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

再生医学是一系列新的新兴疗法的总称，旨在治疗不可治愈的退行性疾病，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类似的活细胞，以及(Ii)将活细胞移植到解剖位置以取代患者的丢失细胞。在帕金森氏症的病例中，丢失的细胞是专门的神经，在大脑的纹状体部分释放多巴胺。第一次CRT治疗帕金森氏症的试验发生在20世纪80年代末，他们试图用来自捐赠的胎儿组织的等同但不成熟的这些细胞版本来取代丢失的多巴胺产生神经。在一些患者中，移植的细胞(I)在纹状体存活和成熟，(II)释放多巴胺，(III)逆转临床运动症状。早期的研究证明CRT治疗帕金森氏症是可能的，但也发现了几个问题。早期CRT试验的一个主要问题是，由于依赖于人类胎儿组织，适合移植的活细胞的质量和数量。当证明人类诱导多能干细胞(IPSCs)可以在实验室中转化为未成熟的产生多巴胺的神经时，这个问题的解决方案就被确定了，这些神经的特征与最初CRT试验中使用的胎儿组织非常相似。从IPSCs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，基于IPSC的CRT治疗帕金森氏症的临床试验于2018年在日本开始。将IPSCs转化为产生多巴胺的神经的过程被称为“分化”。在为期两周的分化过程结束后，活细胞被交给神经外科医生移植到帕金森患者的纹状体中。移植细胞的质量对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IPSCs生产活的专门细胞非常困难，也很难控制。将IPSCs区分为专门化细胞的过程极其复杂，可能会受到许多变量的不利影响。因此，实时监测IPSCs向特殊细胞类型的转化而不干扰细胞(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，以非侵入性、实时监测从IPSCs分化为多巴胺的细胞。我们将通过识别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。由于细胞总是生长在液体溶液(介质)中，我们可以非侵入性地对这种介质进行采样，以测量任何感兴趣的分子的丰度。细胞分泌的分子的特征将反映细胞的身份。由于IPSCs的细胞身份在分化过程中是动态变化的，因此分泌分子的签名也将实时变化。我们将使用一种叫做质谱学的方法来识别分泌分子的“好”和“坏”信号，为帕金森氏症CRT的细胞生产。这些知识将被用来利用Luminex公司的技术构建一个“试剂盒”，它将能够从一小部分介质样本中同时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)协议优化、(Ii)质量控制、(Iii)故障排除以及(Iv)通过和不通过的决定将是极其宝贵的。所有这些要点都对基础研究，特别是临床应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.