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Direct reprogramming for regenerative medicine and disease modelling
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Direct reprogramming of somatic cells into different lineages allows to generate specific cell types that cannot otherwise be easily harnessed, such as human neurons from the central nervous system. This raises the promising possibility of reprogramming-based therapies that may replenish neuronal populations using glial cells within an injured area in vivo. However, the efficiency of conversion and the functionality of the induced neurons (iNs) depend to a large extend on the cellular context where the conversion takes place (i.e. in culture, in the injured brain in vivo, within different areas of the CNS ...). Our team seeks to understand the molecular and cellular mechanisms underpinning fate conversion in order to apply new knowledge on regenerative medicine approaches of cell replacement, as well as to model neurodegenerative diseases in vitro.
Ongoing projects:
Hypothermia to enhance reprogramming of glial cells into induced neurons.
In our previous work we determined that successful neuronal reprogramming requires a metabolic switch from glycolysis (Glyco) to oxidative phosphorylation (OxPhos) (see Gascón, 2016, 2017). But the acquisition of a new oxidative metabolism during neuronal conversion rises reactive oxygen species (ROS) production that lead to cell death by ferroptosis (Gascón et al., 2016). This effect represents a major barrier for the reprogramming procedure, efficiency-wise.
The hypothesis in which this project builds up is that inducing neuronal conversion at low temperature would slow down the speed of transcriptional cascades linked to fate re-specification pathways and provide the cells time enough to adapt to the new oxidative metabolome, without up-regulating ROS production. We have indeed observed that neuronal reprogramming efficiency is potently enhanced by hypothermia in vitro, irrespectively of the transcription factor used for the conversion procedure. Our ultimate goal is to translate these findings to approaches of regenerative medicine in vivo.
Modelling neurodegenerative diseases through direct reprogramming.
An important limitation to study the pathology of the CNS is that living human neural cells are not easy to obtain. To overcome this problem, techniques based on iPSCs reprogramming have recently emerged. However, the acquisition of pluripotency implies resetting of cellular age, which is particularly adverse for exploring molecular and cellular correlations between aging and neurodegeneration. A promising route to circumvent this limitation is the access to neural cells through direct reprogramming strategies that do not imply cell rejuvenation.
Thus, we use direct reprogramming approaches to model human neurodegenerative diseases, such as amyotrophic lateral sclerosis, a fatal condition typically characterized by the selective degeneration of motor neurons, but also affecting the glial and muscular components. In this case, for neuronal reprogramming we use a method based on the retroviral-mediated expression of Neurog2, Isl1 and Bcl-2, which yields a pure population of induced motor neurons (iMNs, β-III-tubulin+, Hb9+ and Isl2+) from healthy control and patient-derived fibroblasts. In addition, reprogramming of fibroblasts into induced myoblasts (iMs) is accomplished by expression of the myogenic factor MyoD. When iMs and iMNs are co-cultured together, they establish bungarotoxin-detectable neuromuscular junctions.
These approaches represent ideal platforms to model neurodegeneration in vitro and our team uses them extensively to study human pathologies of the CNS.
See more information at www.gasconlab.com
Most relevant methodology.
1) Development of neural primary cultures from mouse.
2) Culture and reprogramming of mouse and human cells in vitro.
3) Neural reprogramming techniques within the adult mouse brain in vivo.
4) Oxidative stress and cellular redox analysis.
5) Production and transduction of/with retroviral vectors in vitro and in vivo.
6) Retrograde synaptic tracing with rabies virus.
7) Molecular biology and cell biology techniques, such as plasmid cloning, immunocytochemistry, immunohistochemistry, flow cytometry, and transcriptomic profile analysis.
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