IP Library Granted Patent US 12,454,676
Granted Patent B2
US 12,454,676 · App. 17/405,579 · Granted Oct 28, 2025

Derivation of human microglia from pluripotent stem cells

Inventors: James A. Thomson (Madison, WI); Nicholas E. Propson (Houston, TX); Michael P. Schwartz (Madison, WI); Zhonggang Hou (Madison, WI); Gene I. Uenishi (Madison, WI); Igor I Slukvin (Verona, WI); William L. Murphy (Waunakee, WI); Jue Zhang (Madison, WI)
Assignee: Wisconsin Alumni Research Foundation
C12N5/0622C12N5/0645C12N5/0647C12N5/0697G01N33/5014G01N33/5058C12N5/0062C12N5/0068C12N5/0606C12N2500/25C12N2501/115C12N2501/15C12N2501/22C12N2501/999C12N2506/02C12N2506/45C12N2513/00
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Quick Facts
Patent No.
US 12,454,676
App. No.
17/405,579
Granted
Oct 28, 2025
Kind
B2
Abstract

The present invention relates to methods for deriving human hematopoietic progenitors, primitive macrophages, and microglial cells from human pluripotent stem cells. In particular, provided herein are highly efficient and reproducible methods of obtaining human primitive macrophages and microglia from human pluripotent stem cells, where the primitive macrophages and microglia can be suitable for clinically relevant therapeutic applications.

Claims (23)

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, NaHCO 3 , 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.

2 . The method of claim 1 , wherein the CD45 + /CD11b + /CD14 + primitive macrophages are CD34 low/negative .

3 . The method of claim 1 , wherein the CD45 + /CD11b + /CD14 + primitive macrophages do not express a detectable level of Iba-1.

4 . The method of claim 1 , wherein the hematopoietic cytokine is granulocyte macrophage colony-stimulating factor (GM-CSF).

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.

6 . The method of claim 5 step (d), wherein the hematopoietic cytokine is human granulocyte macrophage colony-stimulating factor (GM-CSF).

7 . The method of claim 5 , wherein the CD45 + /CD11b + /CD14 + primitive macrophages are CD34 low/negative .

8 . The method of claim 5 , wherein the CD45 + /CD11b + /CD14 + primitive macrophages do not express a detectable level of lba-1.

9 . The method of claim 1 , wherein the substrate that promotes cell adhesion comprises Tenascin-C.

10 . The method of claim 9 , wherein the Tenascin-C is recombinant human Tenascin-C.

11 . The method of claim 1 , wherein the ROCK inhibitor is selected from the group consisting of Y-27632 and Blebbistatin.

12 . The method of claim 5 step (a), wherein the growth medium consists essentially of Dulbecco's Modified Eagle Medium (DMEM), nutrient mixture F12, a chemically defined lipid concentrate, L-ascorbic acid-2-phosphate magnesium, monothioglycerol, sodium selenium, polyvinyl alcohol, L-alanyl-L-glutamine, FGF2, bone morphogenetic protein 4 (BMP4), Activin A, and an inhibitor of glycogen synthase 3 (GSK3).

13 . The method of claim 5 , wherein the pluripotent stem cells are initially seeded on the substrate at a cell density between about 2×10 5 cells per cm 2 and about 2.5×10 5 cells per cm 2 .

14 . The method of claim 5 step (a), wherein the substrate that promotes cell adhesion comprises vitronectin.

15 . The method of claim 12 , wherein the inhibitor of GSK3 is selected from the group consisting of CHIR99021, lithium chloride (LiCl), and 6-bromoindirubin-3′-oxime (BIO).

16 . The method of claim 5 step (b), wherein the inhibitor of TGF-mediated signaling is selected from the group consisting of SB431542 and A-83-01.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 5, 2021
From: PROPSON, NICHOLAS; THOMSON, JAMES; SCHWARTZ, MICHAEL; HOU, ZHONGGANG; UENISHI, GENE; SLUKVIN, IGOR; MURPHY, WILLIAM; ZHANG, JUE
To: WISCONSIN ALUMNI RESEARCH FOUNDATION
Reel/Frame 058030/0847 →
Continuity (4)
Division 16138724 · Sep 21, 2018
Division 14986224 · Dec 31, 2015
Provisional Application 62098824 · Dec 31, 2014
Related Publication 20220033773A1 · Feb 3, 2022
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