Kriukov Emil, Mukwaya Anthony, Cullen Paul Francis, Baldwin George, Malechka Volha V, Refaian Nasrin, Bagaev Nikita, Labrecque Everett, Vinjamuri Sthavir, Margeta Milica A, Baranov Petr
Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, MA, USA.
Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.
bioRxiv. 2025 May 21:2025.05.16.654622. doi: 10.1101/2025.05.16.654622.
The host retinal microglia and macrophage activation remains a major challenge for the integration of donor neurons following transplantation. Previously, we and others have shown that it is possible to increase donor retinal ganglion cell (RGC) survival by inhibiting the microglia-RGC interaction with Annexin V or through reprogramming microglia with the soluble Fas ligand. However, the exact mechanisms of the microglia/macrophage activation and their heterogeneity following transplantation remain unknown. To address this question, the donor RGC were differentiated from Brn3b-Tdtomato-Thy1.2 human embryonic stem cells using a 3D protocol, followed by dissociation and RGC purification. RGC were delivered subretinally (1.5×10 viable cells/eye) into 3-6-month-old knock-in mice. Three days after transplantation retinas were dissociated into single-cell suspension and GFP-positive myeloid cells isolated using FACS. Of the sorted cells, up to 10,000 viable cells per sample were used for single-cell RNA library preparation and sequenced using the 10X Genomics Chromium platform. In addition, several retinas were fixed and stained for donor RGC (mCherry) and host microglia/macrophages (Iba1). RNA Velocity was used to reconstruct the myeloid cell population and activation trajectory from scRNAseq data. We observed continuous bi-directional transition of microglia/macrophages from a homeostatic to an activated state. We also observed that the response to the transplant falls into the classic disease-associated-microglia (DAM) activation paradigm with a decrease in expression of the homeostatic gene and an increase in expression of disease-associated genes including and . Our findings show that the host retinal myeloid cell population undergoes activation upon transplantation of stem-cell derived donor RGC, with a molecular profile of the activated cells similar to that of activated myeloid cells associated with neurodegenerative diseases of the brain and the eye. Advanced integrated transcriptomic analysis shows separate activated-to-homeostatic and homeostatic-to-activated trajectories suggesting the reversibility of this process.
宿主视网膜小胶质细胞和巨噬细胞的激活仍然是移植后供体神经元整合的一个主要挑战。此前，我们和其他人已经表明，通过用膜联蛋白V抑制小胶质细胞与视网膜神经节细胞（RGC）的相互作用或通过用可溶性Fas配体重编程小胶质细胞，可以提高供体视网膜神经节细胞的存活率。然而，移植后小胶质细胞/巨噬细胞激活的确切机制及其异质性仍然未知。为了解决这个问题，使用3D方案从Brn3b-tdTomato-Thy1.2人胚胎干细胞中分化出供体RGC，然后进行解离和RGC纯化。将RGC经视网膜下注射（1.5×10个活细胞/眼）到3至6个月大的敲入小鼠中。移植后三天，将视网膜解离成单细胞悬液，并使用荧光激活细胞分选（FACS）分离GFP阳性髓样细胞。对于分选的细胞，每个样本最多10000个活细胞用于单细胞RNA文库制备，并使用10X Genomics Chromium平台进行测序。此外，对几个视网膜进行固定，并对供体RGC（mCherry）和宿主小胶质细胞/巨噬细胞（Iba1）进行染色。使用RNA速度从单细胞RNA测序（scRNAseq）数据重建髓样细胞群体和激活轨迹。我们观察到小胶质细胞/巨噬细胞从稳态到激活状态的持续双向转变。我们还观察到，对移植的反应符合经典的疾病相关小胶质细胞（DAM）激活模式，稳态基因表达降低，包括 和 在内的疾病相关基因表达增加。我们的研究结果表明，在移植干细胞衍生的供体RGC后，宿主视网膜髓样细胞群体发生激活，激活细胞的分子特征与与脑和眼的神经退行性疾病相关的激活髓样细胞相似。先进的综合转录组分析显示了从激活到稳态和从稳态到激活的不同轨迹，表明这一过程具有可逆性。
