Maintaining retinal ganglion cells within human retinal organoids by implementing a vascular system.

通过实施血管系统来维持人视网膜类器官内的视网膜神经节细胞。

• 批准号: 531985111
• 负责人: Professor Dr. Volker Busskamp, Ph.D.
• 金额: --
• 依托单位: Universitäts-Augenklinik Bonn
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/531985111?language=en
• 关键词: Maintaining; retinal; ganglion; cells; within

Stem cell technology has boosted the life sciences over the last decades, especially the bottom-up generation of human tissues from stem cells, so-called organoids, are now enabling new experimental approaches. We are interested in exploiting human retinal organoids for our research. However, we face severe limitations and shortcomings of this technology. As in most brain organoids, only neural cell types, glia and neurons are present. The lack of vascularization leads to the development of a necrotic core. Neighboring cells are negatively impacted and degenerate over time. Within retinal organoids, retinal ganglion cells (RGCs) reside inside the tissue and degenerate shortly after development. RGCs normally form a defined retinal layer and their axons are bundled as the optic nerve, relaying visual information to higher brain areas. However, RGC morphology and function is not recapitulated in retinal organoids. It takes about 30 weeks until the photoreceptors become light-sensitive but RGCs are already degenerated by week 16. To overcome these structural and functional limitations, we hypothesize that the implementation of a vascular system within developing retinal organoids will prevent the formation of the necrotic core and thereby inhibit RGC degeneration. The vascularization will enhance oxygen and nutrient supply to the inner part of the organoids. We take advantage of our previously described transgenic stem cell line in which a transcription factor, ETV2, can be induced leading to almost 100% endothelial differentiation in four days. To this end, we will add stem-cell derived endothelial cells to growing organoids or we will forward program stem cells into endothelial cells in situ. Our preliminary data look very promising. Retinal organoids become significantly larger and apoptotic rates around week 16 are significantly reduced. However, individual parameters of our technology require extensive and systematic modifications to obtain a robust protocol for vascularized retinal organoids. We need to perform in-depth quality control of retinal cell types, cellular morphologies and retinal laminations using imaging techniques. Retinal cell types will also be studied at the transcriptome level using single cell RNA sequencing. Importantly, we are also keen on revealing the functional improvements of RGC survival and function. We will perform micro-electrode array (MEA) recordings over time. We will also develop microchannel devices that are placed on MEAs that RGCs from retinal organoids send their axons via microchannels onto the electrode area. We will record light-evoked neuronal activity originating from the retinal organoids from these postsynaptic neurons, providing a functional simplified assembloid model for functional retinal studies using organoids. Overall, implementing a vascular system within neuronal organoids is an essential step to enlarge the applications of this fascinating system in basic and biomedical research.

在过去的几十年里，干细胞技术推动了生命科学的发展，特别是从干细胞中自下而上生成人体组织，即所谓的类器官，现在正在实现新的实验方法。我们有兴趣利用人类视网膜类器官进行研究。然而，我们面临着这项技术的严重局限性和缺点。与大多数脑类器官一样，只有神经细胞类型，神经胶质和神经元存在。缺乏血管化导致坏死核心的发展。邻近的细胞会受到负面影响，并随着时间的推移而退化。在视网膜类器官内，视网膜神经节细胞（RGC）驻留在组织内并在发育后不久退化。RGC通常形成一个确定的视网膜层，它们的轴突被捆绑成视神经，将视觉信息传递到更高的大脑区域。然而，RGC的形态和功能在视网膜类器官中并不重现。大约需要30周的时间，直到光感受器变得对光敏感，但RGC在第16周已经退化。为了克服这些结构和功能限制，我们假设在发育中的视网膜类器官内实施血管系统将防止坏死核心的形成，从而抑制RGC变性。血管化将增强对类器官内部的氧气和营养供应。我们利用我们先前描述的转基因干细胞系，其中转录因子ETV2可以被诱导，导致在四天内几乎100%的内皮分化。为此，我们将向生长的类器官中添加干细胞衍生的内皮细胞，或者我们将原位将干细胞程序转化为内皮细胞。我们的初步数据看起来很有希望。视网膜类器官变得显著更大，并且在第16周左右凋亡率显著降低。然而，我们的技术的各个参数需要广泛和系统的修改，以获得血管化视网膜类器官的稳健方案。我们需要使用成像技术对视网膜细胞类型、细胞形态和视网膜分层进行深入的质量控制。还将使用单细胞RNA测序在转录组水平上研究视网膜细胞类型。重要的是，我们也热衷于揭示RGC存活和功能的功能改善。我们将随着时间的推移进行微电极阵列（MEA）记录。我们还将开发放置在MEA上的微通道装置，来自视网膜类器官的RGC通过微通道将其轴突发送到电极区域。我们将记录来自这些突触后神经元的视网膜类器官的光诱发神经元活动，为使用类器官的功能性视网膜研究提供功能性简化的类神经元模型。总的来说，在神经元类器官内实现血管系统是扩大这个迷人系统在基础和生物医学研究中应用的重要一步。