Non-invasive monitoring of human pluripotent stem cell differentiation into midbrain dopaminergic neural cells

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Regenerative medicine is an umbrella term for a broad range of novel and emerging therapies designed to tackle incurable degenerative conditions, including neurological conditions such as Parkinson's. A sub-discipline of regenerative medicine is cell replacement therapy or CRT. The underlying concept of CRT is (i) to produce or obtain live cells similar to the cells lost in a disease, and (ii) transplant the live cells into an anatomical location to replace lost cells in patients.In the case of Parkinson's, the lost cells are specialised nerves that release dopamine in a part of the brain called the striatum. The first CRT trials for Parkinson's occurred in the late 1980s where they attempted to replace the lost dopamine-producing nerves with equivalent, but immature, versions of these cells from donated fetal tissue. In some patients the transplanted cells (i) survived and matured in the striatum, (ii) released dopamine, and (iii) reversed clinical motor symptoms. The early studies proved a CRT for Parkinson's is possible, but several problems were identified.A major issue with the early CRT trials was the quality and quantity of suitable live cells for transplantation due to the reliance on human fetal tissue. A solution to this problem was identified when it was demonstrated that human induced pluripotent stem cells (iPSCs) can be transformed into immature dopamine-producing nerves in the laboratory with very similar characteristics to the fetal tissue used for the initial CRT trials. The cells produced from iPSCs produced dopamine and functioned well when transplanted into animal models, and a clinical trial for iPSC-based CRT for Parkinson's began in Japan in 2018.The process of converting iPSCs into dopamine-producing nerves is called "differentiation". At the end of a differentiation procedure, which takes over two weeks, the live cells are given to a neurosurgeon to transplant into the striatum of Parkinson's patients. The quality of the cells transplanted is critically important for the success of the therapy. In contrast to the manufacturing of inanimate objects - electrodes, plates - for transplantation, the production of live specialised cells from iPSCs is very difficult to monitor and challenging to control. The procedure to differentiate iPSCs into specialised cells is incredibly complex and can be adversely affected by a number of variables. Therefore, it would be extremely valuable to monitor the conversion of iPSCs into specialised cell types in real-time and without disturbing the cells (non-invasive). This collaborative proposal aims to provide the tools and knowledge to conduct non-invasive, real-time monitoring of the differentiation of dopamine-producing cells from iPSCs. We will accomplish this by identifying and measuring the unique molecules that the cells secrete during the more than two weeks of differentiation. Since the cells are always grown in a liquid solution (medium), we can non-invasively sample this medium to measure the abundance of any molecules of interest. The signature of molecules secreted by a cell will reflect the cell identity. Since the cell identity of iPSCs is changing dynamically during differentiation, the signature of secreted molecules will also change in real-time. We will use a method called mass spectrometry to identify "good" and "bad" signatures of secreted molecules for the production for cells for Parkinson's CRT. This knowledge will be used to construct a 'kit' with technology from Luminex that will be able to measure the abundance of many informative molecules simultaneously from a small sample of medium. The ability to non-invasively monitor the differentiation of iPSCs in real-time will be extremely valuable for (i) protocol optimisation, (ii) quality control, (iii) trouble-shooting, and (iv) go and no-go decisions. All of these points have significant cost implications for differentiations for basic research and especially for clinical use.