Rapid and deterministic generation of microglia from human pluripotent stem cells - an in vitro platform for disease progression in neurodegeneration and neuroinflammation

小胶质细胞是中枢神经系统（CNS）的常驻免疫细胞，与许多神经系统疾病的发生和发展有关。它们起源于原始的卵黄囊巨噬细胞，在胚胎发育早期定植于发育中的中枢神经系统。一旦胚胎小胶质细胞群建立，它就会通过局部增殖而不是由骨髓来源的髓细胞替代而维持一生。目前正在进行大量的研究工作，以建立忠实的人类小胶质细胞体外模型，作为疾病建模和药物发现的平台。直到最近，才报道了从人类多能干细胞中衍生小胶质样细胞的第一个方案。然而，它们的持续时间长（35-80天，取决于方案），并且需要机械分离步骤来丰富某些发育中间细胞群，可能会禁止它们的广泛应用。在这里，我建议建立一种新的方案，以前所未有的速度和效率从人类多能干细胞中产生纯小胶质细胞群体。我提出的初步数据表明，这一关键步骤是可行的。人类小胶质细胞可以单培养，也可以与人类皮质神经元或3D人脑类器官进行二维共培养，以建立一个多功能的人类小胶质细胞平台。人类小胶质细胞体外模型的不同复杂性水平将相互补充，以解决各种研究问题，从单一培养的还原论研究到类器官环境中复杂细胞相互作用的研究。小胶质细胞表型高度依赖于环境。在目前的应用中，我们将应用人类小胶质细胞体外模型来确定关键的细胞-细胞相互作用，这些相互作用决定了人脑类器官中人类稳态小胶质细胞的表型（作为其体内对应物的代理），而不是人工体外表型。为此，我们将结合新的CITE-seq技术进行单细胞RNA测序，同时进行蛋白质定量。这将使我们能够基于配体受体对分析生成一个全面的细胞相互作用组。最后，我们将与tau突变的皮质神经元和类器官共培养小胶质细胞，以引发和表征与疾病相关的小胶质细胞表型，并揭示与从稳态到疾病相关的小胶质细胞表型转换相关的关键细胞-细胞相互作用。

Microglia are the resident immune cells in the central nervous system (CNS) and implicated in the onset and progression of many neurological diseases. They originate from primitive yolk sac macrophages that colonise the developing CNS during early embryonic development. Once the embryonic microglial population is established, it is maintained throughout life by local proliferation, not replacement by bone-marrow-derived myeloid cells. Enormous research efforts are currently undertaken to establish faithful human microglia in vitro models as platform for disease modelling and drug discovery. Only most recently, the first protocols for the derivation of microglia-like cells from human pluripotent stem cells were reported. However, their long duration (35-80 days, depending on the protocol) and need for mechanical isolation steps to enrich certain developmental intermediate cell populations are likely to prohibit their widespread application. Here, I propose to establish a novel protocol for the generation of pure populations of microglia from human pluripotent stem cells at unprecedented speed and efficiency. I present preliminary data that this key step is feasible. Human microglia will be kept either in monoculture or placed in 2D coculture with human cortical neurons or 3D human brain organoids to establish a versatile human microglia platform. The different levels of complexity of the human microglia in vitro model will complement each other for diverse research questions ranging from reductionist studies in monoculture to studies of complex cellular interactions in an organoid setting. Microglia phenotypes are highly environment dependent. In the present application, we will apply the human microglia in vitro model to determine key cell-cell interactions that determine the human homeostatic microglia phenotype in brain organoids (as proxy of their in vivo counterparts) in contrast to the artificial in vitro phenotype. To this aim we will perform single cell RNA sequencing combined with the novel CITE-seq technology for simultaneous protein quantification. This will allow us to generate a comprehensive cellular interactome based on ligand receptor pair analysis. Finally, we will coculture microglia with tau-mutant cortical neurons and organoids to elicit and characterise a disease-associated microglia phenotype and unveil key cell-cell interactions that are associated with the phenotypic switch from homeostatic to disease-associated microglia.