Since the production of lactic acid seems to be

Since the production of lactic aa dutp seems to be the cause of the genotoxic stress, modifying the culture medium could control its production and its effects. Because hESC do not metabolize carbon sources such as fructose or galactose, which generate less lactate, an option could be to use lower concentrations of glucose in the medium. This seems to lead to lower levels of lactate in hESC cultures, without significantly decreasing cell growth (Chen et al., 2010). A second possibility is to provide a better pH buffer, as we tried to achieve by the addition of HEPES. In this sense, the lack of detailed information of the composition of the KnockOut DMEM culture medium is probably the main reason for our lack of success.
In light of this, the capacity of this culture system to preserve genomic stability, especially during long-term culture, still remains to be assessed. There is currently little information in this regard (Garitaonandia et al., 2015; Rodin et al., 2014); future studies will tell whether these environments are indeed capable of providing the pristine cell cultures necessary for cell-based therapies.

Experimental Procedures

Author Contributions

Acknowledgments
The authors acknowledge Laetitia Gonzalez for her help with the COMET assay, Pedro Couck and Ilse Weets for their assistance in measuring pH and lactic acid concentrations, and their colleagues from the hESC laboratory for the derivation and culture of the cell lines. This work was supported by the Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek [FWO] Vlaanderen) and the Methusalem grant to Karen Sermon of the Research Council of the Vrije Universiteit Brussel. Claudia Spits is a postdoctoral fellow at the FWO Vlaanderen. Lise Barbé is supported by the Leerstoel Mireille Aerens (Vrije Universiteit Brussel).

Introduction
Parkinson\’s disease (PD) is the second most common neurodegenerative disorder, characterized by the preferential degeneration of dopamine neurons in the substantia nigra pars compacta (SNpc). Heterozygous mutations in the glucocerebrosidase gene (GBA) encoding the lysosomal enzyme GCase represent the strongest common genetic risk factor for PD (Sidransky et al., 2009) and have also been associated with other related Lewy body disorders (Goker-Alpan et al., 2006); however, the underlying molecular mechanisms are still poorly understood.
The association of GBA with PD first emerged from clinical studies that demonstrated that relatives of patients with Gaucher\’s disease (GD), a lysosomal storage disease caused by homozygous GBA mutations, had an increased incidence of PD (Goker-Alpan et al., 2004). More recent studies exploring the pathogenic role of homozygous GBA mutations in GD have highlighted a role of GCase in mitochondria function, α-synuclein aggregation, and autophagic machinery (Mazzulli et al., 2011; Sardi et al., 2011). GCase pathology in the context of heterozygous GBA mutations in PD have been addressed in recent postmortem (Gegg aa dutp et al., 2012) and patient fibroblast (McNeill et al., 2014) studies, and recently in a PD patient human neuronal model (Schöndorf et al., 2014). Overall, mutations in GBA and in LRRK2, also known to play a role in regulating autophagy (Alegre-Abarrategui et al., 2009), have emphasized the role for the autophagic/lysosomal pathway as central to the pathogenesis of PD (Tofaris, 2012).
Human induced pluripotent stem cells (iPSCs) derived from patients carrying disease-associated alleles retain the genetic background likely to include disease-permissive genetic modifiers and can be differentiated into highly physiological models of specific cell types to study cellular mechanisms of disease (Kiskinis and Eggan, 2010). Within this paradigm, iPSCs can be differentiated into functional midbrain dopamine neurons to provide a powerful tool to study the genetic contribution to PD (Hartfield et al., 2012). We have previously developed a highly physiological cellular model of differentiated midbrain dopamine neurons that express key dopaminergic markers, exhibit dopamine synthesis, release, and re-uptake, and show autonomous pace-making and spontaneous synaptic activity (Hartfield et al., 2014).