Astrocyte-assisted Neuronal Differentiation of iPS Cells Derived from Skin Biopsies of Parkinson´s Disease Patients with Genetic Alterations

FALOMIR LOCKHART LJ; BOTELHO MG; OLIVEIRA LMA; MAK S; LIN KH; TASCHENBERGER H; ARNDT-JOVIN DJ; SCHUELE B; JOVIN TM

Göttingen

Congreso; German Neuroscience Society Meeting 2013; 2013

German Neuroscience Society

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. Its pathologic hallmark is the loss of dopaminergic neurons and the appearance of intracellular amyloid aggregates known as Lewy Bodies and Lewy Neurites. Although the majority of PD cases are sporadic, some familial mutations are known and they may hold the key to unraveling the initial molecular triggers leading to neurodegeneration. Difficulties with the differentiation and culture of dopaminergic neurons have been mayor impediments to study the biochemistry and biology of PD. The ability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) provides an invaluable disease-relevant tool, especially since animal models do not reproduce essential features of the human disease. We are investigating iPSCs derived from patients with the most common genetic alteration associated with PD, the G2019S mutation of LRRK2, and the very rare triplication mutant of the gene for -synuclein (aSyn), leading to protein overproduction and early onset disease. We are differentiating iPSCs derived from fibroblasts of PD patients and healthy age-matched controls to dopaminergic neurons so as to identify the specific metabolic pathways and cellular processes affected, particularly in early stages. After a first step of partial differentiation into Neuronal Stem Cells (NSCs), iPSCs are further differentiated into functional neurons through a two-step protocol employing defined media that contain recombinant factors and B27 neuronal supplement (Zhang XQ & Zhang SC, 2010 Methods Mol Biol 584: 355-366). Recently, the use of small molecules has been shown to be also beneficial in this process (Mak et al., 2012 Stem Cells Int: Article ID 140427), and supplements like cAMP appear to be essential in the case of LHUMES cells for obtaining a major fraction of neurons with a dopaminergic phenotype (Scholz D et al., 2011 J Neurochem; 119:957-71). In addition, the hypothesis of an immune component in the etiology and progression of PD implies an involvement of accessory glial cells that establish a molecular crosstalk with the affected neurons and mediate propagation of cellular pathology. In this context, co cultures of rat or human astrocytes with the differentiating iPSC lines are of interest because they better approximate the cellular complexity of the central nervous system and reduce the requirements for exogenous growth and differentiation factors. We have used specific antibodies against aSyn, TH, MAP2, III-Tubulin and other markers to compare the differentiation of control and patient derived NSCs lines in the presence and absence of astrocytes. Cellular functionality was assessed by electrophysiology, mitochondrial respiration profile determination (Seahorse XF24) and microscopy utilizing fluorescent probes specific for the subcellular fractions. Preliminary results suggest that astrocyte-conditioned media are beneficial for neuronal differentiation of NSCs. However, supplementation with specific factors or drugs may be required in order to achieve a high yield of dopaminergic neurons, particularly in the case of the lines with PD associated genetic alterations that exhibit difficulties in differentiation.