RESUMO
Parkinson's disease (PD) has been attributed to a combination of genetic and nongenetic factors. We studied a set of monozygotic twins harboring the heterozygous glucocerebrosidase mutation (GBA N370S) but clinically discordant for PD. We applied induced pluripotent stem cell (iPSC) technology for PD disease modeling using the twins' fibroblasts to evaluate and dissect the genetic and nongenetic contributions. Utilizing fluorescence-activated cell sorting, we obtained a homogenous population of "footprint-free" iPSC-derived midbrain dopaminergic (mDA) neurons. The mDA neurons from both twins had â¼50% GBA enzymatic activity, â¼3-fold elevated α-synuclein protein levels, and a reduced capacity to synthesize and release dopamine. Interestingly, the affected twin's neurons showed an even lower dopamine level, increased monoamine oxidase B (MAO-B) expression, and impaired intrinsic network activity. Overexpression of wild-type GBA and treatment with MAO-B inhibitors normalized α-synuclein and dopamine levels, suggesting a combination therapy for the affected twin.
Assuntos
Neurônios Dopaminérgicos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Doença de Parkinson/patologia , Gêmeos Monozigóticos , Biomarcadores/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/enzimologia , Citometria de Fluxo , Glucosilceramidase/genética , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Masculino , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Mutação/genética , Doença de Parkinson/enzimologia , Fenótipo , Análise de Sequência de RNA , alfa-Sinucleína/metabolismoRESUMO
Three-dimensional aggregation cultures allow for complex development of differentiated human induced pluripotent stem cells. However, this approach is not easily amenable to live-cell imaging and electrophysiological applications due to the thickness and the geometry of the tissue. Here, we present an improvement on the traditional aggregation method by combining the use of cell culture inserts with serum-free embryoid bodies (SFEBs). The use of this technique allows the structures to maintain their three-dimensional structure while thinning substantially. We demonstrate that this technique can be used for electrophysiological recodings as well as live-cell calcium imaging combined with electrical stimulation, akin to organotypic slice preparations. This provides an important experimental tool that can be used to bridge 3-D structures with traditional monolayer approaches used in stem cell applications.