Generation of Human Neutrophils from Induced Pluripotent Stem Cells in Chemically Defined Conditions Using ETV2 Modified mRNA

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

doi:10.1016/j.xpro.2020.100075

Generation of Human Neutrophils from Induced Pluripotent Stem Cells in Chemically Defined Conditions Using ETV2 Modified mRNA

STAR Protoc. 2020 Sep 18;1(2):100075. doi: 10.1016/j.xpro.2020.100075. Epub 2020 Jul 27.

Authors

Aditi Majumder¹, Kran Suknuntha^{1

2

3}, David Bennin³, Lucas Klemm⁴, Vera S Brok-Volchanskaya¹, Anna Huttenlocher⁴, Igor Slukvin^{1

2

5

6

7}

Affiliations

¹ Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI 53715, USA.
² Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI 53792, USA.
³ Chakri Naruebodindra Medical Institute, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Samut Prakan 10540, Thailand.
⁴ Department of Pediatrics and Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁵ Department of Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53707-7365, USA.
⁶ Technical Contact.
⁷ Lead Contact.

Abstract

This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using ETV2 modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function. For complete details on the use and execution of this protocol, please refer to Brok-Volchanskaya et al. (2019).

Publication types

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

MeSH terms

Cell Culture Techniques / methods*
Cell Differentiation / genetics
Cells, Cultured
Humans
Induced Pluripotent Stem Cells / cytology*
Neutrophils / cytology*
RNA, Messenger / genetics*
Transcription Factors / genetics*

Substances

ETV2 protein, human
RNA, Messenger
Transcription Factors

Abstract

Publication types

MeSH terms

Substances

Grants and funding