Tolerance, rejection and gender issues

Tuesday September 13, 2022 from 17:35 to 18:35

Room: CF-8

347.2 Multiplexed droplet single-cell sequencing (Mux-Seq) of normal and transplant kidney

Minnie M. Sarwal, United States

Professor in Residence, Surgery, Medicine, Pediatrics
Department of Transplant
UC San Francisco

Abstract

Multiplexed droplet single-cell sequencing (Mux-Seq) of normal and transplant kidney

Priyanka Rashmi1, Swastika Sur1, Tara K Sigdel1, Patrick Boada1, Andrew W Schroeder2, Izabella Damm1, Matthias Kretzler3, Jeff Hodgin3, Chun Jimmie Ye4, George Hartoularos4, Minnie Sarwal1.

1Surgery, UCSF, San Francisco, CA, United States; 2Genomics CoLabs, UCSF, San Francisco, CA, United States; 3Internal Medicine/Nephrology, University of Michigan, Ann Arbor, CA, United States; 4Institute for Human Genetics, UCSF, San Francisco, CA, United States

Maintenance of systemic homeostasis by kidney requires the coordinated response of diverse cell types. Use of single-cell RNA sequencing (scRNAseq) for patient samples remains fraught with difficulties. The ability to characterize immune and parenchymal cells during transplant rejection (some of which may be present at very small numbers due to injury) will be invaluable in defining transplant pathologies. Herein, we present feasibility data for multiplexing approach for droplet scRNAseq (Mux-Seq). Mux-Seq has the potential to minimize experimental batch biases and variations. Explant tissues from 6 normal and 2 transplant recipients after multiple rejection episodes leading to nephrectomy were pooled for Mux-Seq. Subsequently, a computational tool, Demuxlet was applied for demultiplexing the individual cells. Each sample was also applied individually in single microfluidic run (singleplex). We show that data from Mux-Seq correlated highly with singleplex (Pearson coefficient 0.982). Both are able to identify many known kidney cell types including immune cells. Trajectory analysis of proximal tubule and endothelial cells demonstrates separation between healthy and injured kidney from transplant explant suggesting various stages of differentiation. This study provides the technical groundwork for understanding the pathogenesis of alloimmune injury and host tissue response in transplant rejections leading to graft failure in clinical setting.



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