Effective and Rapid Generation of Functional Neutrophils from Induced Pluripotent Stem Cells Using ETV2-Modified mRNA

Vera S Brok-Volchanskaya; David A Bennin; Kran Suknuntha; Lucas C Klemm; Anna Huttenlocher; Igor Slukvin

doi:10.1016/j.stemcr.2019.10.007

Effective and Rapid Generation of Functional Neutrophils from Induced Pluripotent Stem Cells Using ETV2-Modified mRNA

Stem Cell Reports. 2019 Dec 10;13(6):1099-1110. doi: 10.1016/j.stemcr.2019.10.007. Epub 2019 Nov 7.

Authors

Vera S Brok-Volchanskaya¹, David A Bennin², Kran Suknuntha³, Lucas C Klemm², Anna Huttenlocher², Igor Slukvin⁴

Affiliations

¹ Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI 53715, USA.
² Departments of Pediatrics and Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, USA.
³ Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI 53715, USA; Department of Pathology and Laboratory Medicine, Wisconsin National Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715, USA; Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
⁴ Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI 53715, USA; Department of Pathology and Laboratory Medicine, Wisconsin National Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715, USA; Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53707-7365, USA. Electronic address: islukvin@wisc.edu.

Abstract

Human induced pluripotent stem cells (hiPSCs) can serve as a versatile and scalable source of neutrophils for biomedical research and transfusion therapies. Here we describe a rapid efficient serum- and xenogen-free protocol for neutrophil generation, which is based on direct hematoendothelial programming of hiPSCs using ETV2-modified mRNA. Culture of ETV2-induced hematoendothelial progenitors in the presence of GM-CSF, FGF2, and UM171 led to continuous production of generous amounts of CD34⁺CD33⁺ myeloid progenitors which could be harvested every 8-10 days for up to 30 days of culture. Subsequently, myeloid progenitors were differentiated into neutrophils in the presence of G-CSF and the retinoic acid agonist Am580. Neutrophils obtained in these conditions displayed a typical somatic neutrophil morphology, produced reactive oxygen species, formed neutrophil extracellular traps and possessed phagocytic and chemotactic activities. Overall, this technology offers an opportunity to generate a significant number of neutrophils as soon as 14 days after initiation of differentiation.

Keywords: ETV2; hematopoietic differentiation; hemogenic endothelium; human iPSCs; modified mRNA; myeloid progenitors; neutrophils.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Biomarkers
Cell Differentiation / genetics*
Cells, Cultured
Extracellular Traps / genetics
Extracellular Traps / metabolism
Gene Expression Regulation, Developmental
Hematopoiesis
Humans
Immunophenotyping
Induced Pluripotent Stem Cells / cytology*
Induced Pluripotent Stem Cells / metabolism*
Leukopoiesis / genetics
Myeloid Progenitor Cells / cytology
Myeloid Progenitor Cells / metabolism
Neutrophils / cytology
Neutrophils / immunology*
Neutrophils / metabolism*
RNA, Messenger*
Transcription Factors / genetics*

Substances

Biomarkers
ETV2 protein, human
RNA, Messenger
Transcription Factors

Abstract

Publication types

MeSH terms

Substances

Grants and funding