Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique
Graphical abstract
Introduction
Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) are severe and most often rapidly fatal neurodegenerative disorders. ALS is characterized by progressive degeneration of motor neurons in the spinal cord, brainstem and cerebral cortex. ALS can be caused by mutations in more than 25 genes including C9ORF72, SOD1 and TARDBP or occurs sporadically (Robberecht and Philips, 2013). Animal models of ALS and corresponding cellular models have given important insights into disease mechanisms but also bear major limitations. Transgenic mutant SOD1 mice, for instance, develop motor neuron disease only when the mutant enzyme is expressed at high non-physiological levels (Gurney et al., 1994) and their response to numerous pharmacological compounds is not predictive for human ALS (Turner and Talbot, 2008, Mitsumoto et al., 2014).
Human motor neurons generated from induced pluripotent stem cells (iPSc) (Takahashi et al., 2007) offer a new alternative for disease modeling and drug testing in ALS. Recent studies have indeed provided evidence for molecular and functional alterations in motor neurons derived from ALS patients (Bilican et al., 2012, Chen et al., 2014, Egawa et al., 2012, Haeusler et al., 2014, Kiskinis et al., 2014, Sareen et al., 2013). Yet, modeling the two major phenotypic alterations of ALS, motor neuron cell body death and axon degeneration, faces important still unresolved problems in iPSc-derived cultures. First, the efficiency of motor neuron generation remains variable between protocols and from one iPSc clone to another (Amoroso et al., 2013, Qu et al., 2014). Second, motor neuron cell bodies tend to form large clusters which are inaccessible for analysis of cell survival (Chen et al., 2014, Dimos et al., 2008, Ebert et al., 2009, Hu and Zhang, 2009, Kiskinis et al., 2014, Maury et al., 2015, Qu et al., 2014, Sareen et al., 2013). Motor neuron axons often also display extensive criss-crossing and intermingling with axons from other neuronal types thus complicating their identification and analysis (Amoroso et al., 2013, Bilican et al., 2012, Boulting et al., 2011, Burkhardt et al., 2013, Chen et al., 2014, Egawa et al., 2012, Haeusler et al., 2014, Kiskinis et al., 2014, Maury et al., 2015, Qu et al., 2014, Sareen et al., 2013). Third, the proportion of disease-relevant motor neuron subtypes remains most often undetermined in human iPSc-derived cultures (Bilican et al., 2012, Boulting et al., 2011, Egawa et al., 2012, Hester et al., 2011, Karumbayaram et al., 2009, Mitne-Neto et al., 2011, Qu et al., 2014, Serio et al., 2013) but see Amoroso et al. (2013). Fourth, the presence of unwanted cell types such as neural precursors, various types of neurons (Amoroso et al., 2013, Boulting et al., 2011, Chen et al., 2014, Dimos et al., 2008, Ebert et al., 2009, Karumbayaram et al., 2009, Naujock et al., 2014, Qu et al., 2014, Sareen et al., 2012) or astrocytes (Haidet-Phillips et al., 2011, Re et al., 2014) may induce confounding effects on motor neuron survival. Likewise, pharmacological compounds may act indirectly through these cell types rendering difficult the interpretation of their effects. Generating pure and standardized human motor neuron cultures is therefore an important step towards robust ALS modeling and drug testing.
Fluorescence-activated cell sorting (FACS) represents a powerful means to isolate the desired neuronal populations. Yet, there are few well-characterized genetic reporters and surface markers for FACS isolation of motor neurons (Amoroso et al., 2013, Egawa et al., 2012, Kiskinis et al., 2014, Wichterle et al., 2002). In addition, FACS-isolated human motor neurons were reported to survive and grow poorly in culture (Egawa et al., 2012). We here developed a novel procedure for motor neuron isolation by combining a lentiviral reporter vector harboring the 3.6 kb minimal HB9 promoter (Marchetto et al., 2008), which drives RFP expression and a monoclonal antibody against the cell surface receptor p75NTR (Ross et al., 1984). Using p75NTR/HB9::RFP FACS double selection, we succeeded in isolating high numbers of 100% pure and functional human iPSc-derived motor neurons which comprise ALS-relevant motor neuron subtypes. The FACS-isolated motor neurons survive and grow well in low-density cultures without forming clusters or entangled axons. By contrast, they undergo increased cell death and form prominent axon blebs in response to mutant SOD1 astrocytes, indicating their potential for improved ALS modeling.
Section snippets
iPSc reprogramming and characterization
Human skin fibroblasts from two healthy donors aged 33 and 11 years (individuals 1 and 2, respectively) were obtained from the Centre de Ressources Biologiques in Lyon (France) after approval by the competent French authorities. The biological samples were fully anonymized and declared according to French laws from the Ministère de la Recherche. Ethical and regulatory issues were approved by Comité de Protection des Personnes d’Ile-de-France. Fibroblasts were grown in DMEM/Glutamax (Life
Selection of genetic reporters and surface markers for human iPSc-derived motor neurons.
We derived four iPSc clones from two unrelated healthy individuals, characterized them to be fully pluripotent and devoid of chromosomal abnormalities (Supplementary Figs. S1 and S2), and differentiated them towards motor neurons according to the protocol of Hu and Zhang (2009). In line with previous studies, the cell bodies of motor neurons are often clustered and their axons entangled with those of other types of neurons (Fig. 1A), hampering the analysis of degenerative processes. Motor
Discussion
Here, we report cellular ALS modeling using human iPSc-derived motor neurons isolated by a novel p75NTR/HB9::RFP FACS double selection technique. The FACS-isolated motor neurons are absolutely pure (i.e. free of interneurons, neural precursors and glia) and functional (i.e. electrically excitable and able to connect to co-cultured myotubes). In particular, they survive and grow well in low-density cultures where they are devoid of cell body clusters or entangled axons. By contrast, following
Acknowledgements
We gratefully acknowledge the expert help of Marc Barad (Centre for Immunology Marseille-Luminy) and the support of Jean-Gabriel Fernandez and Jean-Marc Sorraing (Becton Dickinson France) on FACS and cytometry. We thank Fred Clotman (University of Leuwen, Belgium) for the generous gift of antibodies, Caroline Chariau (Institut Pasteur, Paris) for technical help with iPSc cultures, Jean Michel Heard, members of the ERANET consortium IPSOALS for helpful discussions and Catherine Rabouille
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