# US 2020/0399602 A1 · Method for Differentiation of Human Pluripotent Stem Cell Lines in Suspension Culture

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- Number: US 2020/0399602 A1
- Document ID: US20200399602A1
- Status: Published application
- Kind: A1
- Application number: 16/962,973
- Filed: 2019-01-15
- Published: 2020-12-24
- Earliest claimed: 2019-01-15
- Assignees: Agency for Science, Technology and Research
- Applicants: Agency for Science, Technology and Research
- Inventors: Jaichandran Sivalingam; Steve Oh; Shaul Reuveny
- Web page: https://patentgrep.com/patent/US20200399602A1

## CPC

- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 5/0641: {Erythrocytes}
- C12N 2501/10: Growth factors
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/14: Erythropoietin [EPO]
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/165: Vascular endothelial growth factor [VEGF]
- C12N 2501/20: Cytokines; Chemokines
- C12N 2501/2303: Interleukin-3 (IL-3)
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2501/39: Steroid hormones
- C12N 2501/392: Sexual steroids
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/602: Sox-2
- C12N 2501/603: Oct-3/4
- C12N 2501/604: Klf-4
- C12N 2501/606: c-Myc
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/00: Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N 2506/02: from embryonic cells
- C12N 2506/11: from blood or immune system cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2510/00: Genetically modified cells
- C12N 2527/00: Culture process characterised by the use of mechanical forces, e.g. strain, vibration
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods of differentiation of pluripotent stem cells into hematopoietic precursor cells, wherein the method is carried out under suspension agitation, and wherein a GSK-3-inhibitor or a Wnt pathway activator is added during a stage of mesoderm induction, and cell culture media for use in the methods, as well as kits for performing the same.

## Claims

### Claim 1

A method of differentiation of pluripotent stem cells into hematopoietic precursor cells, wherein the method is carried out under suspension agitation, and wherein a GSK-3-inhibitor or a Wnt pathway activator is added during a stage of mesoderm induction.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method of differentiation of pluripotent stem cells into hematopoietic precursor cells comprises the following stages:
a. the mesoderm induction stage;
b. a hematopoietic induction stage;
c. an erythroblast induction stage; and
d. an erythroblast maturation stage.

### Claim 3 (depends on 2)

The method of claim 2, wherein during the pluripotent stem cell expansion stage, pluripotent stem cells are expanded to a concentration of between 1.5×105 to 4×106cells/ml.

### Claim 4 (depends on 2, 3)

The method of claim 2 or 3, wherein the mesoderm induction stage results in the induction of mesoderm formation in pluripotent stem cells from the pluripotent stem cell expansion stage, resulting in KDR+PDGFRα− hematopoietic precursor cells.

### Claim 5 (depends on 2, 3, 4)

The method of any one of claims 2 to 4, wherein the hematopoietic induction stage results in an expansion of hematopoietic precursor cells from the mesoderm induction stage, resulting in CD34/CD43/CD45 hematopoietic progenitor cells.

### Claim 6 (depends on 2, 3, 4, 5)

The method of any one of claims 2 to 5, wherein the erythroblast expansion stage results in an expansion of hematopoietic progenitor cells from the hematopoietic induction stage, resulting in CD235a+CD71+ erythroblast cells.

### Claim 7 (depends on 2, 3, 4, 5, 6)

The method of any one of claims 2 to 6, wherein the erythroblast maturation stage results in terminal maturation and enucleation of mature CD235a+ erythroblast cells from the erythroblast expansion stage, resulting in enucleated CD235a+ erythroblast cells.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The method of any of claims 1 to 7, wherein the method comprises use of a cell culture media during the mesoderm induction stage, the cell culture media comprising
a bone morphogenic protein,
GSK3-inhibitor, or a Wnt pathway activator,
Activin A, and
a vascular endothelial growth factor.

### Claim 9

The method of any of the preceding claims, wherein the GSK3-inhibitor is selected from the group consisting of valproic acid sodium salt, staurosporine, KT 5720 (CAS 108068-98-0), GSK-3 Inhibitor IX (CAS 667463-62-9), Ro 31-8220 (CAS 138489-18-6), SB-216763 (CAS 280744-09-4), CID 755673 (CAS 521937-07-5), Kenpaullone (CAS 142273-20-9), lithium chloride, GSK-3beta Inhibitor XII (TWS119; CAS 601514-19-6), GSK-3 Inhibitor XVI (CAS252917-06-9), 10Z-Hymenialdisine (CAS 82005-12-7), Indirubin (CAS 479-41-4), CHIR-98014 (CAS 252935-94-7), GSK-3beta Inhibitor VI (CAS 62673-69-2), Manzamine A (CAS 104196-68-1), Indirubin-3prime-monoxime (CAS 160807-49-8), GSK-3 Inhibitor X (CAS 740841-15-0), GSK-3 Inhibitor XV, SB-415286 (CAS 264218-23-7), 1-Azakenpaullone (CAS 676596-65-9), TWS 119 ditrifluoroacetate (CAS 601514-19-6), 5-lodo-indirubin-3′-monoxime, GSK-3beta Inhibitor I (CAS 327036-89-5), 9-Cyanopaullone, Indirubin-5-sulfonic acid sodium salt, GSK-3beta inhibitor VII (CAS 99-73-0), Cdk1/5 inhibitor (CAS 40254-90-8), Hymenidin (CAS 107019-95-4), bisindolylmaleimide X hydrochloride (CAS 131848-97-0), 3F8 (CAS 159109-11-2), isogranulatimide (CAS 244148-46-7), CR8, (R)-isomer (CAS 294646-77-8) L-779,450 (CAS 303727-31-3), indirubin-3prime-monoxime-5-sulphonic acid (CAS 331467-05-1), GSK-3 Inhibitor II (CAS 478482-75-6), GSK-3beta Inhibitor VIII (CAS 487021-52-3), Aloisine A (CAS 496864-16-5), GSK-3beta Inhibitor XI (CAS 626604-39-5), GSK-3 Inhibitor IX (CAS 710323-61-8), Alsterpaullone, 2-Cyanoethyl (CAS 852529-97-0), TCS 2002 (CAS 1005201-24-0), TCS 21311 (CAS 1260181-14-3), A 1070722 (CAS 1384424-80-9), Ro-31-8220 (CAS 138489-18-6), Enzastaurin (CAS 138489-18-6), MeBIO (CAS 667463-95-8), Cdk2/9 Inhibitor (CAS 507487-89-0), Cdk1/2 Inhibitor III (CAS 443798-55-8), PHA 767491 hydrochloride (CAS 845714-00-3), AR-AO 14418-d3, Indole-3-acetamide (CAS 879-37-8), Hymenialdisine Analogue 1 (CAS 693222-51-4), CHIR-99021 (also known as 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile and CT99021; CAS 252917-06-9), CHIR-98014 (CAS 556813-39-9), (2′Z,3′E)-6-Bromoindirubin-3′-oxime (Bio; CAS 667463-62-9), Bio-Acetoxime (CAS 667463-85-6), SB216763 (CAS 280744-09-4) and combinations thereof.

### Claim 10

The method of any of the preceding claims, wherein the GSK3-inhibitor is CHIR-99021 or a derivative thereof.

### Claim 11

The method of any of the preceding claims, wherein the Wnt pathway activator is selected from the group consisting of IQ-1 and Wnt3a.

### Claim 12 (depends on 8, 9, 10, 11)

The method of any one of claims 8 to 11, wherein the bone morphogenic protein is BMP4.

### Claim 13 (depends on 8, 9, 10, 11, 12)

The method of any one of claims 8 to 12, wherein the vascular endothelial growth factor is VEGF165.

### Claim 14 (depends on 8, 9, 10, 11, 12, 13)

The method of claim any of claims 8 to 13, wherein the cell culture media comprises BMP4, Activin A, CHIR99021 and VEGF165.

### Claim 15 (depends on 8, 9, 10, 11, 12, 13, 14)

The method of any one of claims 8 to 14, wherein BMP4 is present in a concentration of between 26 to 36 ng/ml, wherein Activin A is present in a concentration of between 35 to 46 ng/ml, wherein CHIR-99021 is present in a concentration of between 8 μM to 14 μM, and wherein VEGF165 is present in a concentration of between 48 ng/ml to 51 ng/ml.

### Claim 16

A cell culture media for differentiation of pluripotent stem cells into hematopoietic precursor cells, thereby generating hematopoietic precursor cells from pluripotent stem cells using microcarrier embryoid bodies (EB), the cell culture media comprising
a bone morphogenic protein,
a GSK-3 kinase inhibitor, wherein the inhibitor is selected from the group consisting of CHIR99021, (2′Z,3′E)-6-Bromoindirubin-3′-oxime (Bio; CAS 667463-62-9), Kenpaullone (CAS 142273-20-9), GSK-3beta Inhibitor XII (TWS119; CAS 601514-19-6), Bio-Acetoxime (CAS 667463-85-6), CHIR-98014, SB216763 (CAS 280744-09-4), GSK-3beta Inhibitor VIII (CAS 487021-52-3) and combinations thereof, or a Wnt pathway activator,
Activin A, and
a vascular endothelial growth factor.

### Claim 17 (depends on 16)

The cell culture media of claim 16, wherein the hematopoietic precursor cells are KDR+PDGFRα− hematopoietic precursors cells.

### Claim 18 (depends on 16, 17)

The cell culture media of any one of claims 16 to 17 for use in agitation suspension culture.

### Claim 19 (depends on 16, 17, 18)

The cell culture media of any one of claims 16 to 18, wherein the bone morphogenic protein is BMP4.

### Claim 20 (depends on 16, 17, 18, 19)

The cell culture media of any one of claims 16 to 19, wherein the Wnt pathway activator is selected from the group consisting of IQ-1 and Wnt3a.

### Claim 21 (depends on 16, 17, 18, 19, 20)

The cell culture media of any one of claims 16 to 20, wherein the vascular endothelial growth factor is VEGF165.

### Claim 22 (depends on 16, 17, 18, 19, 20, 21)

The cell culture media of any one of claims 16 to 21, wherein the cell culture media comprises BMP4, Activin A, CHIR99021 and VEGF165.

### Claim 23 (depends on 16, 17, 18, 19, 20, 21, 22)

The cell culture media of any one of claims 16 to 22, wherein BMP4 is present in a concentration of between 26 ng/ml to 36 ng/ml, wherein Activin A is present in a concentration of between 35 to 46 ng/ml, wherein CHIR99021 is present in a concentration of between 8 μM to 14 μM, and wherein VEGF165 is present in a concentration of between 48 ng/ml to 51 ng/ml.

### Claim 24

A cell culture media for differentiation of pluripotent stem cells into hematopoietic precursor cells, thereby generating hematopoietic precursor cells from pluripotent stem cells using microcarrier embryoid bodies (EB) or pluripotent stem cells, the cell culture media comprising
a bone morphogenic protein,
Activin A, and
a vascular endothelial growth factor.

### Claim 25 (depends on 24)

The cell culture media of claim 24, wherein the bone morphogenic protein is BMP4.

### Claim 26 (depends on 24, 25)

The cell culture media of any one of claims 24 to 25, wherein the vascular endothelial growth factor is VEGF165.

### Claim 27 (depends on 24, 25, 26)

The cell culture media of any of claims 24 to 26, wherein the cell culture media comprises BMP4, Activin A, and VEGF165.

### Claim 28 (depends on 24, 25, 26, 27)

The cell culture media of any one of claims 24 to 27, wherein BMP4 is present in a concentration of between 26 to 36 ng/ml, wherein Activin A is present in a concentration of between 35 to 46 ng/ml, and wherein VEGF165 is present in a concentration of between 48 ng/ml to 51 ng/ml.

### Claim 29

A cell culture media for differentiation of pluripotent stem cells into hematopoietic precursor cells, thereby generating hematopoietic precursor cells from pluripotent stem cells using microcarrier embryoid bodies (EB) or pluripotent stem cells, the cell culture media comprising
a bone morphogenic protein,
Activin A,
bFGF (basic fibroblast growth factor) or variant thereof,
a hormone,
a cytokine, and
a vascular endothelial growth factor.

### Claim 30 (depends on 29)

The cell culture media of claim 29, wherein the bone morphogenic protein is BMP4.

### Claim 31 (depends on 29, 30)

The cell culture media of any one of claims 29 to 30, wherein the hormone is beta-estradiol.

### Claim 32 (depends on 29)

The cell culture media of claim 29, wherein basic fibroblast growth factor (bFGF) or variant thereof is a heat-stable chimeric variant of bFGF or a stable chimeric fibroblast growth factor (FGF).

### Claim 33 (depends on 29, 30, 31, 32)

The cell culture media of any one of claims 29 to 32, wherein the cytokine is stem cell factor (SCF).

### Claim 34 (depends on 29, 30, 31, 32, 33)

The cell culture media of any one of claims 29 to 33, wherein the vascular endothelial growth factor is VEGF165.

### Claim 35 (depends on 29, 30, 31, 32, 33, 34)

The cell culture media of any of claims 29 to 34, wherein the cell culture media comprises BMP4, Activin A, bFGF, beta-stradiol, SCF and VEGF165.

### Claim 36 (depends on 29, 30, 31, 32, 33, 34, 35)

The cell culture media of any one of claims 29 to 35, wherein BMP4 is present in a concentration of between 18 ng/ml to 27 ng/ml, wherein Activin A is present in a concentration of between 3 ng/ml to 7 ng/ml, wherein the bFGF is present in a concentration of between 5 ng/ml to 14 g/ml, wherein the beta-estradiol is present in a concentration of between 0.2 ng/ml to 0.8 ng/ml, wherein the SCF is present in a concentration of between 26 ng/ml to 36 g/ml, and wherein VEGF165 is present in a concentration of between 32 to 38 ng/ml.

### Claim 37 (depends on 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76)

A method for differentiation of pluripotent stem cells into hematopoietic precursor cells, wherein the method is carried out under suspension agitation, the method comprising
a. exposing pluripotent stem cells to the cell culture medium as defined in any one of claims 15 to 31 for 24 hours (day 0 to day 1), thereby resulting in T-Brachyury (T-Bra; primitive streak/early mesoderm marker) positive cells;
b. exposing the cells of step a. to the cell culture medium according to any one of claims 48 to 57 for 24 hours (day 1 to day 2);
c. exposing the micro-carrier attached cells of step c. to the cell culture medium according to any one of claims 58 to 76 for 48 hours (day 2 to day 4), whereby steps a. to b. induce mesoderm induction;
d. removing the cell culture medium, and isolating the resulting KDR+PDGFRα− hematopoietic precursor cells of step c.

### Claim 38

A method for differentiation of pluripotent stem cells into hematopoietic precursor cells, wherein the method is carried out under suspension agitation, the method comprising
(a) inducing mesoderm induction in pluripotent stem cells isolated from a cell culture subjected to suspension agitation thereby resulting in KDR+PDGFRα− hematopoietic precursor cells;
(b) inducing hematopoietic induction in the cells isolated from step a, thereby resulting in CD34/CD43/CD45 hematopoietic progenitor cells;
(c) inducing erythroblast expansion in the cells isolated from step b, thereby resulting in CD235a+CD71+ erythroblast cells;
(d) inducing erythroblast maturation in the cells isolated from step c, thereby resulting in CD235a+ DRAQ5-ve enucleated erythroblast cells;
(e) removing the cell culture medium, and isolating the resulting CD235a+ DRAQ5-ve enucleated erythroblast cells of step d.

### Claim 39 (depends on 37, 38)

The method according to any one of claims 37 to 38, wherein the pluripotent stem cells are attached to microcarriers.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The method according to any one of claims 1 to 15, the cell culture media according to any of claims 16 to 36, or the method according to any one of claims 37 to 39, wherein the pluripotent stem cells are induced pluripotent stem cells.

### Claim 41 (depends on 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)

A kit comprising micro-carriers and the cell culture media according to any of claims 16 to 36.