unbiased single-cell analysis of human clinical kidney...
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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 PRECISION MEDICINE PROJECT
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 PRECISION MEDICINE PROJECT
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 PRECISION MEDICINE PROJECT
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
KIDNEY PRECISION MEDICINE PROJECT
snDrop-seq: 18,000 kidney cells
Major and rare cell types identified and correlate with kidney
regions
Lake et al. 2019 (in revision)
KIDNEY PRECISION MEDICINE PROJECT
Stress response activated cell dissociation
Blue Lake & Sanjay Jain
KIDNEY PRECISION MEDICINE PROJECT
Novel proximal tubule(PT) cells
Blue Lake & Sanjay Jain
KIDNEY PRECISION MEDICINE PROJECT
Novel proximal tubule(PT) cells
Blue Lake & Sanjay Jain
• Atypical Hemolytic Uremic Syndrome 1
• C3-glomerulopathy • End-stage kidney disease
KIDNEY PRECISION MEDICINE PROJECT
Novel collecting duct(CD) cells
Cortex Medulla
KIDNEY PRECISION MEDICINE PROJECT
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
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