References of "Jarazo, Javier 50002037"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailAutomated microfluidic cell culture of stem cell derived dopaminergic neurons
Kane, Khalid; Lucumi Moreno, Edinson; Hachi, Siham et al

in Scientific Reports (2019)

Detailed reference viewed: 58 (2 UL)
Full Text
Peer Reviewed
See detail3D Cultures of Parkinson's Disease‐Specific Dopaminergic Neurons for High Content Phenotyping and Drug Testing
Bolognin, Silvia UL; Fossépré, Marie; Qing, Xiaobing et al

in Advanced Science (2018)

Parkinson's disease (PD)‐specific neurons, grown in standard 2D cultures, typically only display weak endophenotypes. The cultivation of PD patient‐specific neurons, derived from induced pluripotent stem ... [more ▼]

Parkinson's disease (PD)‐specific neurons, grown in standard 2D cultures, typically only display weak endophenotypes. The cultivation of PD patient‐specific neurons, derived from induced pluripotent stem cells carrying the LRRK2‐G2019S mutation, is optimized in 3D microfluidics. The automated image analysis algorithms are implemented to enable pharmacophenomics in disease‐relevant conditions. In contrast to 2D cultures, this 3D approach reveals robust endophenotypes. High‐content imaging data show decreased dopaminergic differentiation and branching complexity, altered mitochondrial morphology, and increased cell death in LRRK2‐G2019S neurons compared to isogenic lines without using stressor agents. Treatment with the LRRK2 inhibitor 2 (Inh2) rescues LRRK2‐G2019S‐dependent dopaminergic phenotypes. Strikingly, a holistic analysis of all studied features shows that the genetic background of the PD patients, and not the LRRK2‐G2019S mutation, constitutes the strongest contribution to the phenotypes. These data support the use of advanced in vitro models for future patient stratification and personalized drug development. [less ▲]

Detailed reference viewed: 115 (6 UL)
Full Text
See detailTHE PARKINSON’S DISEASE ASSOCIATED PINK1-PARKIN PATHWAY IN PATHOLOGY AND DEVELOPMENT
Jarazo, Javier UL

Doctoral thesis (2018)

Parkinson’s disease (PD) has an aetiology not completely understood. One of the hypothesis in the field is that many neurodegenerative diseases are influenced by developmental disorders. The underlying ... [more ▼]

Parkinson’s disease (PD) has an aetiology not completely understood. One of the hypothesis in the field is that many neurodegenerative diseases are influenced by developmental disorders. The underlying concept is that already during brain development some processes are deregulated producing a higher degree of susceptibility for neurodegeneration during aging. Two hereditary early onset forms of PD are caused by recessive mutations in PTEN-induced putative kinase 1 (PINK1) and Parkin genes that regulate mitochondrial function and morphology, quarantining damaged mitochondria before their degradation as well as triggering the process of mitophagy. Our hypothesis is that alterations of the Pink1-Parkin pathway have an impact in mitochondrial physiology tempering the differentiation ability of neuroepithelial stem cells into dopaminergic neurons. For evaluating this hypothesis we reprogramed patients’ fibroblasts carrying PINK1 mutations, as well as from healthy individuals, to human induced pluripotent stem cells. We developed a streamlined technique of gene editing (FACE) by using the CRISPR/Cas9 system combined with a composite of fluorescent proteins in the donor template for biallelic gene targeting. Isogenic controls were generated using this technique that allowed us to analyze the contribution of corrected patients’ mutations in the cellular defects observed. Human iPSCs were differentiated into a neuroepithelial stem cell state (NESC) from where the cells were further differentiated into neurons. We established different algorithms for pattern recognition and applied them for image analysis of different features such as mitochondrial morphology, proliferation capacity, apoptosis and differentiation. Patient’s derived cells presented an impaired differentiation efficiency into dopaminergic neurons as well as an imbalanced cell renewal that can be linked to the mitochondrial differences. Using 3D cultures, such as microfluidics and organoids, we were able to recapitulate this differentiation impairment in a system that mimics better the context of an in vivo environment. We evaluated the energetic capabilities of the NESCs and the firing activity of differentiated neurons, which also showed a dysregulation in patient cells. We introduced a new system for large-scale analysis of the autophagy and mitophagy pathways by the combination of stably integrated Rosella constructs in different patients’ lines and an image analysis script for classification of the different subcellular structures involved in these pathways activities. This revealed that the basal activity as well as the response against stressors of these pathways are altered in cells derived from patients having different mutations causative of PD. We performed a screen of repurposed drugs as well as of novel compounds to evaluate their impact in this altered developmental transition identifying a potential candidate to be further analysed in an in vivo context. [less ▲]

Detailed reference viewed: 114 (23 UL)