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

再生医学是一个涵盖广泛的新型和新兴疗法的术语，旨在解决无法治愈的退行性疾病，包括帕金森氏症等神经系统疾病。再生医学的一个分支学科是细胞替代疗法或CRT。CRT的基本概念是（i）产生或获得与疾病中丢失的细胞相似的活细胞，（ii）将活细胞移植到解剖位置以替换患者中丢失的细胞。在帕金森氏症的情况下，丢失的细胞是大脑中称为纹状体的部分中释放多巴胺的专门神经。帕金森氏症的第一次CRT试验发生在20世纪80年代末，他们试图用来自捐赠的胎儿组织的这些细胞的等效但不成熟的版本来取代失去的多巴胺产生神经。在一些患者中，移植的细胞（i）在纹状体中存活并成熟，（ii）释放多巴胺，（iii）逆转临床运动症状。早期的研究证明了CRT治疗帕金森病是可能的，但也发现了一些问题。早期CRT试验的一个主要问题是，由于依赖于人类胎儿组织，移植的合适活细胞的质量和数量。当证明人类诱导多能干细胞（iPSC）可以在实验室中转化为未成熟的多巴胺产生神经时，确定了这个问题的解决方案，该神经具有与用于初始CRT试验的胎儿组织非常相似的特征。从iPSC产生的细胞产生多巴胺，并在移植到动物模型中时功能良好，2018年在日本开始了基于iPSC的CRT治疗帕金森病的临床试验。在分化过程结束时，需要两周多的时间，活细胞被交给神经外科医生移植到帕金森病患者的纹状体中。移植细胞的质量对于治疗的成功至关重要。与制造用于移植的无生命物体-电极，板-相比，从iPSC生产活的特化细胞非常难以监测和控制。将iPSC分化为特化细胞的过程非常复杂，并且可能受到许多变量的不利影响。因此，实时监测iPSC向特化细胞类型的转化并且不干扰细胞（非侵入性）将是非常有价值的。这项合作提案旨在提供工具和知识，以进行非侵入性，实时监测从iPSC分化多巴胺产生细胞。我们将通过鉴定和测量细胞在两周多的分化过程中分泌的独特分子来实现这一点。由于细胞总是在液体溶液（培养基）中生长，因此我们可以非侵入性地对该培养基进行采样，以测量任何感兴趣分子的丰度。由细胞分泌的分子的特征将反映细胞身份。由于iPSC的细胞身份在分化过程中动态变化，因此分泌分子的特征也会实时变化。我们将使用一种称为质谱法的方法来识别分泌分子的“好”和“坏”特征，用于生产帕金森氏CRT细胞。这些知识将被用于构建一个“试剂盒”，该试剂盒将能够同时从培养基的小样本中测量许多信息分子的丰度。实时非侵入性监测iPSC分化的能力对于（i）方案优化、（ii）质量控制、（iii）故障排除和（iv）进行和不进行决策将是非常有价值的。所有这些点都对基础研究，特别是临床使用的差异化具有重大的成本影响。

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.