AUTOMATIC CELL FATE ENGINEERING USING MICROFLUIDICS DEVICES

干细胞是一种多能细胞，既可以无限增殖，产生与它们相同的细胞，又可以专门分化为更成熟的细胞类型。在成人中，干细胞在受损的情况下具有修复功能；成人干细胞目前用于医学治疗。成人干细胞在医学上应用的主要限制是它们的可获得性低，而且难以在培养中扩大它们。诺贝尔奖得主山中伸弥对重新编程的惊人发现被认为克服了这些问题：分化的(即体细胞)细胞可以被编程回到干细胞状态，获得所谓的诱导多能干细胞(IPSCs)。随后，IPSCs可以转化为任何类型的细胞，用于再生和个性化医疗目的。在日本，第一个使用IPSC来源的细胞在人类身上进行的临床试验正在治疗老年性黄斑变性。然而，IPSC治疗仍然面临着重大挑战：很难对体细胞进行重新编程并保持IPSC处于多能性状态；此外，IPSC的分化通常效率低下。在这项研究中，我们旨在应用最先进的合成生物学和控制工程工具来自动化和优化IPSC来源细胞的制造。我们将使用小鼠细胞系证明，如果在提供触发多能性或分化的输入的同时，持续观察细胞，并因此对输入进行调整以获得目标表型，那么上述三个挑战中的每一个都可以得到解决。这一闭环策略将通过微流体和显微镜来实现，这使得能够在实时活细胞中进行监测，将相关的细胞输出与目标细胞进行比较，并应用控制算法，允许对细胞进行作用，以将误差降至最低。在证明通过“闭合回路”可以自动控制干细胞命运的同时，我们将提供一个平台，允许在实验结束时从微流体设备中高效和可重复性地检索所需的细胞类型。

Stem cells are pluripotent cells that can both proliferate indefinitely producing cells identical to them, and specialise into more mature cells types. In adults, stem cells have a repair function in case of damage; adult stem cells are currently used in medical therapy. The major limitation of adult stem cells' medical applications is their low availability, and the difficulty to expand them in culture.Such issues were thought to be overcome thanks to the astonishing discovery of reprogramming by the Nobel Prize-winning Shinya Yamanaka: differentiated (i.e. somatic) cells can be programmed back to a stem-like state, obtaining the so-called induced Pluripotent Stem Cells (iPSCs). iPSCs can be subsequently converted into any cell type, to be used for regenerative and personalised medicine purposes. In Japan, the first clinical trial using iPSC-derived cells in humans is on going to cure age-related macular degeneration.iPSC therapy still faces, however, major challenges: it is difficult to reprogram somatic cells and maintain iPSCs in the pluripotent state; also, iPSC differentiation is often inefficient.In this research, we aim at applying state-of-the-art Synthetic Biology and Control Engineering tools to automatize and optimise the manufacturing of iPSC-derived cells. We will prove, using mouse cell lines, that each of the 3 mentioned challenges can be addressed if, while providing inputs that trigger pluripotency or differentiation, cells are continuously observed and inputs are consequently "adjusted" to obtain the target phenotype. This closed-loop strategy will be implemented by means of microfluidics and microscopy, that allow monitoring in real-time living cells, comparing relevant cellular outputs to the target one and applying control algorithms that allow acting on the cells to minimise the error. While proving that, by "closing the loop", it is possible to automatically control stem cell fate, we will provide a platform that allows, at the end of the experiment, to retrieve from the microfluidics device the desired cell type with high efficiency and reproducibility.