Modeling neurological disorders using human induced Pluripotent Stem cells-derived neurons D. Buttigieg1; E. Gras-Lavigne1; Lydia Thon1 ; C. Badja2 ; M. Ouamer1 ; R. Steinschneider1 ; F. Magdinier2

1 Neuron Expert, 51 Bd Pierre Dramard, 13015; 2 Equipe "Epigénétique, Chromatine & Maladies" Laboratoire INSERM UMR S_910, Faculté de Médecine de la Timone, 27 Bd Jean Moulin, 13005 Marseille, France

INTRODUCTION

MATERIALS AND METHODS

RESULTS

The aims of this study were multiple. First, it was to generated iPS derived - cortical and dopaminergic neurons, second to establish robust and relevant models of Alzheimer and Parkinson disease based on human neurons and finally to validate these models by using several reference compounds. We showed (a) the efficient generation of human cortical and dopaminergic neurons; (b) the responsiveness of the generated human neurons to different well known toxins as rodent neurons (c) the accurateness of our cellular model by using several neurotrophic factors able to rescue induced cell death. In conclusion, these new in vitro cellular models, based on human neurons appeared to be predictive, reproducible and relevant that could be used for the evaluation of new therapeutic compounds in high content screening.

Neurodegenerative diseases are a major health problem and the number of patients is predicted to quadruple by 2050. Animal models and their corresponding cellular models have given important insight in the understanding of disease mechanisms and have greatly facilitated the assessment of new therapeutic strategies. However, these models also display major limitations such as the inability to reproducibly mimic human pathology and the often unpredictable effect of test compounds on humans. The use of human induced Pluripotent Stem cells (iPS) - derived neurons is an attractive approach to get closer to human physiological conditions and to propose relevant in vitro cellular models for drug discovery.

For these reasons, we attempt to produce two kinds of neurons impaired in two major neurodegenerative diseases. Firstly, we differentiated human iPS cells into cortical neurons in order to develop Alzheimer’s disease cellular models. By employing phenotypic and molecular markers, we characterized and tested their responsiveness against exitotoxic stress and β-amyloid insults. Secondly, we produced dopaminergic neurons in order to develop Parkinson’s disease cellular models. We confirmed their molecular identity by immunolabeling and submitted them to mimicking models of Parkinson’s disease particularly an impairment of mitochondria by a MPP+ intoxication and an α-synuclein injury.

Reprogramming of human fibroblasts and production of hiPSCs

REFERENCES

Cortical neurons Differentiation, Characterization and Injury

A

Badja C., Malleva G., El-yazidi C., Barruet E., Lasserre M., Tropel P., Binetruy B., Bregestovski P., Magdinier F.; Efficient and Cost-Effective Generation of Mature Neurons From Human Induced Pluripotent Stem Cells. Stem Cells Trans Med. 2014 ;3:1–6 Callizot N, Combes M, Steinschneider R, Poindron P. Operational dissection of β-amyloid cytopathic effects on cultured neurons.J Neurosci Res. 2013, May;91(5):706-16. Hargus G, Cooper O, Deleidi M, Levy A, Lee K, Marlow E, Yow A, Soldner F, Hockemeyer D, Hallett PJ, Osborn T, Jaenisch R, Isacson O.; Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats. Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15921-6.

For differentiation, mTeSR1 medium was replaced with Neurobasal®-A Medium supplemented with 1X N2 Supplement; 1X B27 Supplement; 1X ITS-A; Stable Glutamine; 20 ng/ml bFGF and 20 ng/ml EGF and cells were maintained for 15 days on BD Matrigel-coated. Cells are then transferred onto Fibronectin-coated plates where neuronal precursors grow and become highly confluent in about 15 days. At this step, cells can be expanded for several passages and frozen.

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Imunolabeling and analyses

Cells were fixed by a solution of 4% paraformaldehyde, permeabilized and non-specific sites were blocked with phosphate buffered saline containing 0.1% of saponin and 4% goat serum, 1% BSA. Dopaminergic neurons were stained against-Tyrosine Hydroxylase (TH) /and β-tubulin whereas cortical neurons against Microtubule Associated Protein (MAP2) and NMDA-R receptor. These antibodies were revealed with a combination of Alexa Fluor 488 / Alexa Fluor 568 goat anti-mouse or anti-rabbit. For each condition, 20 pictures per well were taken using InCell AnalyzerTM 2000 (GE Healthcare).

B Effect of β-amyloid

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CONCLUSION

Cortical neuron differentiation, culture and treatment

For cortical neurons differentiation, NSC were plated on laminin coated 96 wells plate in the same medium as used for NSC but without bFGF and EGF during 11 days before treatment. -amyloid peptide was reconstituted in define culture medium at 40µM and slowly shacked at 37°C for 3 days in dark. -amyloid preparation was incubated 24H on cells at the desired concentrations. Glutamate was incubated for 20min, washed out and cells were fixed 24H later. Brain-derived neurotrophic factor (BDNF) and Oestradiol were incubated in the same time than toxins.

Dopaminergic neurons Differentiation, Characterization and Injury

H: Images showing iPS - derived dopaminergic neurons in absence (upper panel) of presence (lower panel) of MPP+ 50µM (x20, scale bar: 100 µm) I: MPP+ is able to alter dopaminergic neuron survival in a significant and dose dependent manner. J: Protective effect of BDNF on dopaminergic neuron survival after a MPP+ injury (20µM or 50µM) depends of its concentration. K: Images showing iPS derived dopaminergic neurons in absence (upper panel) of presence (lower panel) of 500nM α-synuclein (x20, scale bar: 100 µm). a-syn toxicity was evaluated after 3 days of agitation. L: α-syn is able to alter dopaminergic neuron survival in a significant and dose dependent manner. M: Protective effect of BDNF on dopaminergic neuron survival after a α-syn injury depends of its concentration. Data was expressed in percentage of control (mean sem. * p<0.05; ** p<0.01; *** p<0.001; MPP+ or α-synuclein vs control ; one way Anova followed by Dunnett’s test).

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Effect of MPP+

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K L Effect of Glutamate

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β-tubuline

Dopaminergic neuron differentiation, culture and treatment

For dopaminergic neurons differentiation, NSC were plated on laminin coated 96 wells plate in the same medium as used for NSC but without FGF and EGF and in presence of 100ng/mL of FGF8/SHH during 8 days.

Neural stem cells (NSC) induction and culture

Human iPSCs were generated after infection of primary skin fibroblasts with a lentivirus encoding KLF4, OCT4, SOX2 and c-MYC. HiPS colonies were picked about 5 weeks after infection based on ES cell-like morphology. The picked colonies were grown and expanded on mTeSR1 medium in BD Matrigel™ coated dishes. The clone used here was fully characterized using classical procedures.

α-syn peptide was reconstituted in define culture medium at 4µM and slowly shacked at 37°C for 3 days in dark. α-syn was incubated 4 days on cells. MPP+ was incubated at the desired concentration for 48h. BDNF was incubated in the same time than toxins.

Protective Effect of BDNF on iPS - derived

dopaminergic neuron survival against MPP+

NMDA-R

A: Images showing iPS - derived cortical neurons in absence (upper panel) of presence (lower panel) of β-amyloid 20µM (x20, scale bar: 100 µm) B-C: β-amyloid is able to alter cortical neuron survival and neurite length in a significant and dose dependent manner. D: Protective effect of Oestradiol on cortical neuron survival after a β-amyloid injury depends of its concentration. E: Images showing iPS - derived cortical neurons in absence (upper panel) of presence (lower panel) of Glutamate (x20, scale bar: 100 µm) F: Glutamate is able to alter cortical neuron survival in a significant and dose dependent manner. G: Protective effect of BDNF on cortical neuron survival after a glutamate injury (60µM or 100µM, 24H) depends of its concentration. Data was expressed in percentage of control (mean sem. * p<0.05; ** p<0.01; *** p<0.001; β-amyloid or glutamate vs control ; one way Anova followed by Dunnett’s test).

Protective Effect of BDNF on iPS - derived cortical

neuron survival against Glutamate injury

Protective Effect of BDNF on iPS - derived dopaminergic

neuron survival against α-synuclein (500nM or 250nM, 4d)

TH DAPI MERGE -tubuline

MAP-2 DAPI MERGE D Effect of β-amyloid

on iPS - dervied cortical neuron neurite length

Protective Effect of Oestradiol on iPS - derived cortical

neuron survival against β-amyloid injury

Thanks to Nathalie Py for her participation

UMR - GMGF