Maintenance of systemic homeostasis by kidney requires the coordinated response of diverse cell types. The use of single-cell RNA sequencing (scRNAseq) for patient tissue samples remains fraught with difficulties with cell isolation, purity, and experimental bias. The ability to characterize immune and parenchymal cells during transplant rejection will be invaluable in defining transplant pathology where tissue availability is restricted to needle biopsy fragments. Herein, we present feasibility data for multiplexing approach for droplet scRNAseq (Mux-Seq). Mux-Seq has the potential to minimize experimental batch bias and variation even with very small sample input. In this first proof-of-concept study for this approach, explant tissues from six normal and two transplant recipients after multiple early post-transplant rejection episodes leading to nephrectomy due to aggressive antibody mediated rejection, were pooled for Mux-Seq. A computational tool, Demuxlet was applied for demultiplexing the individual cells from the pooled experiment. Each sample was also applied individually in a single microfluidic run (singleplex) to correlate results with the pooled data from the same sample. Our applied protocol demonstrated that data from Mux-Seq correlated highly with singleplex (Pearson coefficient 0.982) sequencing results, with the ability to identify many known and novel kidney cell types including different infiltrating immune cells. Trajectory analysis of proximal tubule and endothelial cells demonstrated separation between healthy and injured kidney from transplant explant suggesting evolving stages of cell- specific differentiation in alloimmune injury. This study provides the technical groundwork for understanding the pathogenesis of alloimmune injury and host tissue response in transplant rejection and normal human kidney and provides a protocol for optimized processing precious and low input human kidney biopsy tissue for larger scale studies.

中文翻译：

---

正常和移植肾的多重液滴单细胞测序（Mux-Seq）

肾脏维持全身稳态需要不同细胞类型的协调反应。对患者组织样本使用单细胞 RNA 测序 (scRNAseq) 仍然充满了细胞分离、纯度和实验偏差方面的困难。在移植排斥过程中表征免疫和实质细胞的能力对于定义移植病理学非常宝贵，其中组织可用性仅限于穿刺活检碎片。在此，我们展示了液滴 scRNAseq (Mux-Seq) 多路复用方法的可行性数据。Mux-Seq 有可能最大限度地减少实验批次偏差和变异，即使样本输入非常小。在这种方法的第一个概念验证研究中，由于侵袭性抗体介导的排斥而导致肾切除术的多次早期移植后排斥事件后，来自六个正常和两个移植受者的外植体组织被合并用于 Mux-Seq。Demuxlet 是一种计算工具，用于从合并实验中分离单个细胞。每个样本也被单独应用于单次微流体运行（单重），以将结果与来自同一样本的合并数据相关联。我们应用的协议表明，来自 Mux-Seq 的数据与单重（Pearson 系数 0.982）测序结果高度相关，能够识别许多已知和新型肾细胞类型，包括不同的浸润免疫细胞。近端小管和内皮细胞的轨迹分析表明，移植外植体的健康肾脏和受损肾脏之间存在分离，表明同种免疫损伤中细胞特异性分化的进化阶段。本研究为了解移植排斥反应和正常人肾脏中同种免疫损伤和宿主组织反应的发病机制提供了技术基础，并为优化处理珍贵和低输入的人肾活检组织提供了更大规模研究的方案。