US 12,410,401 B2
Generation of functional neutrophils and macrophages from induced pluripotent stem cells in chemically defined conditions using transient expression of ETV2
Igor I. Slukvin, Verona, WI (US); Vera Sergeyevna Brok Volchanskaya, Singapore (SG); Kran Suknuntha, Madison, WI (US); Anna Huttenlocher, Madison, WI (US); David Alfred Bennin, Mount Horeb, WI (US); and Lucas Klemm, Madison, WI (US)
Assigned to Wisconsin Alumni Research Foundation (WARF), Madison, WI (US)
Filed by Wisconsin Alumni Research Foundation, Madison, WI (US)
Filed on Jun. 5, 2020, as Appl. No. 16/894,569.
Claims priority of provisional application 62/858,173, filed on Jun. 6, 2019.
Prior Publication US 2020/0385676 A1, Dec. 10, 2020
Int. Cl. C12N 5/0783 (2010.01); A61K 40/10 (2025.01); A61K 40/42 (2025.01); A61K 40/45 (2025.01); C12N 5/0786 (2010.01)
CPC C12N 5/0636 (2013.01) [A61K 40/10 (2025.01); A61K 40/4242 (2025.01); A61K 40/4544 (2025.01); C12N 5/0645 (2013.01); C12N 2500/02 (2013.01); C12N 2501/113 (2013.01); C12N 2501/155 (2013.01); C12N 2501/165 (2013.01); C12N 2501/23 (2013.01); C12N 2501/26 (2013.01); C12N 2501/599 (2013.01); C12N 2506/45 (2013.01)] 6 Claims
 
1. A population of in vitro derived CD 16+ CD 10− neutrophils obtained by an in vitro method of producing CD 16+ CD 10− neutrophils from pluripotent stem cells (PSCs), the method comprising:
(a) introducing exogenous ETS Variant Transcription Factor 2 (ETV2) in the PSCs and culturing the ETV2-induced PSCs in, xenogen- and serum-free medium comprising fibroblast growth factor receptor 2 (FGF-2) to produce a population of ETV2-induced CD144+hematoendothelial progenitor cells (ETV2-induced HEPs);
(b) culturing the ETV2-induced CD 144+hematoendothelial progenitor cells in xenogen- and serum-free medium comprising granulocyte-macrophage colony-stimulating factor (GM-CSF) and FGF-2 for a sufficient time to produce non-adherent myeloid progenitors; and
(c) culturing the myeloid progenitors in xenogen- and serum-free medium comprising G-CSF and retinoic acid agonist to differentiate the non-adherent myeloid progenitors into CD16+ CD10− neutrophils,
wherein the CD16+ CD10− neutrophils exhibit impaired neutrophil extracellular trap (NET) production in response to phorbol 12-myristate 13-acetate (PMA),
wherein the neutrophils have a CD66blow phenotype,
wherein greater than 50% of the neutrophils are CD 16+, and
wherein the pluripotent stem cell is genetically modified to obtain the neutrophil population of interest.