Generation of functional neutrophils and macrophages from induced pluripotent stem cells in chemically defined conditions using transient expression of etv2
AssigneeWisconsin Alumni Research Foundation (WARF)
InventorsIgor I. Slukvin, Vera Sergeyevna Brok Volchanskaya, Kran Suknuntha, Anna Huttenlocher, David Alfred Bennin, Lucas Klemm
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Abstract
The present invention provides methods of producing in vitro derived neutrophils or macrophages in xenogen- and serum-free conditions from pluripotent stem cells and in vitro derived populations of neutrophils and macrophages. Methods of treatment using in vitro derived neutrophils or macrophages are also contemplated.
Claims37
An in vitro method of producing CD16+ CD10−neutrophils from pluripotent stem cells (PSCs), the method comprising:
- (a) transiently introducing exogenous ETV2 in the PSCs and culturing the ETV2-induced PSCs in, xenogen- and serum-free medium comprising FGF-2 to produce a population of ETV2-induced CD144+hematoendothelial progenitor cells (ETV2-induced HEPs);
- (b) culturing the ETV2-induced CD144+hematoendothelial progenitor cells in xenogen- and serum-free medium comprising GM-CSF and FGF2 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.
A method of producing exogenous CD16+CD10− neutrophils, the method comprising:
- (a) culturing CD144+hematoendothelial progenitor cells derived from PSCs in xenogen- and serum-free medium comprising GM-CSF and FGF2 for a sufficient time to produce non-adherent myeloid progenitors;
- (b) isolating the non-adherent myeloid progenitors from the adherent cells;
- and
- (c) culturing the non-adherent myeloid progenitors in xenogen- and serum-free medium comprising G-CSF and retinoic acid agonist for a time sufficient to differentiate the myeloid progenitors into CD16+CD10− neutrophils.
An in vitro method of producing CD14+CD16+ macrophages from pluripotent stem cells (PSCs), the method comprising:
- (a) transiently introducing exogenous ETV2 into pluripotent stem cells and culturing the ETV2-induced PSCs in xenogen-free medium comprising FGF2 to produce ETV2-induced CD144+hematoendothelial progenitor cells;
- (b) culturing the ETV2-induced CD144+hematoendothelial progenitor cells in xenogen- and serum-free medium comprising GM-CSF and FGF-2 for about 4 days;
- (c) isolating the adherent cells from the culture;
- and
- (d) culturing the adherent cells of step (b) in medium comprising IL-3 and M-CSF to produce the CD14+CD16+ macrophages/monocyte.
Dependencies are inferred from the claim text.