A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery

Background

Amyotrophic lateral sclerosis (ALS) is a neurological disorder resulting in degeneration of both upper and lower motor neurons, resulting in the progressive failure of the neuromuscular system. Death typically occurs 2-3 years post-symptom onset, due to a lack of effective therapies. Although death of motor neurons is a key feature of ALS, the disease is non-cell autonomous with neighboring cells such as astrocytes, microglia and oligodendrocytes playing a key role (Ferraiuolo et al., 2011b; Ferraiuolo et al., 2016; Frakes et al., 2017; Vandoorne et al., 2018). Astrocytes play a crucial metabolic role in the CNS as they are the major source of brain glycogen, and astrocyte lactate can be taken up by motor neurons and used as a source of energy (Pellerin and Magistretti, 1994). Several mechanisms have been implicated in astrocyte-mediated motor neuron death, including release of nitric oxide and prostaglandin E2, altered glutamate transporter expression, reduced lactate release and reduced extracellular vesicle mediated release of miRNA (Lin et al., 1998; Ferraiuolo et al., 2011a; Ferraiuolo et al., 2011b; Allen et al., 2019; Varcianna et al., 2019).

Until recently, the ability to study how patient derived astrocytes affect motor neuronal function has been hampered by technological limitations. Initial pioneering studies (Haidet-Phillips et al., 2011, Re et al., 2014) demonstrated that post-mortem human sporadic and familial patient-derived astrocytes could induce motor neuron death in vitro. Although elegant, this methodology presents with limitations in terms of limited availability, scalability and represents end stage of disease. The development of techniques to reprogram fibroblasts into pluripotent stem cells (iPSCs) by Yamanaka and colleagues (Takahashi and Yamanaka, 2006) has radically improved our ability to study human CNS disorders in vitro, for a recent review see (Myszczynska and Ferraiuolo, 2016). This technological advancement was followed up by methodologies to directly reprogram mouse fibroblasts into induced neuronal progenitor cells (iNPCs) and concomitantly by Meyer and Ferraiuolo who developed novel methodologies to reprogram adult human fibroblasts into iNPCs from control and ALS subjects (Kim et al., 2011; Meyer et al., 2014). This advancement in direct reprogramming, has overcome the challenge of phenotypic inconsistency caused by clonal variation in iPSCs and has significantly shortened the reprogramming timescales, reducing costs and increasing throughput (Myszczynska and Ferraiuolo, 2016). Furthermore, an additional beneficial feature of the direct reprogramming approach is that the epigenetic state of the cell is not reset, so any aging phenotype inherent in the donor fibroblasts should be retained (Mertens et al., 2015). This subsequent development has enabled ALS researchers to assess the effect of disease on patient derived cells in vitro, reducing the reliance on post-mortem tissue and animal models of disease that may lack translational efficacy.

In the co-culture approach described in this paper, iNPCs were derived from ALS patient fibroblasts and healthy donor control fibroblasts as described previously (Meyer et al., 2014). iNPCs were differentiated into iAstrocytes by culturing in iAstrocyte media for at least 5 days (Figure 1). Mouse motor neurons expressing the green fluorescent protein (GFP) under the Hb9 motor neuron-specific promoter (called from now on Hb9-GFP+ MN) were differentiated from murine embryonic stem cells (mESCs) via embryonic bodies (mEBs), as previously described (Wichterle et al., 2002; Haidet-Phillips et al., 2011). The advantage of this approach over existing co-culture methods using post-mortem material or iPSCs, is that iAstrocyte support of motor neurons can be measured in a high-throughput, cost-effective fashion, under a number of conditions including drug treatment or nutritional supplementation. Furthermore, traditional cell-based high throughput screening methodologies that have been utilized in the ALS field have focused on animal or human cell models of the disease in monoculture, for a recent review see (McGown and Stopford, 2018). In the methodological approach adopted here, iAstrocytes can be reproducibly differentiated within a week from iNPCs and their effect on motor neuron survival can be measured in co-culture. Survival can be assessed by monitoring fluorescence of the motor neurons over time and counting the number of viable motor neurons (Meyer et al., 2014; Hautbergue et al., 2017; Allen et al., 2019). Moreover, the effect of treated or untreated iAstrocyte conditioned media on motor neuron survival can also be assessed (Varcianna et al., 2019). Therefore, this approach has the advantage over traditional high throughput monoculture methodologies of being more physiologically representative of the in vivo state and therefore has greater translational potential.