Derivation of human microglia from pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-2016186137-A1
Application number	US-201514986224-A
Country	US
Kind code	A1
Filing date	Dec 31, 2015
Priority date	Dec 31, 2014
Publication date	Jun 30, 2016
Grant date	—

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.

First claim

We claim:

1 . A method of obtaining human hematopoietic precursor cells, comprising 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 (TGFβ1), and a Rho kinase (ROCK) inhibitor, whereby the cultured pluripotent stem cells differentiate into hematopoietic precursor cells (HPCs).

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

3 . The method of claim 2 , wherein the Tenascin-C is recombinant human Tenascin-C.

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

5 . A method of obtaining human myeloid progenitors, comprising culturing human HPCs obtained according to the method of claim 1 for about 3 to about 5 days in a culture medium comprising FGF2, a vascular endothelium growth factor (VEGF), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6 (IL-6), and interleukin-3 (IL-3), wherein the hematopoietic progenitor cells differentiate into myeloid progenitors.

6 . A method of obtaining human primitive macrophages, comprising culturing human myeloid progenitors obtained according to the method of claim 3 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.

7 . The method of claim 6 , wherein the CD45 + /CD11b + /CD14 + primitive macrophages are CD34 low/negative .

8 . The method of claim 6 , wherein the CD45 + /CD11b + /CD14 + primitive macrophages do not express a detectable level of Iba-1.

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

10 . A method of obtaining human hematopoietic precursor cells, comprising the steps of:
(a) culturing human pluripotent stem cells under hypoxic conditions on substrate that promotes cell adhesion and in a growth medium consisting 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) for a length of time between about 40 and about 48 hours, 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 ; (b) further culturing the cultured cells of step (a) under hypoxic conditions in a culture medium comprising FGF2, a VEGF, and an inhibitor of TGFβ-mediated signaling, whereby the further cultured cells differentiate into hematopoietic progenitor cells.

11 . The method of claim 10 , wherein the substrate that promotes cell adhesion comprises vitronectin.

12 . The method of claim 10 , wherein the inhibitor of GSK3 is selected from the group consisting of CHIR99021, lithium chloride (LiCl), and 6-bromoindirubin-3′-oxime (BIO).

13 . The method of claim 10 , wherein the inhibitor of TGFβ-mediated signaling is selected from the group consisting of SB431542 and A-83-01.

14 . A method of obtaining human myeloid progenitors, comprising culturing human HPCs obtained according to the method of claim 10 under normoxic conditions in a culture medium comprising FGF2, a VEGF, TPO, SCF, IL-6, and IL-3 until the cultured hematopoietic progenitor cells differentiate into myeloid progenitors.

15 . A method of obtaining human primitive macrophages, comprising culturing human myeloid progenitors obtained according to the method of claim 14 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.

16 . The method of claim 15 , wherein the hematopoietic cytokine is human granulocyte macrophage colony-stimulating factor (GM-CSF).

17 . The method of claim 15 , wherein the CD45 + /CD11b + /CD14 + primitive macrophages are CD34 low/negative .

18 . The method of claim 15 , wherein the CD45 + /CD11b + /CD14 + primitive macrophages do not express a detectable level of Iba-1.

19 . A method of making a composition comprising human microglial cells, the method comprising contacting human pluripotent stem cell-derived primitive macrophages to a chemically defined, xenogen-free three-dimensional tissue construct comprising stratified layers of human neurons and glia, thereby producing a composition comprising human microglial cells.

20 . The method of claim 19 , wherein the primitive macrophages are obtained according to the method of claim 6 .

21 . The method of claim 19 , wherein the tissue construct comprises a hydrogel.

22 . The method of claim 19 , wherein the microglial cells are Iba-1 + .

23 . The method of claim 19 , wherein, prior to the contacting step, the human primitive macrophages are cultured for about 5 days in a culture medium consisting essentially of DMEM/F12, interleukin-1-beta (IL-1β), serum, and a hematopoietic growth factor.

24 . The method of claim 19 , wherein the hematopoietic growth factor is macrophage colony-stimulating factor (M-CSF).

25 . A method of screening a compound for toxicity, comprising exposing a test compound to a composition obtained according to the method of claim 19 and assaying for an effect of the compound on one or more aspects of human microglial growth or development.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Thomson James A
• Propson Nicholas E
• Schwartz Michael P
• Hou Zhonggang
• Uenishi Gene I
• Slukvin Igor I
• Murphy William L
• Zhang Jue

Classifications

• C12N5/0647

  Haematopoietic stem cells; Uncommitted or multipotent progenitors · CPC title

• G01N33/5058

  Neurological cells · CPC title

• G01N33/5088

  of vertebrates · CPC title

• C12N5/0622 Primary

  Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells · CPC title

• C12N2501/22

  Colony stimulating factors (G-CSF, GM-CSF) · CPC title