A revolutionary tool to find new therapeutic avenues
The innovative project based on iPS cells proposed by Delphine Bohl was selected in 2010 Project. Her very interesting results have been recently published, she comments them for the Foundation
Human induced pluripotent stem cells, called iPSc, are since their discovery in 2007 a revolutionary tool in order to study human diseases affecting the brain and the spinal cord. Indeed from a skin biopsy it is now possible to reprogram cultured fibroblasts from the biopsy and generate iPSc. These iPSc being pluripotent, they can be differentiated into any body cell type, including neurons impossible to obtain from humans.
Delphine Bohl’s team, from the Inserm Unit U1115 at the Pasteur Institute (Paris, France), has used this technology to generate iPSc from healthy subjects and patients with various ALS forms. These iPSc were then differentiated into motor neurons which are the primary target cells dying in patients. However, in order to study mechanisms leading to the selective death of ALS motor neurons, a problem arose. Indeed irrespective of the iPSc differentiation protocol, human motoneuron cultures are not pure, and it is known that other cells surrounding the motor neuron, such as astrocytes, are involved in their destiny during the course of the disease and therefore their presence in cultures can impact analyzes. Thus, to study the intrinsic mechanisms leading to the death of motor neurons in cultures containing exclusively these types of neurons, Delphine Bohl’s team worked with the team of Dr. G. Haase, of the Timone Institute of Neuroscience in Marseille (France), to develop a unique technique for sorting motoneurons by flow cytometry.
First, authors showed that the use of a viral vector – commonly used by many teams – to mark motor neurons and sort them, was not entirely specific and led to the co-purification of motoneurons and interneurons. They then searched for and identified a second marker, the low affinity neurotrophin receptor, p75NTR, and applying the double selection in iPSc-derived cultures, they were able to obtain cultures containing 100% motor neuron cells. They finely characterized these motoneurons and showed that they were functional, that is to say electrically excitable and capable of forming neuromuscular junctions.
Secondly, they cultured pure iPS-derived motoneurons with supernatants of astrocytes carrying a mutation in the gene encoding the superoxide dismutase 1 enzyme, responsible for 10% of familial forms of ALS. They then demonstrated the toxicity of astrocytes carrying the mutation towards pure human motoneurons, thus validating their new purification tool.
Future aims of Delphine Bohl’s team is now to differentiate iPSc from healthy subjects and ALS patients into motoneurons, to purify these cells and perform transcriptomic analyzes to identify between different forms of familial and sporadic ALS common or divergent pathways that would be affected, in the perspective to identify new therapeutic avenues.