Neural cell cultures to study spinal cord injury

Drug Discovery Today: Disease Models

DRUG DISCOVERY

TODAY

DISEASE

MODELS

Vol. 25–26, 2017

Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA

Models of Neuroimmune and Neurodegenerative Diseases

Neural cell cultures to study spinal cord injury George A. McCanney, Michael J. Whitehead, Michael A. McGrath, Susan L. Lindsay, Susan C. Barnett* Institute of Infection, Immunity and Inflammation, University of Glasgow, 120 University Place, Glasgow, G12 8TA, UK

There are great challenges involved in identifying potential therapies for the repair of spinal cord injury (SCI). It is well accepted that not one, but a combination of therapeutic strategies will be required to effec-

Section editor: Professor Sandra Amor – Department of Pathology, Amsterdam UMC, VUMC site, 1081 HV Amsterdam, The Netherlands.

tively repair the damage. However, identifying novel therapeutics is hindered by the lack of reliable methods

Neural cell cultures can be used to model different

available that facilitate high throughput screening of

aspects of spinal cord injury.In vitro models range from

numerous compounds. While the use of animals pro-

individual cell types through to complex co-cultures.

vides an important means for testing new therapies, in

Simple cultures may be easier to scale up to high

vivo models of SCI can be time consuming and require

throughput, however lack the cell-cell cross talk and

the use of large cohorts of animals. In this review, we

3D architecture found in the central nervous system.

have focused on three aspects of repair following SCI

paralysis. The initial primary injury causes mechanical disruption of axons and cell death due to ischemia and cell membrane rupture. Moreover, during this stage vascular damage, changes in oxidative stress, excitotoxicity and inflammation occurs, initiating a secondary injury cascade of cellular and molecular processes [1]. Secondary injury, which can last for several weeks, results in glial and neuronal cell death, axonal degeneration, disrupted nerve pathways, cystic cavitation and the formation of a glial scar [2]. The inflammatory cascade is one of the main attributors to secondary damage by releasing mediators such as, IL-1b, TNFa and IL-6. Cytokine release leads to the activation of resident CNS immune cells (microglia) [3–5] resulting in oligodendrocyte loss/demyelination of spared axons [6]. Consequently, both primary and secondary SCI contribute to a hostile inhibitory environment that requires therapeutic intervention to promote repair and functional recovery. Due to this apparent complexity of SCI, therapies would be multifaceted and need

(1) neurite outgrowth, (2) glial scar and (3) remyelination. No in vitro model encapsulates all the features of SCI and we discuss the limitations and virtues of the various cultures, which range from individual cell-types through to complex co-cultures. We discuss how these cultures can be used as a moderate throughput screen to identify novel therapeutics for CNS repair before being verified in animal models.

Introduction Spinal cord injury (SCI) is a devastating condition, which can result from trauma to the cord and often leads to permanent *Corresponding author: S.C. Barnett ([email protected])

1740-6757/$ Crown Copyright © 2018 Published by Elsevier Ltd. All rights reserved. https://doi.org/10.1016/j.ddmod.2018.10.005

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to target many aspects of the pathology [7]. Ideal strategies would decrease the inhibitory molecules associated with astrocytosis and prevent the formation of the glial scar, fill cysts, promote neurite outgrowth/plasticity and ultimately promote re/myelination (Fig. 1). The complex nature of SCI has created many challenges in the pursuit of identifying potential therapies. Animal models have been developed with the aim of providing a platform to study SCI and are used routinely to determine the therapeutic potential of novel compounds. However, using such models are time-consuming and require large cohort of animals; significantly decreasing the number of compounds that can be screened. In addition, experimentation on animals does not follow the NC3Rs principles of replacement, reduction and refinement, which provide a framework for performing more humane animal research (www.nc3rs.org.uk). Consequently, several in vitro models of SCI injury have been developed for pre-screening of potential therapeutics. In this way, in vitro pre-screening identifies the most promising novel therapeutics, before committing to in vivo experiments. Interestingly, many potential candidates have been identified, however, few have entered the clinic [8]. This could be attributed to the inability of the models used to effectively validate these candidates.

In vitro approaches to study SCI In vitro approaches to study SCI can offer a more controlled environment to study the cellular and molecular aspects of the injury and repair process over time [9–12]. The composition of in vitro cultures can be in the form of monocultures, which

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allow detailed changes in a defined cell to be observed, or cocultures that can range from two cell types to complex dissociated neural tissue and slice cultures. Variations of such cultures have allowed the study of specific aspects of injury and repair after SCI such as (1) neurite outgrowth, (2) glial scar and (3) remyelination, which we will now discuss in detail:

Neurite outgrowth Neurite outgrowth assays have provided a relatively easy way to determine the effect of a therapeutic drug or compound on neuronal function and their cellular properties. It is well recognised that after SCI, injured axons have a limited capacity to regenerate [1]. The identification of the molecular processes involved in axon regeneration is, therefore, an attractive area of research [13]. Neurite outgrowth assays commonly use either dissociated or explanted dorsal root ganglions (DRGs) [14–16] (Fig. 2C–D), although neuronal cell lines such as the PC12 and F11 have also been used [17–19] (Fig. 2B). Alternatively, organotypic spinal cord slices can be used, in which intact tissue sections placed as explants allow the outgrowth of sensory and motor neurons to be individually assessed [20] (Fig. 2E). In these assays, typical parameters measured include neurite number, length and morphology, growth cone collapse and neurotoxicity of compounds. Assays, such as DRG explants, have identified neurite outgrowth inhibitor molecules found at the site of SCI, such as myelin-associated inhibitors and chondroitin sulphate proteoglycans (CSPGs) [14,21,22]. Furthermore, the growth-inhibitory effect of other glial scar secreted proteins, such as semaphorin 3A [24] or NG2 [23,25] have also been identified

In vivo SCI Axotomised nerve fibres

Spared nerve fibre

Demyelinated nerve fibre

White matter

Grey matter

Fluid filled cavity

Glial scar

Reactive Astrocytes Microglia/Macrophages Oligodendrocytes Myelin debris

Drug Discovery Today: Disease Models

Fig. 1. Pathological changes in the spinal cord after injury.

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(a) Axonal transection (1) and Neurite outgrowth (2)

2

1

(b) PC12/F11 cell lines

(c) Dissociated DRGs

(d) Explanted DRGs

(e) Spinal cord slices

(f) Injured myelinating culture (g) Injured organotypic slice array

Key: Neurite

Coated nanofiber

Myelin debris Drug Discovery Today: Disease Models

Fig. 2. In vitro neurite outgrowth models. Schematic of axonal transection, which occurs during SCI (1) with minimal neurite outgrowth occurring spontaneously (2). (B–D) Neurite outgrowth assays. Parameters for assessment include length and number of neurites. (E–G) Complex mixed cell or organotypic cultures used to study SCI after injury. White dotted line depicts lesion edge after cutting with a scalpel blade.

in this way. Therefore, neuronal cultures can be used to screen single or combinational treatments to evaluate their ability to overcome the inhibitory environment around SCI. Importantly, neurite outgrowth assays of this nature can be easily scaled up to be a high throughput method. Neurite outgrowth assays have been used to screen FDAapproved therapeutics for other diseases to test whether they could be repurposed as a SCI treatment. For example, Epothilone B (EpoB), a microtubule-stabilising drug similar to the FDA-approved anti-cancer drug Paclitaxel, has shown encouraging results [26]. EpoB can inhibit the abhorrent polarisation of fibroblasts following injury and cause rapid

polymerisation of microtubules at the growth cone leading to axon elongation [26]. In addition, simple neurite outgrowth assays have been used to assess the potential of novel biomaterials which have been proposed to fill the lesion or ‘‘bridge the gap’’ after SCI [27–29]. Polarized matrices have been found to promote guidance and directed growth of neurites [30]. While different variations of hydrogels, including PEGylated fibrinogen hydrogels, laminin modified hydrogels and positively charged hydrogels, support DRG survival and attachment, and promote extensive outgrowth of sprouting neurites [27,31–33]. www.drugdiscoverytoday.com

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[26,36–38]. However, establishing an effect on neurite outgrowth using a single cell type does not necessarily infer the same effect will be present in the complex injury environment created during SCI. It is therefore necessary to screen potential therapeutics in other more complex in vitro SCI models to determine therapeutic efficacy.

Combining neurite outgrowth assays with biomaterial screening means a number of potential biomaterials are now being developed for SCI repair [29,34]. Furthermore, the development of organotypic slice arrays enables the incorporation of coated nanofibers into a lesion, thus facilitates the screening of potentially neuroregenerative biomaterials in an injury setting [35] (Fig. 2G). Innovative studies have used organotypic cultures of spinal cord slices together with dorsal root ganglia explants to explore the interface between the PNS and CNS and make comparison of motor and sensory neuron outgrowth. In this example, organotypic slice cultures are plated on gelled collagen droplets next to DRG explants [20]. Compounds and biomaterials which directly enhance neurite outgrowth in vitro can then be tested in animal models to assess functional outcomes. Several of the targets discussed previously have already been tested using animal models of SCI and have shown promising results, providing a proof-of-concept for these cultures as drug discovery screens

Glial scar It is well accepted that astrocytes exert a detrimental effect in the chronic injury phase by the formation of a glial scar; a physical border encapsulating the injury site through newly proliferated elongated astrocytes [1,39]. A hallmark feature is localised astrocytosis, characterised by cellular hypertrophy with increased expression of intermediate filaments including GFAP, vimentin and nestin [40] (Fig. 3C). Reactive astrocytes also display increased CSPG secretion and extracellular proteins such as tenascin C and semaphorin 3A [41,42]. The glial scar inhibits axonal outgrowth as the growth cone of extending neurites form dystrophic ends upon entry into the

(a) Scratch-wound assay i

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(b) Astrocyte-Fibroblast Stretch

ii

i

ii

(c)

Glial Scar in vivo Astrocytosis Increased CSPG Increased tenascin C Inhibition of axonal outgrowth

(e) Neuron-Glia Microwires

(d) Astrocyte-Cell candidate Mingling ii

i

Key:

Astrocyte

i

Fibroblast

ii

Cell candidate

Microwire

Drug Discovery Today: Disease Models

Fig. 3. In vitro glial scar models. Several cultures are depicted which mimic characteristics of the glia scar. (A) Schematic of a scratch assay using (i) monolayer of astrocytes (ii) after an injury is induced using a pipette tip. (B) Schematic of a stretch assay (i) co-culture of astrocytes and fibroblasts (ii) injury is induced using Flexplates. (C) Schematic of the in vivo glial scar including features which can be recapitulated using in vitro models. (D) Confrontation/Mingling assays (i) co-culture of astrocytes and cell candidates, (i.e. Schwann cells) with a boundary forming at the cell interface. (ii) Olfactory ensheathing cells can mingle with astrocytes (E) Co-cultures with microwires (i) neuronal/glial cell cultures (astrocytes labelled) (ii) aspects of the glial scar can be modelled by the addition of several microwires into the cultures.

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lesion [43]. Therefore, it is not surprising that there exist numerous in vitro models which aim to recapitulate this facet of the injury response. The simplest model is the scratch-wound assay where a monolayer of astrocytes is mechanically injured using a pipette tip to produce a cell-free lesion [44] (Fig. 3A). This model simulates the proliferation, migration and activation of astrocytes, which are the key responses involved in the initiation of the glial scar. It has been used to demonstrate numerous potential therapies targeted to reducing glial scar formation. It has revealed the importance of voltage-gated sodium channels [45], identified PD168393, the EGF receptor inhibitor as a potent modulator of astrogliosis [46] and EphA4 kinase inhibitors have been shown to have a potential role in the inhibition of scar formation [47]. Recently, the plant flavonol, Fisetin has been shown to prevent astrocyte migration and glial scar formation using in vitro scratch assays [48]. Reactive astrogliosis has also been induced through mechanical astrocyte stretching, which induces astrocyte stiffness with an accompanying increase in GFAP expression, akin to that found in vivo [49,50]. Although these simple monocultures allow for the elucidation of astrocyte specific mechanisms there are obvious limitations, most notably the loss of cell-cell cross talk. During the formation of the glial scar, meningeal fibroblasts infiltrate and occupy the lesion site and so an astrocytefibroblast interface is a fundamental component of the scar [51]. This important interaction has been recapitulated in vitro using meningeal fibroblasts co-cultured with astrocytes [52]. Mechanical stretching of astrocyte-fibroblast co-cultures induced increased GFAP and tenascin C expression analogous to the monoculture stretch and also to that seen in vivo [52] (Fig. 3B). This model was developed further to mimic additional features of the glial scar by culturing astrocytes alongside cortical or spinal neurons. These assessments of neurite outgrowth have led to the identification of numerous compounds which increase neurite outgrowth [52,53]. Such ‘‘hit’’ compounds could now be validated in vivo and potentially improve functional outcomes for SCI. Confrontation or mingling assays have also been used to mimic aspects of the glial scar [54]. In these assays, astrocytes are cultured aligned against cell candidates for transplantmediated repair e.g. Schwann cells or olfactory ensheathing cells, and allowed to grow towards each other. Results have shown differences in their ability to mingle or form cell boundaries [52,55] (Fig. 3D). This assay has been refined to examine fibroblast-astrocyte cultures which form an in vitro fibrotic scar upon addition of TGF-b (a known contributor to glial scar formation) with concomitant upregulation of scarassociated factors [41,56]. Thus demonstrating this models capacity to recapitulate the glial scar in vivo. Glial scarring has also been modelled in vitro by placing stainless steel microwires into neuronal-glia mixed cultures [57] (Fig. 3E). The

injury is quantified using a scarring index based on GFAP intensity plot across the wire. This model identified that silk coating of the microwire reduces gliosis, proposing the use of this culture to screen different novel biomaterials for SCI repair [58]. It can be seen that such astrocyte models are useful since they can identify compounds relatively quickly and easily. In particular, they can ascertain which potential candidates elicit their effect by overcoming inhibition; rather than a generic promotion of neurite outgrowth, which may have no relevance in an injury environment like the glial scar. Therefore, glial scar models could lead to the development of therapeutics which overcome repair inhibition. It is evident even from the few examples discussed here that the models used can be attuned dependent on the therapeutic approach, for example the microwire injury would be more relevant for testing a potential biomaterial for SCI repair, while the boundary assays would be more relevant to test cell candidates for transplant mediated repair.

Remyelination During SCI chronic focal demyelination occurs proximal to the injury site leaving denuded axons susceptible to degradation [59,60]. Remyelination is the process whereby oligodendrocyte precursor cells (OPCs) differentiate and ensheath demyelinated axons forming new compact myelin (Fig. 4A). Transplanting rat OPCs into a demyelinated lesion in rat spinal cord resulted in remyelination [61]. Moreover, the transplantation of OPCs derived from human embryonic stem cells into an in vivo contusion injury promoted remyelination, resulting in improved functional recovery [62]. These data suggest remyelination of axons in and around the injury site is an important facet of SCI repair and therapeutics which promote this could have beneficial clinical outcomes for patients. Indeed it has been shown in clinical cases of SCI that remyelination of spared axons is incomplete in post-mortem tissue [60,63,64]. However, whether exogenous OPC transplantation is a worthy therapeutic approach is currently under debate [65,66]. Myelination has been studied in vitro using several culture methods. Indeed, in vitro models which screen chemical libraries have been used to identify potential therapeutics that promote OPC remyelination [67]. However, when selecting the most suitable model for any given experiment there is a trade-off between culture complexity and throughput, and is therefore dependent upon the number of compounds to be tested and the time-frame available. OPCs in combination with DRG cultures allow myelination to be examined in the absence of any other glial cells (Fig. 4C) and have been used to demonstrate fibrinogen as an inhibitor of myelination through BMP signalling pathways [68]. Chan and colleagues showed that electro-spun polystyrene nanofibers can be myelinated by OPCs, uncoupling any www.drugdiscoverytoday.com

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(a) Re/Myelinaon

(b) OPC monocultures

i

(c)

DRG/OPC cultures

ii

(d) Myelinang co-cultures

i

ii

(e) Organotypic slice cultures

i

ii

Key: Myelinang oligodendrocyte

Myelinated micropillar

Nanofiber

Neurite

Myelinated neurite

Drug Discovery Today: Disease Models

Fig. 4. In vitro models to study remyelination. (A) Schematic of re/myelination with an oligodendrocyte precursor cell (OPC) differentiating and ensheathing the denuded axon to form compact internodes of myelin with nodes of Ranvier. (B–E) in vitro tools used to study remyelination with increasing complexity. (B) OPC monocultures can be grown on nanofibers (i) or on micropillars (ii) and will form myelin sheaths around the inert fibers or pillars respectively. (C) OPC/dorsal root ganglion cocultures to investigate axon ensheathment. (D) Myelinating dissociated spinal cord cultures allow developmental myelination to be observed in a complex mixed CNS cell environment (i). Injury can be induced on these cultures using a scalpel blade (ii) allowing examination of remyelination and neurite outgrowth after injury. (E) Organotypic slice cultures provide intact CNS tissue in which developmental myelination and remyelination can be studied (i). An injury can be modelled through a partial transection using a scalpel blade (ii).

axonal guidance cues (Fig. 4Bi). This model can be used to screen compounds which directly promote myelination [69]. The concept of myelinating inert scaffolds was developed further through culturing OPCs on 96 micropillar-well plates (Fig. 4Bii). The subsequent high throughput assay facilitated screening of a chemical library pulling out eight FDA-approved compounds with anti-muscarinic properties that enhanced oligodendrocyte differentiation and membrane wrapping [67]. Although the cultures discussed above allow the direct investigation of myelination and could be used to identify 16

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compounds which modulate it, they offer no information on how a compound will affect myelination in the presence of the immune response and other glial cells (namely microglia and astrocytes). For instance, Fibroblast Growth Factor (FGF) 9 is an astrocyte secreted protein which inhibits myelination and could subsequently be a therapeutic target [70]. This and similar potential candidates could not be discovered using astrocyte lacking cultures, highlighting the need for a more representative CNS environment. One such system is the use of myelinating cultures, which are generated from dissociated rodent spinal cords and comprise of all CNS cell types [71]

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(Fig. 4Di). These cultures, which form myelinated fibres, have been modified to model aspects of SCI with an injury being created using a flat edge scalpel blade. These injured cultures recapitulate many of the features of SCI in vivo, including astrocytosis, focal demyelination, loss of neurite density and lack of spontaneous neurite outgrowth [38] (Fig. 2F; Fig. 4Dii). This injury environment provides several quantifiable parameters including myelination adjacent to the lesion and neurite outgrowth. Substantiation of this culture as an in vitro SCI model was performed with the Rho-ROCK inhibitors, C3 and Y23627 which have been tested in animal models of SCI, as well as testing the growth supporting properties of biomaterials [72]. As seen in animal models of SCI, these inhibitors also promoted repair after injury in myelinating cultures, leading to both increased myelination and promotion of neurite outgrowth [9]. An advantage of these more complex cultures is that they can easily be used as a low-moderate throughput screen.

Limitations in current in vitro methodologies A limitation of using cultured cells is that the typical 3D physiological architecture formed in vivo is lost. This may alter cellular interactions and therefore questions the extent to which such models truly represent the in vivo environment. The use of ex vivo organotypic slice cultures from the brain or spinal cord somewhat negates this issue providing intact complex CNS tissue for study [73] (Fig. 4Ei). These slice cultures can be demyelinated using lysophosphatidyl choline allowing subsequent remyelination to be investigated. This culture system facilitated the identification of several factors that promote CNS remyelination, including CCN3, endothelin 2 and Retinoid X receptor gamma agonist [74–76]. Moreover, slice cultures have been adapted to model SCI with the

induction of a partial transection injury using a scalpel blade [77] (Fig. 3Eii). This successfully determined the optimal wavelengths for phototherapy to reduce oxidative stress after CNS injuries [77]. While slice cultures may produce an improved representation of the CNS compared to cell culture, there are limitations in reproducibility as well as the need for high powered confocal imaging of the thick slices.

Future perspectives The future direction of neural cell cultures for the study of SCI should be focussed on the development of human-derived complex co-cultures. This is now becoming a possibility with the identification of factors that can induce cell-type specific differentiation of induced hiPSCs into astrocytes, neurons oligodendrocytes and microglia [78]. Recent developments are paving the way for in vitro SCI screens using human hiPSC derived neurons in a high throughput screen for neurite outgrowth [79]. Future assay development using hiPSC could be used for human glial scar assays, as well as the development of in vitro SCI models similar to what we have previously developed in rodents, where there is cross-talk between the many cell types of the CNS including astrocytes, neurons, microglia, oligodendrocytes and pericytes [9,80–82]. This cellular cross-talk will be understood in much greater detail with the current advancements in single cell omics technology. In addition to the use of human cells, future assays should aim to model the extracellular matrix, using hydrogels for example, and attempt to mimic the changes seen in pathology after SCI. All of the cell culture models discussed here attempt to mimic the changes seen in pathology after a transection SCI (Table 1). While other types of SCI (e.g. contusion) have been modelled in vitro, as reviewed in [83],

Table 1. In vitro models of spinal cord injury.A list of in vitro models of SCI indicating the feature which they represent, the cellular complexity of the model (on a scale 1 monocultures 5 ex vivo tissue) and the potential use for low to high throughput screen. Model

Neurite outgowth

PC12/F11 cell line outgrowth Dissociated DRGs outgrowth Explantated DRGs outgrowth Astrocyte scratch Astrocyte stretch Astrcoyte-fibroblast stretch Astrocyte-fibroblast-neuron stretch Neuronal-glia culture microwires Astrcoyte-fibroblast mingling Astrocyte-Schwann cell mingling OPC-DRG cocultures OPC nanofibers OPC micropillars Myelinating co-cultures Organotypic slice cultures

  

 

 

Glial scar

Remyelination

Complexity

Throughput

Citation

    

1 1 5 1 1 2 3 4 2 2 2 1 1 4 5

High Moderate Moderate Moderate Moderate Moderate Moderate Low Moderate Moderate Moderate Moderate High Moderate Low

[17–19] [14,16] [15] [45–48] [49,50] [52] [53] [57] [41] [55] [68] [69] [67] [71,9] [20,74–77]

      

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these techniques have not been widely adopted because they are difficult to scale up.

Conclusion The pathology of SCI is complex and treatment strategies will involve more than one therapeutic. As discussed above, there are many in vitro options to study characteristics of SCI, which offer different levels of complexity with varying efficacy as a throughput screen. Simple monocultures allow the screening of large compound libraries, however, may lack the complex cellular architecture provided by low throughput organotypic cultures or animal models of SCI. However, they do allow a more detailed study of the cellular, molecular and biochemical changes which occur after injury, which are not as easily made using in vivo models of SCI. Furthermore, the fundamental cross-talk between astrocytes, neurons, microglia, oligodendrocytes and pericytes must be taken into account when testing therapeutics, as they could have important biological effects on repair mechanisms. Future development of drug discovery screens for SCI should focus on including cellular cross-talk, more accurately recapitulating the 3D extracellular environment, human induced pluripotent stem cell-derived neuronal/glial cells, and attempt to create techniques for modelling different types of SCI.

Acknowledgments This study was funded by the National Centre for Replacement, Refinement and Reduction, of Animals in Research (MAM) and a project grant ETM/439 from CSO (SL) and MS Society (56, SL), Medical Research Scotland (MRS; GM) and a Doctoral Training Grant to MJW provided jointly by the UK MRC (MR/K501335/1) and UK BBSRC (BB/J013854/1).

Conflict of interest The authors declare that they have no conflict of interest.

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