Automated cell type classification in intact tissues by single-cell molecular profiling

Abstract
Data availability
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Abstract

A major challenge in biology is identifying distinct cell classes and mapping their interactions in vivo. Tissue-dissociative technologies enable deep single cell molecular profiling but do not provide spatial information. We developed a proximity ligation- in situ hybridization technology (PLISH) with exceptional signal strength, specificity, and sensitivity in tissue. Multiplexed data sets can be acquired using barcoded probes and rapid label-image-erase cycles, with automated calculation of single cell profiles, enabling clustering and anatomical re-mapping of cells. We apply PLISH to expression profile ~2,900 cells in intact mouse lung, which identifies and localizes known cell types, including rare ones. Unsupervised classification of the cells indicates differential expression of 'housekeeping' genes between cell types, and re-mapping of two sub-classes of Club cells highlights their segregated spatial domains in terminal airways. By enabling single cell profiling of various RNA species in situ, PLISH can impact many areas of basic and medical research.

Data availability

The following previously published data sets were used

(2014) Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq
Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE52583).

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52583

Article and author information

Author details

Monica Nagendran

Department of Internal Medicine, Division of Pulmonary & Critical Care, Stanford University School of Medicine, Stanford, United States

Competing interests
Monica Nagendran, MN has filed a provisional patent for PLISH.(Application # 62/475,090).
Daniel P Riordan

Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

Competing interests
Daniel P Riordan, DR has filed a provisional patent for PLISH. (Application # 62/475,090).
Pehr B Harbury

Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

For correspondence
harbury@stanford.edu

Competing interests
Pehr B Harbury, PH has filed a provisional patent for PLISH. (Application # 62/475,090).
Tushar J Desai

Department of Internal Medicine, Division of Pulmonary & Critical Care, Stanford University School of Medicine, Stanford, United States

For correspondence
tdesai@stanford.edu

Competing interests
Tushar J Desai, TD has filed a provisional patent for PLISH. (Application # 62/475,090).

"This ORCID iD identifies the author of this article:" 0000-0002-8794-5319

Funding

National Heart, Lung, and Blood Institute (5U01HL09999507)

Pehr B Harbury
Tushar J Desai

National Heart, Lung, and Blood Institute (1R56HL1274701)

Tushar J Desai

Stanford University School of Medicine (BIO-X IIP-130)

Pehr B Harbury
Tushar J Desai

Stanford University School of Medicine (ChEM-H)

Monica Nagendran
Pehr B Harbury
Tushar J Desai

Stanford University School of Medicine (Discovery Innovation Fund Award)

Pehr B Harbury

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#22988) of Stanford University. The protocol was approved by the Administrative Panel on Laboratory Animal Care (APLAC) of Stanford University. Every effort was made to minimize suffering.

Human subjects: Adult human lung was obtained from Stanford Healthcare with patient informed consent and consent to publish in strict accordance with protocol 18891, approved by the Institutional Review Board Administrative Panel on Human Subjects in Medical Research of Stanford University, in compliance with requirements for protection of human subjects.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.