Biomedical research aims to understand the pathological and pathophysiological mechanisms that cause disease. Neurodegenerative diseases, such as Parkinson’s disease (PD), are major contributors to the burden of disease across the globe. PD is an age-related, progressive neurodegenerative disease. The pathological hallmarks are a selective loss of dopaminergic neurons from the substantia nigra in conjunction with the presence of protein aggregates involving α-synuclein in the residual neurons. Cystatin C expression has been shown to become upregulated in brain injuries, neurological disorders and in animal models of neurodegenerative states, which suggests it could play a part in neurodegenerative disorders. The main function of this primarily secreted protein is the inhibition of cysteine proteases. Various tools are available to researchers to study diseases, ranging from animal models, human biospecimens and human in vitro models. Regardless of the model selected, reproducibility is crucial to ensure meaningful research. To maximise the quality of biomedical research, biobanks work to ensure the biospecimens they issue are compromised as little as possible as a consequence of the unavoidable preanalytical variables occurring during their collection, processing and storage. The scientific discipline that studies preanalytical variables and how they affect biospecimens is called biospecimen science. In this thesis, biospecimen science was applied to patient specific stem cells and cystatin C in the scope of PD research. A standardized research-grade human induced pluripotent stem cell (iPSC) workflow was established for use as an in vitro PD model, which encompasses both iPSC generation and cryostorage. Controlled-rate freezing of iPSCs using three different dimethyl sulfoxide-based cryosolutions containing ice recrystallization inhibitors was evaluated and optimized to achieve efficient iPSC cryopreservation. A double, indirect sandwich ELISA was established to quantify the concentration and the degradative state of secreted cystatin C. The ELISA was validated using well-defined and standardized cerebrospinal fluid (CSF) biospecimens, then applied as a tool to retrospectively identify CSF biospecimens that had been stored in suboptimal conditions. Secreted cystatin C was quantified and compared in blood derivatives (plasma and serum) and in the culture media of derived models (iPSCs, neuroepithelial stem cells and midbrain organoids) from three idiopathic PD patients and age-matched healthy controls. The standardized in vitro PD models, novel quality control and cryopreservation methods not only demonstrate the critical importance of preanalytical standardization but open the way to future biomedical research.
|Award date||18 Feb 2022|
|Publication status||Published - 18 Feb 2022|