Methods and compositions for inducing hematopoietic cell differentiation

• US 10,626,372 B1
• Filed: 01/26/2016
• Issued: 04/21/2020
• Est. Priority Date: 01/26/2015
• Status: Active Grant

• Current Assignee: Fate Therapeutics Inc.
• Original Assignee: Fate Therapeutics Inc.
• Inventors: Valamehr, Bahram, Clarke, Raedun
• Primary Examiner(s): Bunner, Bridget E
• Application Number:
  US15/545,243
• Time in Patent Office: 1,547 Days
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/10 Growth factors
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/2302 Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/2303 Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/2307 Interleukin-7 (IL-7)
• CPC Class Codes: C12N 2501/2311 Interleukin-11 (IL-11)
• CPC Class Codes: C12N 2501/2315 Interleukin-15 (IL-15)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/26 : Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/40 : Regulators of development
• CPC Class Codes: C12N 2501/415 : Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 : Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/727 : Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 : Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 : Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 : from embryonic cells
• CPC Class Codes: C12N 2506/11 : from blood or immune system cells
• CPC Class Codes: C12N 2506/1369 : from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
• CPC Class Codes: C12N 2506/45 : from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/52 : Fibronectin; Laminin
• CPC Class Codes: C12N 2533/90 : Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0018 : Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0607 : Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0636 : T lymphocytes
• CPC Class Codes: C12N 5/0646 : Natural killers cells [NK], NKT cells
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0662 : Stem cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The invention provides culture platforms, cell media, and methods of differentiating pluripotent cells into hematopoietic cells. The invention further provides pluripotent stem cell-derived hematopoietic cells generated using the culture platforms and methods disclosed herein, which enable feed-free, monolayer culturing and in the absence of EB formation. Specifically, pluripotent stem cell-derived hematopoietic cell of this invention include, and not limited to, iHSC, definitive hemogenic endothelium, hematopoietic multipotent progenitors, T cell progenitors, NK cell progenitors, T cells, and NK cells.

Claims

1. An in vitro method for generating human pluripotent stem cell-derived Natural Killer (NK) lineage cells comprising:

(i) contacting human pluripotent stem cells with a composition comprising a BMP activator, to allow cell differentiation and expansion to obtain human pluripotent stem cell-derived mesodermal cells;

(ii) contacting the human pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, to allow cell differentiation and expansion, to obtain human pluripotent stem cell-derived mesodermal cells having definitive hemogenic endothelium (HE) potential;

(iii) contacting the human pluripotent stem cell-derived mesodermal cells having definitive HE potential with a composition comprising a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6 and IL11, to allow cell differentiation and expansion, wherein the composition is free of TGFβ

receptor/ALK inhibitor to obtain human pluripotent stem cell-derived definitive hemogenic endothelium; and

(iv) contacting the human pluripotent stem cell-derived definitive hemogenic endothelium with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the composition is free, or essentially free, of one or more of VEGF, bFGF, BM′

activators and ROCK inhibitors for generating the human pluripotent stem cell-derived NK lineage cells.

2. The method of claim 1, wherein the method further comprises subjecting human pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, mesodermal cells with hemogenic endothelium potential, and/or definitive hemogenic endothelium under low oxygen tension between about 2% to about 10%.

3. The method of claim 2, wherein the human pluripotent stem cell-derived NK lineage cells comprise pluripotent stem cell-derived (i) pre-NK cell progenitors, (ii) NK cell progenitors or (iii) NK cells.

4. The method of claim 1, wherein the human pluripotent stem cells are iPSCs or naï

ve iPSCs.

5. The method of claim 1, wherein the composition contacting the human pluripotent stem cells further comprises bFGF.

6. The method of claim 1, wherein the composition contacting the human pluripotent stem cell-derived mesodermal cells is free of TGFβ

receptor/ALK inhibitor.

7. The method of claim 1, wherein the method further comprises contacting human pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, to seed and expand the cells, wherein the composition is free of TGFβ

receptor/ALK inhibitors.

8. The method of claim 1, wherein generating human pluripotent stem cell-derived NK lineage cells is void of generation of embryoid bodies.

9. The method of claim 1, wherein generating human pluripotent stem cell-derived NK lineage cells is under monolayer culturing.

10. The method of claim 1, wherein generating human pluripotent stem cell-derived NK lineage cells is under feeder-free condition.

11. The method of claim 1, wherein generating human pluripotent stem cell-derived NK lineage cells is under stromal-free condition.

12. A differentiation platform for generating human pluripotent stem cell-derived NK cells comprising:

(i) a culture medium that comprises a BMP activator, and optionally bFGF, wherein the medium is suitable for differentiating and expanding human pluripotent stem cell-derived mesodermal cells from human pluripotent stem cells;

(ii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, wherein the medium is suitable for obtaining definitive hemogenic endothelium potential in the human pluripotent stem cell-derived mesodermal cells;

(iii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, wherein the medium is suitable for differentiating human pluripotent stem cell-derived definitive hemogenic endothelium from human pluripotent stem cell-derived mesodermal cells having definitive hemogenic endothelium potential; and

(iv) a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the medium is free of one or more of VEGF, bFGF, BMP activators and ROCK inhibitors and is suitable for differentiating human pluripotent stem cell-derived definitive hemogenic endothelium to human pluripotent stem cell-derived NK cell progenitors or NK cells.

13. The differentiation platform of claim 12, further comprising:

(v) a culture medium that comprises a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and is free of TGFβ

receptor/ALK inhibitors, wherein the medium is suitable for seeding and expanding human pluripotent stem cells.

14. The differentiation platform of claim 12, wherein the culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, is free of TGFβ

receptor/ALK inhibitor.

15. The differentiation platform of claim 12, wherein the culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, and IL11, is free of TGFβ

receptor/ALK inhibitor.

16. The differentiation platform of claim 12, wherein the human pluripotent stem cells are iPSCs or naive iPSCs.