Method for Differentiation of Human Pluripotent Stem Cell Lines in Suspension Culture

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.

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.

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.

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.

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.