transplant rejection in a viral model of multiple sclerosis.

Human embryonic stem cell-derived oligodendrocyte progenitors (OPCs) were transplanted into mice persistently infected with the neurotropic JHM strain of mouse hepatitis virus with established demyelination. Engrafted cells did not survive past 2 weeks following transplantation despite treatment with high dose cyclosporine A. While T cell infiltration into the CNS was dampened, elevated numbers of macrophage/microglia and endogenous OPCs were evident surrounding the implantation site and this was associated with increased remyelination. These data suggest that remyelination was initiated by the local response to xenograft transplantation. These findings illustrate the complexities of OPC transplantation into areas of robust immune-mediated pathology.

IFN-γ-induced apoptosis of human embryonic stem cell derived oligodendrocyte progenitor cells is restricted by CXCR2 signaling

Engraftment of human embryonic stem cell (hESC)-derived OPCs in animal models of demyelination results in remyelination and clinical recovery, supporting the feasibility of cell replacement therapies in promoting repair of damaged neural tissue. A critical gap in our understanding of the mechanisms associated with repair revolves around the effects of the local microenvironment on transplanted cell survival. We have determined that treatment of human ESC-derived OPCs with the pleiotropic cytokine IFN-γ promotes apoptosis that is associated with mitochondrial cytochrome c released into the cytosol with subsequent caspase 3 activation. IFN-γ-induced apoptosis is mediated, in part, by secretion of the CXC chemokine ligand 10 (CXCL10) from IFN-γ-treated cells. Signaling through the chemokine receptor CXCR2 by the ligand CXCL1 functions in a tonic manner by muting apoptosis and this is associated with reduced levels of cytosolic cytochrome c and impaired cleavage of caspase 3. These findings support a role for both IFN-γ and CXCL10 in contributing to neuropathology by promoting OPC apoptosis. In addition, these data suggest that hOPCs used for therapeutic treatment for human neurologic disease/damage are susceptible to death through exposure to local inflammatory cytokines present within the inflammatory milieu.

Derivation of Oligodendrocyte Progenitor Cells from Human Embryonic Stem Cells

The directed differentiation of human pluripotent stem cells into specific, determined, and high-purity cell types can provide a means to study the cellular and molecular mechanisms of development and to generate cells for potential therapeutic applications. The ability to derive homogeneous cell populations obviates the need for transgene expression or cell sorting methods and can improve selection efficiency, lineage differentiation, cell viability, and clinical utility. Compared to undifferentiated pluripotent stem cells, high-purity cell phenotypes for clinical therapeutic strategies are expected to enhance engraftment, potentiate clinical efficacy, and decrease the risk of adverse effects such as dedifferentiation or teratoma formation. Clinical interest in the derivation of oligodendrocyte progenitor cells from pluripotent stem cells is based on research that demonstrates the effectiveness of progenitor cell transplants to improve outcomes after spinal cord injury. Here, we describe a protocol to generate oligodendroglial lineage-specific cells in high purity from human embryonic stem cells.

Oligodendrocyte Differentiation from Human Embryonic Stem Cells

Publisher Summary Oligodendrocytes are glial cells that play a critical role in supporting the central nervous system (CNS). Specifically, they insulate axons and nerve cells within the CNS by wrapping them with myelin sheaths. The myelin sheath enables fast, saltatory conduction of impulses along the axons of neurons, controlling functions such as walking, perception of visual stimuli, and cognitive processes. When axons become demyelinated (i.e. lose their myelin sheath), as occurs in multiple sclerosis (MS) and spinal cord injury (SCI), axons cannot properly function. It may be due to loss and/or damage of oligodendrocytes. Therefore, replacement of oligodendrocytes or oligodendrocyte progenitor cells (OPCs) by cellular replacement therapies may in part restore axonal conduction and normal neuronal function. One approach to produce oligodendrocytes is through differentiation from embryonic stem cells (ESCs). This chapter describes an efficient way to produce OPCs from human embryonic stem cells (hESCs); specific examples are given for the WA01 and WA07 lines. Differentiation into oligodendroglial progenitors is attained by using specialized media supplemented with specific growth and differentiation factors at key time points. The resulting oligodendroglial progenitors are then amplified and positively selected using mechanical enrichment.

Neuropathic pain and SCI: Identification and treatment strategies in the 21st century

Pain is a common complication in patients following spinal cord injury (SCI), with studies citing up to 80% of patients reporting some form of pain. Neuropathic pain (NP) makes up a substantial percentage of all pain symptoms in patients with SCI and is often complex. Given the high prevalence of NP in patients with SCI, proper identification and treatment is imperative. Indeed, identification of pain subtypes is a vital step toward determining appropriate treatment. A variety of pharmacological and non-pharmacological treatments can be undertaken including antiepileptics, tricyclic antidepressants, opioids, transcranial direct current stimulation, and invasive surgical procedures. Despite all the available treatment options and advances in the field of SCI medicine, providing adequate treatment of NP after SCI continues to be challenging. It is therefore extremely important for clinicians to have a strong foundation in the identification of SCI NP, as well as an understanding of appropriate treatment options. Here, we highlight the definitions and classification tools available for NP identification, and discuss current treatment options. We hope that this will not only provide a better understanding of NP for physicians in various subspecialties, but that it will also help guide future research on this subject.

Demystifying Poststroke Pain: From Etiology to Treatment

Pain after stroke is commonly reported but often incompletely managed, which prevents optimal recovery. This situation occurs in part because of the esoteric nature of poststroke pain and its limited presence in current discussions of stroke management. The major specific afflictions that affect patients with stroke who experience pain include central poststroke pain, complex regional pain syndrome, and pain associated with spasticity and shoulder subluxation. Each disorder carries its own intricacies that require specific approaches to treatment and understanding. This review aims to present and clarify the major pain syndromes that affect patients who have experienced a stroke in order to aid in their diagnosis and treatment.

Disease-Specific Induced Pluripotent Stem Cell Modeling: Insights into the Pathophysiology of Valosin Containing Protein (VCP) Disease

Valosin Containing Protein (VCP) disease is an autosomal dominant disorder caused by mutations in the VCP gene and is associated with progressive muscle weakness and atrophy. Affected individuals exhibit striking scapular winging due to shoulder girdle weakness. Currently, there are no treatments available and patients are dying early from cardiac and respiratory failure, typically in their 40’s and 50’s. The generation of disease-specific induced pluripotent stem cells (iPSC) offers a novel platform to investigate mechanisms of VCP disease and potential treatments similar to other disease models including Amyotrophic Lateral Sclerosis (ALS), Duchenne muscular dystrophy (DMD), Parkinson’s disease, Alzheimer’s disease (AD), Best Disease (BD), and type I juvenile diabetes mellitus (T1DM). Herein, we report the generation and characterization of a human iPSC line to examine the cellular and molecular processes underlying VCP disease. The VCP iPSC line expressed specific pluripotency markers NANOG, SSEA4, OCT-4, TRA-1-81 and exhibited characteristic morphology. We differentiated the human iPSC cell line into a neuronal lineage confirmed by TUJ-1 staining, a neuronal class III β-tubulin marker. We detected higher protein expression levels of ubiquitin (Ub), TAR DNA binding protein-43 (TDP-43), Light Chain 3-I/II (LC3), p62/SQSTM1, and optineurin (OPN) in the iPSC neural lineage compared to the control neural line. Collectively, our results demonstrate that patient-specific iPSC technology may provide useful disease modeling for understanding the complex mechanisms and for novel treatments of VCP and related disorders.

Chemokines and Spinal Cord Injury

The immune system plays a critical role in CNS disorders and spinal cord injury (SCI). Primary trauma to the adult mammalian spinal cord is immediately followed by secondary degeneration in which the inflammatory response is thought to be detrimental. This inflammatory response is mediated by small, chemotropic cytokines, called chemokines, which are secreted by a variety of cell types in the CNS including neurons, glia, and vascular cells. Here, we review studies which provide insight into the functional role of chemokines in neuroinflammation and disease, with an emphasis on SCI. More specifically, this review emphasizes studies which indicate that ablation of the T cell chemotactic CXC chemokine ligand 10 (CXCL10) results in diminished neuropathology associated with decreased immune cell infiltration into the CNS. Importantly, these findings reveal that targeting chemokines such as CXCL10 may offer a powerful therapeutic approach for the treatment of neuroinflammatory diseases. 1 Spinal Cord Injury Traumatic injury to the spinal cord is a combination of primary and secondary phases resulting from some form of mechanical insult. The direct tissue damage caused by the injury, known as the primary phase, initiates a cascade of events that lead to a detrimental secondary phase. Secondary degeneration is defined by a cascade of chemical and physiological events which include activation of voltage (dependent or agonist) gated channels, ion leaks, activation of calcium-dependent enzymes such as proteases, lipases, and nucleases, mitochondrial dysfunction, vascular changes and cellular infiltration and activation (Hausmann, 2003; Ramer et al., 2005). These processes lead to cell death, progressive tissue loss and cystic cavitation evolving away from the initial trauma site (Schwab and Bartholdi, 1996; Beattie et al., 2002) which can ultimately result in the functional deficits seen after SCI. Primary tissue damage is often irreversible, whereas secondary degeneration is amenable to pharmacological treatment. Although it is unclear which particular component of secondary degeneration is most significant to SCI severity, it is widely accepted that attenuating secondary degeneration would be beneficial. T.E. Lane et al. (eds.), Central Nervous System Diseases and Inflammation. 221