US 11,773,375 B2
In vivo method for differentiating human pluripotent stem cells into atrial cardiomyocytes
Stefan Robbert Braam, Leiden (NL); Ana Catarina Martins Grandela, Leiden (NL); and Karin Langenberg, Leiden (NL)
Assigned to Ncardia B.V., Leiden (NL)
Appl. No. 16/88,227
Filed by Ncardia B.V., Leiden (NL)
PCT Filed Mar. 27, 2017, PCT No. PCT/NL2017/050190
§ 371(c)(1), (2) Date Sep. 25, 2018,
PCT Pub. No. WO2017/164746, PCT Pub. Date Sep. 28, 2017.
Claims priority of application No. NL2016496 (NL), filed on Mar. 25, 2016.
Prior Publication US 2020/0216812 A1, Jul. 9, 2020
Int. Cl. C12N 5/077 (2010.01)
CPC C12N 5/0657 (2013.01) [C12N 2500/38 (2013.01); C12N 2501/155 (2013.01); C12N 2501/999 (2013.01); C12N 2506/45 (2013.01)] 13 Claims
 
1. An in vitro method of differentiating human pluripotent stem cells into mature atrial cardiomyocytes expressing COUPTFII and COUPTFI, the method comprising the steps of:
a) culturing the human pluripotent stem cells in an aqueous media containing 1-7 μM of a Wnt-signaling agonist and/or a BMP-signaling agonist for a period of about 24-54 hours;
b) culturing the Wnt-signaling agonist and/or BMP-signaling agonist-treated cells obtained from step (a) in an aqueous media containing 0.1-10 μM of a Wnt-signaling antagonist for a period of about 24-54 hours,
wherein during step b) the Wnt-signaling agonist and/or BMP-signaling agonist-treated cells are provided with 2-10 μM of retinoic acid or all-trans retinoic acid within the first 72 hours of step a) and b) and wherein the cells are treated with retinoic acid or all-trans retinoic acid for a period of at least 24 hours; and
c) replacing the aqueous medium of the cells obtained after step (b) with an aqueous medium devoid of the Wnt-signaling agonist and/or BMP-signaling agonist and devoid of the Wnt-signaling antagonist such that mature atrial cardiomyocytes expressing COUPTFII and COUPTFI are obtained.