# US 11,439,667 B2 · Compositions and methods for expansion of embryonic hematopoietic stem cells

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- Number: US 11,439,667 B2
- Document ID: US11439667B2
- Status: Granted
- Kind: B2
- Application number: 16/656,342
- Filed: 2019-10-17
- Published: 2020-02-13
- Granted: 2022-09-13
- Earliest claimed: 2013-01-08
- Assignees: Fred Hutchinson Cancer Center
- Applicants: Fred Hutchinson Cancer Research Center
- Inventors: Irwin D. Bernstein; Brandon K. Hadland
- Web page: https://patentgrep.com/patent/US11439667B2

## CPC

- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- C07K 14/52: Cytokines; Lymphokines; Interferons
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- C07K 14/475: Growth factors; Growth regulators
- C07K 14/54: Interleukins [IL]
- C07K 14/5403: {IL-3}
- C07K 14/5412: {IL-6}
- C07K 14/5431: {IL-11}
- C12N 2501/10: Growth factors
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/2301: Interleukin-1 (IL-1)
- C12N 2501/2303: Interleukin-3 (IL-3)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/2311: Interleukin-11 (IL-11)
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2501/42: Notch; Delta; Jagged; Serrate
- C12N 2533/52: Fibronectin; Laminin
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to methods, kits and compositions for expansion of embryonic hematopoietic stem cells and providing hematopoietic function to human patients in need thereof. In one aspect, it relates to kits and compositions comprising a Notch agonist, one or more growth factors, and, optionally, an inhibitor of the TGFβ pathway. Also provided herein are methods for expanding embryonic hematopoietic stem cells using kits and compositions comprising a Notch agonist, one or more growth factors, and, optionally, an inhibitor of the TGFβ pathway. The embryonic hematopoietic stem cells expanded using the disclosed kits, compositions and methods include cells derived from an embryo (e.g., aorta-gonad-mesonephros region of the embryo), embryonic stem cells, induced pluripotent stem cells, or reprogrammed cells of other types. The present invention also relates to administering the embryonic hematopoietic stem cells expanded using a combination of a Notch agonist, one or more growth factors, and, optionally, an inhibitor of the TGFβ pathway to a patient for short-term and/or long-term in vivo repopulation benefits.

## Claims

### Claim 1

A method of expanding embryonic hematopoietic stem cells, comprising:
culturing the embryonic hematopoietic stem cells ex vivo in the presence of
(i) a composition comprising an immobilized Delta or Jagged Notch agonist; and
(ii) a fluid medium comprising an antibody that inhibits TGFβ and growth factors comprising stem cell factor (SCF), Flt-3 ligand (Flt-3L), Interleukin-3 (IL-3), thrombopoietin (TPO), Interleukin-11 (IL-11), and Interleukin-6 (IL-6);
thereby producing an expanded embryonic hematopoietic stem cell sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the culturing is not in the presence of an aryl hydrocarbon receptor antagonist.

### Claim 3 (depends on 1)

The method of claim 1, wherein the immobilized Delta Notch agonist is an extracellular domain of a Delta protein.

### Claim 4 (depends on 3)

The method of claim 3, wherein the extracellular domain is fused to an Fc region of an IgG.

### Claim 5 (depends on 3)

The method of claim 3, wherein the Delta protein is a human Delta protein.

### Claim 6 (depends on 1)

The method of claim 1, wherein the immobilized Notch agonist is Delta1ext-IgG and wherein Delta1ext-IgG has been applied to a solid phase at a concentration of about 2.5 μg/ml.

### Claim 7 (depends on 1)

The method of claim 1, wherein the immobilized Jagged Notch agonist is an extracellular domain of a Jagged protein.

### Claim 8 (depends on 1)

The method of claim 1, wherein the SCF and Flt-3L are each in the fluid medium at a concentration between about 10 and 200 ng/ml, and wherein the IL-6 is in the fluid medium at a concentration between about 10 and 100 ng/ml.

### Claim 9 (depends on 1)

The method of claim 1, wherein the composition further comprises fibronectin or a fragment thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the composition further comprises CH-296, and wherein in said culturing step, CH-296 is immobilized on a solid phase.

### Claim 11 (depends on 1)

The method of claim 1, wherein the fluid medium further comprises fetal bovine serum (FBS).

### Claim 12 (depends on 1)

The method of claim 1, wherein the embryonic hematopoietic stem cells are derived from an embryo, from embryonic stem cells (ESC), from induced pluripotent stem cells (iPSC), or from reprogrammed non-pluripotent cells.

### Claim 13 (depends on 1)

The method of claim 1, wherein the embryonic hematopoietic stem cells are derived from induced pluripotent stem cells (iPSC).

### Claim 14 (depends on 1)

The method of claim 1, wherein the embryonic hematopoietic stem cells are derived from a single human or more than one human.

### Claim 15 (depends on 1)

The method of claim 1, wherein the embryonic hematopoietic stem cells cultured in said culturing step are derived from cells of a patient with a genetic disorder associated with a gene having a sequence defect.

### Claim 16 (depends on 1)

The method of claim 1, wherein the embryonic hematopoietic stem cells are derived from the aorta-gonad-mesonephros (AGM) region of an embryo.

### Claim 17 (depends on 1)

The method of claim 1, wherein the cells cultured in said culturing step are a population of cells enriched for CD45 and/or VE-Cadherin, in which said embryonic hematopoietic stem cells are present.

### Claim 18

A method of expanding human embryonic hematopoietic stem cells, comprising culturing the human embryonic hematopoietic stem cells ex vivo on a solid phase coated with Delta1ext-IgG and CH-296, and in the presence of a fluid medium comprising:
(i) an antibody that inhibits TGFβ; and
(ii) growth factors comprising SCF, Flt-3L, IL-3, TPO, IL-11, and IL-6; thereby producing an expanded embryonic hematopoietic stem cell sample.

### Claim 19 (depends on 18)

The method of claim 18, wherein the Delta1ext-IgG comprises a human Delta1 fragment and a human IgG fragment, the CH-296 is a human fibronectin fragment, and the SCF, Flt-3L and IL-6 are all human.