Unbiased single-cell analysis of human clinical kidney...
Transcript of 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
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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
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RNA in nuclei vs. whole cellsKCNC2
NeuN+ nuclei
Brain section
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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
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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
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UCSD-WU TIS
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KIDNEY PRECISION MEDICINE PROJECT
snDrop-Seq pipeline for clinical tissues
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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
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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
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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
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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)
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KIDNEY PRECISION MEDICINE PROJECT
Stress response activated cell dissociation
Blue Lake & Sanjay Jain
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KIDNEY PRECISION MEDICINE PROJECT
Novel proximal tubule(PT) cells
Blue Lake & Sanjay Jain
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KIDNEY PRECISION MEDICINE PROJECT
Novel proximal tubule(PT) cells
Blue Lake & Sanjay Jain
• Atypical Hemolytic Uremic Syndrome 1
• C3-glomerulopathy • End-stage kidney disease
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KIDNEY PRECISION MEDICINE PROJECT
Novel collecting duct(CD) cells
Cortex Medulla
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KIDNEY PRECISION MEDICINE PROJECT
Cell clusters -> kidney diseases & hypertension
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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.
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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