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

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.

An emerging regenerative medicine therapy for Parkinson's is cell replacement therapy (CRT) with midbrain dopaminergic (mDA) cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). Clinical trials have started in Japan with iPSC-derived mDA cells in 2018. The process of hESC/hiPSC differentiation is complex and heterogeneous. Furthermore, it is well described that different iPSC lines can respond differently to the same differentiation protocol.

Here we propose to establish tools for non-invasive monitoring for mDA differentiation by interrogation the secretome of cells as they are converting from pluripotent cells to committed mDA progenitor cells. We have identified a number of secreted molecules, including TFF3, CORIN, PDGFC, SERPIN F1, and NRP1, that increase in conditioned medium during the early stages of mDA differentiation. In this project we will determine how robust these, and other markers, are at predicting mDA differentiation efficiency across two laboratories (Edinburgh and Kyoto) and for multiple hESC and hiPSC cell lines. We will further use unbiased proteomic methods to discover novel biomarkers for mDA cells, and importantly markers of non-mDA cells (Kyoto). Single-cell RNAseq will be use to define the heterogeneity of cells present in a transplantable mDA population with a known biomarker profile (Edinburgh), and the transplantation of these cell populations with be assessed a rat lesion model of Parkinson's (Cardiff). One of the deliverables of this project is a multiplexed assay system to simultaneously measure multiple positive and negative biomarkers of mDA differentiation that will be of high value to academic and commercial efforts toward cell replacement therapy for Parkinson's.

This research is specifically aimed at improving the production of a cell replacement therapy (CRT) for Parkinson's. Therefore, the major impact will be for people living with this incurable neurodegenerative disorder. In 2020 the global incidence of Parkinson's is over 8 million people, and this number is expected increase with the aging population. If CRT is successful, it has the potential to reverse motor symptoms, reduce reliance on dopamine-based medications, and significantly improve quality of life. The research on non-invasive real-time monitoring of the manufacture of cells for CRT could significantly lower the cost of production, thereby making the therapy more widely available. The tools and knowledge generated from this work could also significantly accelerate the adoption of dopaminergic cell production by industry, and reduce the failure rate of the production process. Taken together this research could accelerate the delivery of a life-changing treatment to a larger number of Parkinson's patients.

Other impacts include significant economic savings to cell manufacturing companies, due to decreased production costs.
Significant savings to healthcare services, such as the NHS, is another potential impact due to decrease medication costs, and other types of care, including palliative care.
Finally, successful CRT for younger Parkinson's patients could significantly reduce or eliminate adverse effects on their employment.