	US 12,454,676 B2
	Derivation of human microglia from pluripotent stem cells
James A. Thomson, Madison, WI (US); Nicholas E. Propson, Houston, TX (US); Michael P. Schwartz, Madison, WI (US); Zhonggang Hou, Madison, WI (US); Gene I. Uenishi, Madison, WI (US); Igor I Slukvin, Verona, WI (US); William L. Murphy, Waunakee, WI (US); and Jue Zhang, Madison, WI (US)
Assigned to Wisconsin Alumni Research Foundation, Madison, WI (US)
Filed by Wisconsin Alumni Research Foundation, Madison, WI (US)
Filed on Aug. 18, 2021, as Appl. No. 17/405,579.
Application 17/405,579 is a division of application No. 16/138,724, filed on Sep. 21, 2018, granted, now 11,124,765.
Application 16/138,724 is a division of application No. 14/986,224, filed on Dec. 31, 2015, granted, now 10,081,792, issued on Sep. 25, 2018.
Claims priority of provisional application 62/098,824, filed on Dec. 31, 2014.
Prior Publication US 2022/0033773 A1, Feb. 3, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 5/079 (2010.01); C12N 5/00 (2006.01); C12N 5/071 (2010.01); C12N 5/0735 (2010.01); C12N 5/0786 (2010.01); C12N 5/0789 (2010.01); G01N 33/50 (2006.01)

CPC C12N 5/0622 (2013.01) [C12N 5/0645 (2013.01); C12N 5/0647 (2013.01); C12N 5/0697 (2013.01); G01N 33/5014 (2013.01); G01N 33/5058 (2013.01); C12N 5/0062 (2013.01); C12N 5/0068 (2013.01); C12N 5/0606 (2013.01); C12N 2500/25 (2013.01); C12N 2501/115 (2013.01); C12N 2501/15 (2013.01); C12N 2501/22 (2013.01); C12N 2501/999 (2013.01); C12N 2506/02 (2013.01); C12N 2506/45 (2013.01); C12N 2513/00 (2013.01)]

16 Claims

1. A method of obtaining human primitive macrophages, comprising

(a) culturing human pluripotent stem cells under normoxic conditions for about 24 hours, wherein the pluripotent stem cells are cultured on a substrate that promotes cell adhesion and in a culture medium consisting essentially of L-ascorbic acid-2-phosphate magnesium, sodium selenium, transferrin, insulin, NaHCO₃, fibroblast growth factor 2 (FGF2), transforming growth factor beta 1 (TGF 1), and a Rho kinase (ROCK) inhibitor, whereby the cultured pluripotent stem cells differentiate into hematopoietic precursor cells (HPCs),

(b) culturing the HPCs of step (a) in a culture medium comprising FGF2, a VEGF, TPO, SCF, IL-6, and IL-3 until the cultured HPC differentiate into myeloid progenitors, and

(c) further culturing the human myeloid progenitors in the presence of a culture medium comprising insulin and a hematopoietic cytokine, whereby the cultured myeloid progenitors differentiate into a cell population comprising at least 80% CD45⁺/CD11b⁺/CD14⁺ primitive macrophages.

5. A method of obtaining human primitive macrophages, comprising culturing human myeloid progenitors obtained by:

(a) culturing human pluripotent stem cells under hypoxic conditions on a substrate that promotes cell adhesion and in a growth medium for between about 40 and about 48 hours; and

(b) further culturing the human pluripotent stem cells of step (a) under hypoxic conditions in a culture medium comprising FGF2, VEGF, and an inhibitor of TGF-mediated signaling, whereby the further cultured cells differentiate into hematopoietic progenitor cells (HPCs); and

(c) culturing the HPC of step (b) under normoxic conditions in a culture medium comprising FGF2, a VEGF, TPO, SCF, IL-6, and IL-3 until the cultured HPC differentiate into myeloid progenitors; and

(d). culturing the myeloid progenitors of step (c) under normoxic conditions in the presence of a culture medium comprising insulin and a hematopoietic cytokine, whereby the cultured myeloid progenitors differentiate into a cell population comprising at least 80% CD45⁺/CD11b⁺/CD14⁺ primitive macrophages.