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.
Vera S Brok-Volchanskaya  1 ,  David A Bennin  2 ,  Kran Suknuntha  3 ,  Lucas C Klemm  2 ,  Anna Huttenlocher  2 ,  Igor Slukvin  4
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: [email protected].
PMID: 31708474 DOI: 10.1016/j.stemcr.2019.10.007
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.
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