Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory

Daniel J. Beisang; Karen Smith; Libang Yang; Alexey Benyumov; Adam Gilbertsen; Jeremy Herrera; Eric Lock; Emilian Racila; Colleen Forster; Brian J. Sandri; Craig A. Henke; Peter B. Bitterman

doi:10.1038/s41598-020-66630-5

Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory

Daniel J. Beisang, Karen Smith, Libang Yang, Alexey Benyumov, Adam Gilbertsen, Jeremy Herrera, Eric Lock, Emilian Racila, Colleen Forster, Brian J. Sandri, Craig A. Henke, Peter B. Bitterman

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Abstract

In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44^hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44^lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.

Original language	English (US)
Article number	11162
Journal	Scientific reports
Volume	10
Issue number	1
DOIs	https://doi.org/10.1038/s41598-020-66630-5
State	Published - Jul 7 2020

Bibliographical note

Funding Information:
The authors would like to thank the University of Minnesota Genomics Center, the UMGC Pilot Grant Program, Minnesota Supercomputing Institute, Flow Cytometry core, and BioNet Histology core for their technical assistance. We thank Mark Peterson for his technical assistance, and Matthew Parker for preliminary analysis. We thank Dr. Teschendorff for his personal correspondence regarding network entropy analysis. This work was funded by R01HL125236 to P.B, R01HL125227A1 to C.H., and 5T32HL7741 to D.B.

Publisher Copyright:
© 2020, The Author(s).

Keywords

Case-Control Studies
Cell Differentiation/genetics
Fluorescent Antibody Technique
Gene Expression Profiling
Humans
Hyaluronan Receptors/metabolism
Idiopathic Pulmonary Fibrosis/metabolism
Lung/metabolism
Mesenchymal Stem Cells/metabolism
Sequence Analysis, RNA

PubMed: MeSH publication types

Journal Article
Research Support, N.I.H., Extramural

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10.1038/s41598-020-66630-5

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Beisang, D. J., Smith, K., Yang, L., Benyumov, A., Gilbertsen, A., Herrera, J., Lock, E., Racila, E., Forster, C., Sandri, B. J., Henke, C. A., & Bitterman, P. B. (2020). Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory. Scientific reports, 10(1), Article 11162. https://doi.org/10.1038/s41598-020-66630-5

Beisang, DJ , Smith, K , Yang, L, Benyumov, A, Gilbertsen, A, Herrera, J, Lock, E , Racila, E , Forster, C , Sandri, BJ , Henke, CA & Bitterman, PB 2020, 'Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory', Scientific reports, vol. 10, no. 1, 11162. https://doi.org/10.1038/s41598-020-66630-5

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title = "Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory",

abstract = "In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.",

keywords = "Case-Control Studies, Cell Differentiation/genetics, Fluorescent Antibody Technique, Gene Expression Profiling, Humans, Hyaluronan Receptors/metabolism, Idiopathic Pulmonary Fibrosis/metabolism, Lung/metabolism, Mesenchymal Stem Cells/metabolism, Sequence Analysis, RNA",

author = "Beisang, {Daniel J.} and Karen Smith and Libang Yang and Alexey Benyumov and Adam Gilbertsen and Jeremy Herrera and Eric Lock and Emilian Racila and Colleen Forster and Sandri, {Brian J.} and Henke, {Craig A.} and Bitterman, {Peter B.}",

note = "Funding Information: The authors would like to thank the University of Minnesota Genomics Center, the UMGC Pilot Grant Program, Minnesota Supercomputing Institute, Flow Cytometry core, and BioNet Histology core for their technical assistance. We thank Mark Peterson for his technical assistance, and Matthew Parker for preliminary analysis. We thank Dr. Teschendorff for his personal correspondence regarding network entropy analysis. This work was funded by R01HL125236 to P.B, R01HL125227A1 to C.H., and 5T32HL7741 to D.B. Publisher Copyright: {\textcopyright} 2020, The Author(s).",

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doi = "10.1038/s41598-020-66630-5",

language = "English (US)",

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AU - Beisang, Daniel J.

AU - Smith, Karen

AU - Yang, Libang

AU - Benyumov, Alexey

AU - Gilbertsen, Adam

AU - Herrera, Jeremy

AU - Lock, Eric

AU - Racila, Emilian

AU - Forster, Colleen

AU - Sandri, Brian J.

AU - Henke, Craig A.

AU - Bitterman, Peter B.

N1 - Funding Information: The authors would like to thank the University of Minnesota Genomics Center, the UMGC Pilot Grant Program, Minnesota Supercomputing Institute, Flow Cytometry core, and BioNet Histology core for their technical assistance. We thank Mark Peterson for his technical assistance, and Matthew Parker for preliminary analysis. We thank Dr. Teschendorff for his personal correspondence regarding network entropy analysis. This work was funded by R01HL125236 to P.B, R01HL125227A1 to C.H., and 5T32HL7741 to D.B. Publisher Copyright: © 2020, The Author(s).

PY - 2020/7/7

Y1 - 2020/7/7

N2 - In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.

AB - In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.

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KW - Lung/metabolism

KW - Mesenchymal Stem Cells/metabolism

KW - Sequence Analysis, RNA

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Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory

Abstract

Bibliographical note

Keywords

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