Unbiased single-cell analysis of human clinical kidney ...s770312771.onlinehome.us/Presentations/Zhang2019.pdf · •Yan Wu •Song Chen •Nongluk Plongthongkum •Derek Gao Funding

Unbiased single-cell analysis of human clinical kidney specimens

Kun Zhang, PhD

Professor, Department of BioengineeringUniversity of California, San Diego, USA

Western Society of Pediatric Nephrology Conference San Diego

January 26, 2019

Single Nucleus Sequencing (SNS)• Enables cellular level analyses on

challenging tissue sources– e.g. Human postmortem tissue

repositories• No cellular cross contamination

– e.g. Highly adherent oligodendrocytes in neuronal isolates

• Stabilized RNA – Nuclear RNA that is not reliant on

live/dead state• Scalable

– Cryopreservation of sorted nuclei for deeper sampling

RNA in nuclei vs. whole cellsKCNC2

NeuN+ nuclei

Brain section

First brain: 6 areas, 51-yr female

86 C1 chips53% single capture4,488 single sequenced8.3M reads per cell3,227 passed filter

• Nuclear RNA seq• Nuclei preservation• SmartSeq Plus with

poly(dIdC)

Lake et al. Science, 2016

Transcriptome/Chromatin Integrative Analysis

Lake et al. Nature Biotechnology, 2018

22-yr, male (BA17/BA6)20-yr, male (BA17)49-yr, female (BA17/BA10/CBM)48-yr, male (BA17/BA10/CBM)35-yr, female (BA17/BA10/CBM)49-yr, male (CBM)

35k brain cells from 6 individuals

UCSD-WU TIS

KIDNEY PRECISION MEDICINE PROJECT

snDrop-Seq pipeline for clinical tissues


Kidney Tissue Optimization

Establish a single-nucleus RNA-seq pipeline optimized for processing adult kidney tissue

Goal 1:

19 Experiments: Nuclei > 400 transcripts

Identified snDrop-seq compatible tissue processing methods:

• Frozen tissue - cryosections• Fresh Tissue – Dissociated

Outcome 1:

Biopsy Scale:Average size: 6 mm3

Average yield: 100K Nuclei/mm3

Average # cell types detected: 12


Kidney Tissue Optimization (cont.)

Optimize cryosection quality for snDrop-seqGoal 2:

RNAlater treatment of cryosections improved:

1) Quality/Viability (lower % mitochondrial transcripts)

2) Outcome (higher postQC yields)

3) Experiment Pass Rates (92% vs 35% for CryoF)

Outcome 2:

25 Experiments, 5 Pilot samples (U Mich), 6 Local samples (WU):% Mitochondrial Transcripts (MT) / Post-QC Nuclei / Exp. Pass Rates


Kidney Tissue Optimization (cont.)

Tissue validation for snDrop-seq cryosections

Goal 3:

Histological assessment of sections adjacent to those used for snDrop-seq allow QA/QC of tissue composition

Outcome 3:

5 Pilot samples (U Mich), 10 Local samples (WU):

H & E stain


snDrop-seq: 18,000 kidney cells

Major and rare cell types identified and correlate with kidney

regions

Lake et al. 2019 (in revision)


Stress response activated cell dissociation

Blue Lake & Sanjay Jain


Novel proximal tubule(PT) cells



Novel proximal tubule(PT) cells


• Atypical Hemolytic Uremic Syndrome 1

• C3-glomerulopathy • End-stage kidney disease


Novel collecting duct(CD) cells

Cortex Medulla


Cell clusters -> kidney diseases & hypertension

Summary1. Single-cell analysis of archived human tissue

requires a robust tissue processing and preservation method, for uniform sampling and avoiding preparation artifacts.

2. From ~18,000 snDrop-seq data, we identified 30 cell types or subtypes with novel biological and histopathlogical insights.

AcknowledgementsUCSD BE (Zhang)•Blue Lake•Yan Wu•Song Chen•NonglukPlongthongkum•Derek Gao

Funding supportsNIDDK UG3/UH3 (KPMP), HuBMAP U54, BICCN U01s, NIH R01s

Harvard•Peter Kharchenko

WashU Urology/KTRC•Sanjay Jain•Joe Gaut•Anitha Vijayan•Diane Salamon•Masato Hoshi•Amanda Knoten

Documents

Unbiased single-cell analysis of human clinical kidney ...s770312771.onlinehome.us/Presentations/Zhang2019.pdf · •Yan Wu •Song Chen •Nongluk Plongthongkum •Derek Gao Funding