Modeling demyelination and endogenous remyelination in spinal cord ex vivo rat organotypic slice cultures.

Introduction: Demyelination of the spinal cord is a prominent feature of multiple sclerosis (MS) and spinal cord injuries (SCI), where impaired neuronal communication between the brain and periphery has devastating consequences on neurological function. Demyelination precedes remyelination, an endogenous process in which oligodendrocyte precursor cells (OPCs) differentiate into mature, myelinating oligodendrocytes with the ability to restore the myelin sheath and reinstate functional nerve signaling. However, in MS or SCI, demyelination is more severe, persistent, and inhibitory to OPC-mediated remyelination, leading to a permanent loss of neuronal function. Currently, there are no effective treatments for demyelination, and existing pre-clinical models typically focus on brain tissue with little characterization of demyelination within the spinal cord. Organotypic slice cultures are a useful tool to study neurological disease, providing a more complex 3-dimensional system than standard 2-dimensional in vitro cell cultures. Methods: Building on our previously developed rat brain slice culture protocol, we have extended our findings to develop a rat longitudinal spinal cord ex vivo model of demyelination. Results: We generated rat longitudinal spinal cord slice cultures that remain viable for up to 6 weeks in culture and retain key anatomical features of the spinal cord's cytoarchitecture. We show that treating longitudinal spinal cord slices with lysolecithin (LPC) induced robust demyelination with some endogenous remyelination, which was not seen following exposure to lipopolysaccharide (LPS). Discussion: Our ex vivo organotypic spinal cord slice culture system provides a platform to model demyelination and endogenous remyelination long-term, mimicking that observed in LPC-induced rodent models of demyelination. This platform is suitable for the development and testing of novel therapeutic strategies with ease of manipulation prior to in vivo experimentation.

GelMA hydrogel as a scaffold to enhance the survival and differentiation of human induced lateral ganglionic eminence precursor cells.

Cell reprogramming holds enormous potential to revolutionize our understanding of neurological and neurodevelopmental disorders, as well as enhance drug discovery and regenerative medicine. We have developed a direct cell reprogramming technology that allows us to generate lineage-specific neural cells. To extend our technology, we have investigated the incorporation of directly reprogrammed human lateral ganglionic eminence precursor cells (hiLGEPs) in a 3-dimensional (3D) matrix. Hydrogels are one of the most promising bio-scaffolds for 3D cell culture, providing cells with a supportive environment to adhere, proliferate, and differentiate. In particular, gelatin methacryloyl (GelMA) hydrogels have been used for a variety of 3D biomedical applications due to their biocompatibility, enzymatic cleavage, cell adhesion and tunable physical characteristics. This study therefore investigated the effect of GelMA hydrogel encapsulation on the survival and differentiation of hiLGEPs, both in vitro and following ex vivo transplantation into a quinolinic acid (QA) lesion rat organotypic slice culture model. We demonstrate, for the first time, that the encapsulation of hiLGEPs in GelMA hydrogel significantly enhances the survival and generation of DARPP32+ striatal neurons both in vitro and following ex vivo transplant. Furthermore, GelMA-encapsulated hiLGEPs were predominantly located away from the reactive astrocyte network that forms following QA lesioning, suggesting GelMA provides a protective barrier for cells in regions of inflammatory activation. Overall, these results indicate that GelMA hydrogel has the potential to act as a 3D bio-scaffold to augment the viability and differentiation of hiLGEPs for research and translation of pharmaceutical development and regenerative medicine.

Directly reprogrammed fragile X syndrome dorsal forebrain precursor cells generate cortical neurons exhibiting impaired neuronal maturation.

Introduction: The neurodevelopmental disorder fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability associated with autism spectrum disorder. Inaccessibility to developing human brain cells is a major barrier to studying FXS. Direct-to-neural precursor reprogramming provides a unique platform to investigate the developmental profile of FXS-associated phenotypes throughout neural precursor and neuron generation, at a temporal resolution not afforded by post-mortem tissue and in a patient-specific context not represented in rodent models. Direct reprogramming also circumvents the protracted culture times and low efficiency of current induced pluripotent stem cell strategies. Methods: We have developed a chemically modified mRNA (cmRNA) -based direct reprogramming protocol to generate dorsal forebrain precursors (hiDFPs) from FXS patient-derived fibroblasts, with subsequent differentiation to glutamatergic cortical neurons and astrocytes. Results: We observed differential expression of mature neuronal markers suggesting impaired neuronal development and maturation in FXS- hiDFP-derived neurons compared to controls. FXS- hiDFP-derived cortical neurons exhibited dendritic growth and arborization deficits characterized by reduced neurite length and branching consistent with impaired neuronal maturation. Furthermore, FXS- hiDFP-derived neurons exhibited a significant decrease in the density of pre- and post- synaptic proteins and reduced glutamate-induced calcium activity, suggesting impaired excitatory synapse development and functional maturation. We also observed a reduced yield of FXS- hiDFP-derived neurons with a significant increase in FXS-affected astrocytes. Discussion: This study represents the first reported derivation of FXS-affected cortical neurons following direct reprogramming of patient fibroblasts to dorsal forebrain precursors and subsequently neurons that recapitulate the key molecular hallmarks of FXS as it occurs in human tissue. We propose that direct to hiDFP reprogramming provides a unique platform for further study into the pathogenesis of FXS as well as the identification and screening of new drug targets for the treatment of FXS.

Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures.

Introduction: With the increase in aging populations around the world, the development of in vitro human cell models to study neurodegenerative disease is crucial. A major limitation in using induced pluripotent stem cell (hiPSC) technology to model diseases of aging is that reprogramming fibroblasts to a pluripotent stem cell state erases age-associated features. The resulting cells show behaviors of an embryonic stage exhibiting longer telomeres, reduced oxidative stress, and mitochondrial rejuvenation, as well as epigenetic modifications, loss of abnormal nuclear morphologies, and age-associated features. Methods: We have developed a protocol utilizing stable, non-immunogenic chemically modified mRNA (cmRNA) to convert adult human dermal fibroblasts (HDFs) to human induced dorsal forebrain precursor (hiDFP) cells, which can subsequently be differentiated into cortical neurons. Analyzing an array of aging biomarkers, we demonstrate for the first time the effect of direct-to-hiDFP reprogramming on cellular age. Results: We confirm direct-to-hiDFP reprogramming does not affect telomere length or the expression of key aging markers. However, while direct-to-hiDFP reprogramming does not affect senescence-associated ß-galactosidase activity, it enhances the level of mitochondrial reactive oxygen species and the amount of DNA methylation compared to HDFs. Interestingly, following neuronal differentiation of hiDFPs we observed an increase in cell soma size as well as neurite number, length, and branching with increasing donor age suggesting that neuronal morphology is altered with age. Discussion: We propose direct-to-hiDFP reprogramming provides a strategy for modeling age-associated neurodegenerative diseases allowing the persistence of age-associated signatures not seen in hiPSC-derived cultures, thereby facilitating our understanding of neurodegenerative disease and identification of therapeutic targets.

Cell reprogramming for oligodendrocytes: A review of protocols and their applications to disease modeling and cell-based remyelination therapies.

Oligodendrocytes are a type of glial cells that produce a lipid-rich membrane called myelin. Myelin assembles into a sheath and lines neuronal axons in the brain and spinal cord to insulate them. This not only increases the speed and efficiency of nerve signal transduction but also protects the axons from damage and degradation, which could trigger neuronal cell death. Demyelination, which is caused by a loss of myelin and oligodendrocytes, is a prominent feature of many neurological conditions, including Multiple sclerosis (MS), spinal cord injuries (SCI), and leukodystrophies. Demyelination is followed by a time of remyelination mediated by the recruitment of endogenous oligodendrocyte precursor cells, their migration to the injury site, and differentiation into myelin-producing oligodendrocytes. Unfortunately, endogenous remyelination is not sufficient to overcome demyelination, which explains why there are to date no regenerative-based treatments for MS, SCI, or leukodystrophies. To better understand the role of oligodendrocytes and develop cell-based remyelination therapies, human oligodendrocytes have been derived from somatic cells using cell reprogramming. This review will detail the different cell reprogramming methods that have been developed to generate human oligodendrocytes and their applications to disease modeling and cell-based remyelination therapies. Recent developments in the field have seen the derivation of brain organoids from pluripotent stem cells, and protocols have been devised to incorporate oligodendrocytes within the organoids, which will also be reviewed.

Rat cortico-striatal sagittal organotypic slice cultures as ex vivo excitotoxic striatal lesion models.

Organotypic brain slice cultures are a useful tool to study neurological disease as they provide a 3-dimensional system which more closely recapitulates the in vivo cytoarchitectural complexity than standard 2-dimensional in vitro cell cultures. Building on our previously developed rat brain slice culture protocol, we have extended our findings to develop ex vivo excitotoxic lesion models by treatment of rat sagittal organotypic slices with AMPA or quinolinic acid (QA). We show that treatment of rat sagittal cortico-striatal organotypic slices with 8µM AMPA or 50µM QA causes striatal cell loss with a reduction in neuronal nuclei (NeuN)+ cells and an increase in ethidium homodimer-1 (EthD-1)+ dead cells compared to untreated slices. More specifically, following treatment with QA, we observed a reduction in medium spiny neuron DARPP32 + cells in the striatum and cortex of slices. Treatment of the slices with AMPA does not alter glial fibrillary acidic protein (GFAP) expression, while we observed an acute increase in GFAP expression 1-week post-QA exposure both in the cortex and striatum of slices. This recapitulates the excitotoxic and striatal degeneration observed in rat AMPA and QA lesion models in vivo. Our slice culture platform provides an advance over other systems with the ability to generate acute AMPA- and QA-induced striatal excitotoxicity in sagittal cortico-striatal slices which can be cultured long-term for at least 4 weeks. Our ex vivo organotypic slice culture system provides a long-term cellular platform to model neuronal excitotoxicity, with QA specifically modelling Huntington's disease. This will allow for mechanistic studies of excitotoxicity and neuroprotection, as well as the development and testing of novel therapeutic strategies with reduced cost and ease of manipulation prior to in vivo experimentation.

Development of agarose-gelatin bioinks for extrusion-based bioprinting and cell encapsulation.

Three-dimensional bioprinting continues to advance as an attractive biofabrication technique to employ cell-laden hydrogel scaffolds in the creation of precise, user-defined constructs that can recapitulate the native tissue environment. Development and characterisation of new bioinks to expand the existing library helps to open avenues that can support a diversity of tissue engineering purposes and fulfil requirements in terms of both printability and supporting cell attachment. In this paper, we report the development and characterisation of agarose-gelatin (AG-Gel) hydrogel blends as a bioink for extrusion-based bioprinting. Four different AG-Gel hydrogel blend formulations with varying gelatin concentration were systematically characterised to evaluate suitability as a potential bioink for extrusion-based bioprinting. Additionally, autoclave and filter sterilisation methods were compared to evaluate their effect on bioink properties. Finally, the ability of the AG-Gel bioink to support cell viability and culture after printing was evaluated using SH-SY5Y cells encapsulated in bioprinted droplets of the AG-Gel. All bioink formulations demonstrate rheological, mechanical and swelling properties suitable for bioprinting and cell encapsulation. Autoclave sterilisation significantly affected the rheological properties of the AG-Gel bioinks compared to filter sterilisation. SH-SY5Y cells printed and differentiated into neuronal-like cells using the developed AG-Gel bioinks demonstrated high viability (>90%) after 23 d in culture. This study demonstrates the properties of AG-Gel as a printable and biocompatible material applicable for use as a bioink.

Small Molecules Enhance Reprogramming of Adult Human Dermal Fibroblasts to Dorsal Forebrain Precursor Cells.

The development of human cell-based platforms for disease modeling, drug discovery, and regenerative therapy relies on robust and practical methods to derive high yields of relevant neuronal subtypes. Direct reprogramming strategies have sought to provide a means of deriving human neurons that mitigate the low conversion efficiencies, and protracted timing of human embryonic stem cell and induced pluripotent stem cell-derived neuron specification in vitro. However, few studies have demonstrated the direct conversion of adult human fibroblasts into multipotent neural precursors with the capacity to differentiate into cortical neurons with high efficiency. In this study, we demonstrate a reprogramming strategy using chemically modified mRNA encoding the proneural genes SOX2 and PAX6 coupled with small molecule supplementation to enhance the derivation of human-induced dorsal forebrain precursors directly from adult human dermal fibroblasts (aHDFs). Through transcriptional and phenotypic analysis of lineage-specific precursor and cortical neuron markers, we have demonstrated that this combined strategy significantly enhances the direct derivation of dorsal forebrain precursors from aHDFs, which, after timely exposure to defined differentiation media, gives rise to high yields of functional glutamatergic neurons. We propose that this combined strategy provides a highly tractable and efficient human cell-based platform for disease modeling and drug discovery.

Clozapine administration enhanced functional recovery after cuprizone demyelination.

The atypical antipsychotic agent, clozapine, is used to treat a variety of neurological disorders including schizophrenia and Parkinson's disease and readily crosses the blood brain barrier to interact with a wide range of neuroreceptors including those for dopamine and serotonin. Recent work has shown that clozapine can reduce neuroinflammation in experimental autoimmune encephalomyelitis, a neuroinflammatory model of multiple sclerosis (MS) and mediates its effects in the central nervous system. To further characterise the protection provided by clozapine, the cuprizone model of demyelination was used to assess the effect of clozapine treatment on the cellular events surrounding demyelination and remyelination. Using this model of non-immune demyelination, we found that clozapine administration was unable to prevent demyelination, but when administered post demyelination, was able to enhance the rate of functional recovery. The more rapid improvement of clozapine-treated mice correlated with a decreased level of astrocyte and microglial activation but only modestly enhanced remyelination. Together, these studies highlight the potential of clozapine to support enhanced functional recovery after demyelination, such as that occurring during MS.

Conversion of adult human fibroblasts into neural precursor cells using chemically modified mRNA.

Direct reprogramming offers a unique approach by which to generate neural lineages for the study and treatment of neurological disorders. Our objective is to develop a clinically viable reprogramming strategy to generate neural precursor cells for the treatment of neurological disorders through cell replacement therapy. We initially developed a method for directly generating neural precursor cells (iNPs) from adult human fibroblasts by transient expression of the neural transcription factors, SOX2 and PAX6 using plasmid DNA. This study advances these findings by examining the use of chemically modified mRNA (cmRNA) for direct-to-iNP reprogramming. Chemically modified mRNA has the benefit of being extremely stable and non-immunogenic, offering a clinically suitable gene delivery system. The use of SOX2 and PAX6 cmRNA resulted in high co-transfection efficiency and cell viability compared with plasmid transfection. Neural positioning and fate determinant genes were observed throughout reprogramming with ion channel and synaptic marker genes detected during differentiation. Differentiation of cmRNA-derived iNPs generated immature GABAergic or glutamatergic neuronal phenotypes in conjunction with astrocytes. This represents the first time a cmRNA approach has been used to directly reprogram adult human fibroblasts to iNPs, potentially providing an efficient system by which to generate human neurons for both research and clinical application.

Human Cortical Neuron Generation Using Cell Reprogramming: A Review of Recent Advances.

The study and treatment of neurological disorders have been hampered by a lack of access to live, healthy, or disease-affected human neurons. The recent advances in the field of cell reprogramming offer exciting new possibilities for disease modeling, drug development, and cell-based therapies. Since the derivation of human embryonic stem cells (hESCs) and their differentiation into neurons, cell reprogramming technologies have built on these protocols to generate mature human neurons of disease-associated phenotypes from somatic cells. Mechanistic knowledge of neural patterning and neurogenesis has been essential for the establishment of reprogramming strategies that employ a combination of transcription factors and small molecules selected due to their critical role in brain development. The generation of reprogrammed human neurons has the potential to further enhance our knowledge of pathways underlying the developmental process of the human brain, the current knowledge of which has predominantly come from animal studies, postmortem tissue, and most recently hESCs. Somatic cell reprogramming began in 2006 with the first report of induced pluripotent stem cell (iPSC) derivation from mouse fibroblasts. This has now expanded to direct-to-induced neuron and direct-to-induced neural stem or precursor reprogramming using a variety of viral and nonviral delivery methods. Most recently, iPSC technology has been extended to the development of three-dimensional brain structures referred to as brain spheroids or organoids. This review will discuss the reprogramming strategies that have been formulated to generate cortical neurons that are associated with many diseases, including autism spectrum disorders and schizophrenia.

Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system.

BACKGROUND: Organotypic brain slice cultures are a useful tool to study neurological function as they provide a more complex, 3-dimensional system than standard 2-dimensional in vitro cell cultures. NEW METHOD: Building on a previously developed mouse brain slice culture protocol, we have developed a rat sagittal brain slice culture system as an ex vivo model of dopamine cell loss. RESULTS: We show that rat brain organotypic slice cultures remain viable for up to 6 weeks in culture. Using Fluoro-Gold axonal tracing, we demonstrate that the slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period, with particular focus on the nigrostriatal pathway. Treatment of the cultures with 6-hydroxydopamine and desipramine induces a progressive loss of Fluoro-Gold-positive nigral cells with a sustained loss of tyrosine hydroxylase-positive nigral cells. This recapitulates the pattern of dopaminergic degeneration observed in the rat partial 6-hydroxydopamine lesion model and, most importantly, the progressive pathology of Parkinson's disease. COMPARISON WITH EXISTING METHODS: Our slice culture platform provides an advance over other systems, as we demonstrate for the first time 3-dimensional cytoarchitecture maintenance of rat nigrostriatal sagittal slices for up to 6 weeks. CONCLUSION: Our ex vivo organotypic slice culture system provides a long term cellular platform to model Parkinson's disease, allowing for the elucidation of mechanisms involved in dopaminergic neuron degeneration and the capability to study cellular integration and plasticity ex vivo.