US 12,104,173 B2
Method for differentiation of human pluripotent stem cell lines in suspension culture
Jaichandran Sivalingam, Singapore (SG); Steve Oh, Singapore (SG); and Shaul Reuveny, Singapore (SG)
Assigned to AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH, Singapore (SG)
Appl. No. 16/962,973
Filed by AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH, Singapore (SG)
PCT Filed Jan. 15, 2019, PCT No. PCT/SG2019/050023
§ 371(c)(1), (2) Date Jul. 17, 2020,
PCT Pub. No. WO2019/143292, PCT Pub. Date Jul. 25, 2019.
Claims priority of application No. 10201800488W (SG), filed on Jan. 18, 2018.
Prior Publication US 2020/0399602 A1, Dec. 24, 2020
Int. Cl. C12N 5/0789 (2010.01); C12N 5/078 (2010.01)
CPC C12N 5/0647 (2013.01) [C12N 5/0641 (2013.01); C12N 2501/10 (2013.01); C12N 2501/115 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/20 (2013.01); C12N 2501/392 (2013.01); C12N 2501/415 (2013.01); C12N 2501/727 (2013.01); C12N 2501/999 (2013.01); C12N 2506/00 (2013.01); C12N 2506/11 (2013.01); C12N 2527/00 (2013.01)] 12 Claims
 
1. A method for differentiation of pluripotent stem cells into hematopoietic precursor cells, wherein the method is carried out under continuous suspension agitation, the method comprising the steps of:
(a) culturing pluripotent stem cells in a cell culture medium comprising BMP4, CHIR99021, Activin A and VEGF165 for 24 hours to produce T-Brachyury (T-Bra) positive cells,
(b) culturing the cells obtained in step (a) in a cell culture medium comprising BMP4, Activin A and a vascular endothelial growth factor for 24 hours,
(c) culturing the cells of step (b) in a cell culture medium comprising BMP4, Activin A, bFGF or a variant thereof, a hormone, a cytokine and a vascular endothelial growth factor for 48 hours to produce KDR+PDGFRα-hematopoietic precursor cells; and
(d) removing the cell culture medium from step (c) and isolating the KDR+PDGFRα− hematopoietic precursor cells.